Download Transfer of DNA from Genetically Modified Organisms (GMOs)

Transfer of DNA from Genetically Modified
Organisms (GMOs)
A review on present scientific achievements
Prepared at the
Biotechnological Institute
Kogle Allé 2
DK-2970 Hørsholm
By
Mads Grønvald Johnsen
Flemming Jørgensen
Lars Hagsholm Pedersen
Holger Rieman
Peter Stougaard
YEAR 2000
TRANSFER OF DNA
TABLE OF CONTENTS
TABLE OF CONTENTS ............................................................................................................I
PREFACE.................................................................................................................................IV
FORORD ..................................................................................................................................IV
SUMMARY............................................................................................................................... V
RESUMÉ ..................................................................................................................................VI
1
INTRODUCTION AND DEFINITIONS......................................................................1
1.1
1.2
Definitions ..............................................................................................................1
Dissemination of genes...........................................................................................1
1.2.1
1.2.2
1.2.3
1.3
2
References ..............................................................................................................4
SELECTABLE DNA MARKERS .................................................................................5
2.1
2.2
2.3
Summary.................................................................................................................5
Recombinant DNA and cells ..................................................................................5
Organisation of Antibiotic Resistance Genes .........................................................6
2.3.1
2.3.2
Structure of gene cassettes .............................................................................................................. 7
Structure of integrons...................................................................................................................... 8
2.4.1
Naturally occurring antibiotic resistance........................................................................................ 9
2.4
Antibiotic resistance in natural environments ........................................................9
2.5
3
References ............................................................................................................11
CONTAINMENT SYSTEMS ......................................................................................13
3.1
3.2
3.3
3.4
3.5
3.6
3.7
4
Summary...............................................................................................................13
Containment of organisms....................................................................................13
Suicide or killing functions...................................................................................14
Controlled expression of killing functions ...........................................................15
Containment efficacy............................................................................................17
In vivo efficiency of biological containment systems ..........................................17
References ............................................................................................................18
ENVIRONMENTAL ASPECTS OF DNA TRANSFER...........................................21
4.1
4.2
4.3
Summary...............................................................................................................21
Introduction ..........................................................................................................21
Natural competence ..............................................................................................22
4.3.1
4.3.2
4.3.3
4.3.4
4.4
4.5
DNA uptake by Streptococcus ....................................................................................................... 23
DNA uptake by Bacillus ................................................................................................................ 23
DNA uptake by Neisseria............................................................................................................... 24
DNA uptake by Acinetobacter ....................................................................................................... 24
DNA transfer and bacterial evolution...................................................................25
Persistence of naked and recombinant DNA........................................................26
4.5.1
4.5.2
4.5.3
Fate of naked DNA in soil ............................................................................................................. 26
Fate of naked DNA in aquatic systems.......................................................................................... 27
Persistence of recombinant DNA .................................................................................................. 27
4.6.1
4.6.2
4.6.3
4.6.4
Bacterial DNA uptake in soil......................................................................................................... 28
DNA uptake from water................................................................................................................. 30
DNA uptake from food................................................................................................................... 30
Miscellaneous data on transformation .......................................................................................... 31
4.6
4.7
4.8
Transformation................................................................................................................................ 3
Transduction.................................................................................................................................... 3
Conjugation..................................................................................................................................... 3
Natural transformation in the environment ..........................................................28
Conclusions ..........................................................................................................31
References ............................................................................................................32
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GENETICALLY MODIFIED ORGANISMS
5
TRANSFER OF DNA FROM LIVE GMO ................................................................38
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
6
Summary...............................................................................................................38
Conjugative plasmid transfer and DNA transposition..........................................38
Which bacteria can transfer and receive genetic information ..............................38
Factors of importance for the transfer process .....................................................39
Conjugational transfer rates..................................................................................39
Molecular phylogenetic evidence for transfer ......................................................39
Transfer of DNA from Ti and Ri plasmids...........................................................39
Conclusions ..........................................................................................................40
References ............................................................................................................40
DNA TRANSFER FROM FUNGI TO NON-FUNGAL HOSTS .............................42
6.1
6.2
6.3
6.4
6.5
6.6
7
Summary...............................................................................................................42
Exchange of genetic material between fungi........................................................42
Transposons ..........................................................................................................42
Transfer of genetic information to fungi from other organisms ...........................43
Conclusions ..........................................................................................................43
References ............................................................................................................43
DNA TRANSFER FROM FOOD AND NON-BACTERIA......................................45
7.1
7.2
7.3
7.4
7.5
7.6
8
Summary...............................................................................................................45
Genetically modified foods ..................................................................................45
Uptake of DNA from recombinant foods .............................................................45
Evidence for horizontal transfer of DNA .............................................................45
Conclusions ..........................................................................................................46
References ............................................................................................................46
DNA UPTAKE AND TRANSFER IN EUKARYOTES............................................48
8.1
8.2
8.3
8.4
8.5
9
Summary...............................................................................................................48
Exposure to and ingestion of naked DNA. ...........................................................48
DNA uptake and transfer by mammalian microflora. ..........................................50
Naked DNA as vaccine delivery systems.............................................................50
References ............................................................................................................52
DNA TRANSFER BY BACTERIOPHAGES ............................................................54
9.1
9.2
9.3
Summary...............................................................................................................54
Introduction ..........................................................................................................54
Phages in the environment: Presence and transduction........................................54
9.3.1
9.3.2
9.3.3
9.3.4
9.3.5
9.3.6
9.3.7
9.3.8
Biological barriers to transduction ............................................................................................... 55
Physiochemical barriers to transduction ...................................................................................... 55
Transduction in situ....................................................................................................................... 55
Phage transduction: Plants ........................................................................................................... 56
Phage transduction: soils.............................................................................................................. 56
Phage transduction: Animals ........................................................................................................ 57
Phage transduction: The human intestine ..................................................................................... 57
Phage transduction: Comestibles.................................................................................................. 57
9.4
9.5
Conclusions ..........................................................................................................57
References ............................................................................................................58
10
DNA TRANSFER BY EUKARYOTIC VIRUSES ....................................................60
10.1
10.2
10.3
10.4
- II -
Summary...............................................................................................................60
Viral gene therapy.................................................................................................60
Viral therapy systems ...........................................................................................60
Viral containment .................................................................................................61
TRANSFER OF DNA
10.5
Unintended presence of therapeutic viruses. ........................................................63
10.5.1 Replication competent viruses....................................................................................................... 63
10.5.2 General concerns. ......................................................................................................................... 66
10.6
10.7
10.8
Environmental risk assessment.............................................................................67
Conclusion and suggestions..................................................................................67
References ............................................................................................................68
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GENETICALLY MODIFIED ORGANISMS
PREFACE
This report on transfer of DNA from genetically modified organisms (GMOs) to other
organisms is a literary review resulting from information collected during the spring of year
2000. The Danish Ministry of Environment and Energy has requested and financed the report.
The contributing authors are: Mads Grønvald Johnsen, Flemming Jørgensen, Lars Hagsholm
Pedersen, Holger Rieman and Peter Stougaard, Biotechnological Institute, Denmark. Heidi
K. Rønnest kindly helped editing the final manuscript.
A steering committee was appointed to follow and counsel the authors. The members of the
steering committee were:
Finn Bech:
The Danish Ministry of Environment and Energy; The National Forest and Nature Agency.
Bodil Jacobsen:
The Danish Veterinary and Food Administration; Institute of Food Safety and Toxicology.
Ilona Sørensen:
The Danish Veterinary and Food Administration; Institute of Food Safety and Toxicology.
Niels Bohse Hendriksen:
The Danish Ministry of Environment and Energy; The National Environmental Research
Institute.
FORORD
Denne rapport omfatter et litteraturstudie gennemført i foråret år 2000 og omhandler mulig
overførsel af DNA fra genetisk modificerede organismer (GMOer). Rapporten er bestilt og
finansieret af Miljø- og Energiministeriet i Danmark.
Rapporten er forfattet af Mads Grønvald Johnsen, Flemming Jørgensen, Lars Hagsholm
Pedersen, Holger Rieman og Peter Stougaard, Bioteknologisk Institut, Danmark. Heidi K.
Rønnest var behjælpelig med redigering af rapporten.
Under projektforløbet har en styregruppe fulgt og rådgivet forfatterne. Styregruppens
medlemmer var:
Finn Bech,
Miljø- og Energiministeriet, Skov- og Naturstyrelsen.
Bodil Jacobsen,
Fødevaredirektoratet, Institut for Fødevaresikkerhed og Toksikologi.
Ilona Sørensen,
Fødevaredirektoratet, Institut for Fødevaresikkerhed og Toksikologi.
Niels Bohse Hendriksen,
Miljø- og Energiministeriet, Danmarks Miljøundersøgelser.
- IV -
TRANSFER OF DNA
SUMMARY
From the literature presented in this review it can be concluded that DNA from genetically
modified organisms (GMOs) can be shuttled between organisms. Especially bacteria are well
documented to have natural competence for uptake of foreign DNA. However, this
competence only allows for a limited number of transformation events to occur. The numbers
describing the frequency of transformation events are important in the evaluation of
environmental impact as well as risk assessment. It has become clear that only few studies
describe transformation during field conditions. Previously, the inability to cultivate a large
proportion of microorganisms, which are present in many habitats, has been a major obstacle
in the evaluation of DNA shuffling. With the introduction of polymerase chain reaction (PCR)
technology, it should now be possible to investigate the extension and impact of gene transfer
within the group of ‘un-culturable’ microorganisms. In bacteria, the ability of bacteriophages
to transduce DNA between strains has been shown to work effectively, but the influence on
field populations is a topic that has not yet received much attention in research.
The genetic material of recombinant DNA is often engineered with genes that express
resistance against antibiotics. As antibiotics still have profound clinical value it is feared that
resistance may be transferred from GMOs to pathogen microorganisms. Field results have
revealed that the number of ‘natural’ resistance genes can be enormous and therefore, it is
assumed that the resistance introduced by GMOs may be practically undetectable. There are
several technological solutions that can improve containment of GMOs. Even systems that
allow GMOs to function in the environment and then selfdestruct are being developed.
The public debate on the use of GMOs is very concerned about the risk of transfer of
manipulated genetic material from GMOs to humans. This ability has been evaluated in a
series of experiments. However, no toxic or documented genetic effects have been reported. At
present, it is tested if recombinant DNA constructions can be administrated to humans in
order to obtain vaccine response or to supplement genes with abnormal functionality. This
genetic supplementation process that is introduced with the gene therapy methods, is made
possible with modified viruses as vectors. Gene therapy systems are still at an early
experimental stage, but restrictions and risk assessment has to be applied already at this
point. Detection systems that allow for standardized evaluation of the specific systems need to
be developed.
-V-
GENETICALLY MODIFIED ORGANISMS
RESUMÉ
Fra den litteratur som er samlet i denne gennemgang, kan det konkluderes, at genetisk
modificerede organismer (GMOer) kan udveksle DNA med andre organismer. Især har
bakterier en veldokumenteret evne til at optage fremmed DNA. Denne kompetence er
imidlertid begrænset og tillader kun få transformations begivenheder. Det er vigtigt, at der
sættes tal på omfanget af transformationen således, at der kan fortages en vurdering af den
miljømæssige betydning og risiko. Det er blevet klart, at der kun er gennemført ganske få
forsøg med transformation under ’felt’ forhold. Det har tidligere været en stor hindring for
undersøgelse af DNA spredning mellem mikroorganismer, at hovedparten af de
mikroorganismer, der findes i mange habitater, ikke kan kultiveres. Med udviklingen af
polymerase chain reaction teknikken (PCR) er det imidlertid blevet muligt at gennemføre
undersøgelser, der kan belyse udbredelsen og betydningen af genoverførsel mellem
organismer, der normalt er ’ikke-kultiverbare’. I bakterier kan DNA overføres effektivt
mellem stammer via transducerende bakteriofager. Betydningen af denne overførselsform på
udviklingen af bakteriepopulationer er et emne, som imidlertid ikke er grundigt udforsket.
Genetisk materiale bestående af rekombinant DNA indeholder ofte konstruktioner, hvor der
indgår resistens rettet mod antibiotika. Da antibiotika stadig er en vigtig del af kliniske
behandlinger, har der været udtrykt stigende bekymring for, hvorvidt resistensgener kan
overføres fra GMOer til patogene organismer. Feltforsøg har vist, at antallet af ’naturlige’
resistensgener kan være enormt, og det antages derfor, at resistensen fra introducerede
GMOer i mange tilfælde overdøves. Der findes flere systemer, der kan bidrage til at GMOer
ikke spredes. På nuværende tidspunkt udvikles systemer, der tillader udsættelse af GMOer,
som er selvdestruktive efter at deres funktion er fuldbragt.
Gennem den offentlige debat vedrørende GMOer er der endvidere udtrykt bekymring over
risikoen for overførsel af manipuleret genetisk materiale til mennesker. Denne risiko er blevet
vurderet i en række forsøg, hvor resultaterne ikke viste toxiske effekter eller dokumenteret
genetiske ændringer. Der afprøves for tiden nye behandlingssystemer, hvor rekombinant DNA
tilføres mennesker for at opnå vaccination eller for at udbedre unormale genfunktioner. Den
genetiske supplering som kendetegner genterapi, er blevet mulig ved brug af modificeret virus
som vektorer. Genterapi er stadig på et tidligt udvikligsstadie, men det er vigtigt at inddrage
restriktioner og risikovurdering allerede på dette tidspunkt. Der er behov for at udvikle
standardiserede systemer til detektion og evaluering af de specifikke systemer.
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TRANSFER OF DNA
1
INTRODUCTION AND DEFINITIONS
by Peter Stougaard
1.1
Definitions
Genetically Modified Organisms (GMOs) have been defined as plants, animals, micro-organisms, cell cultures,
and virus containing new combinations of genetic material, which can not arise naturally (Lov om miljø og
genteknologi nr 356 af 06/06/1991).
Recently, GMOs have been the subject of both ethical, political, environmental, and health
debates. Since GMOs may interact with other organisms in the environment while alive or as
dead material, the potential impact of GMOs on the environment or on human health is very
complex. Due to this complexity it is not surprising that risk assessment of GMOs is very
difficult and that it has been hard for politicians and their government officers to acquire the
necessary overview of all possible problems associated with e.g. deliberate release of GMOs
into the environment or release of GMOs for food use. One of the major public concerns is
the undeliberate transfer of recombinant DNA from GMOs, being alive or dead, to other
organisms in the environment or to humans. However, the potential dissemination of DNA
from GMOs has been the subject of several recent reviews and research papers. In this report,
we shall accumulate and discuss these publications, we will compile the state of the art on
transfer of DNA from GMOs, and we shall make recommendations on additional future
research areas that should be launched in order to establish the necessary technical platform
for future risk assessment.
In this report, we shall review transfer of DNA from GMOs from an environmental point of
view and from human health associated perspectives. We will focus on the potential uptake of
naked DNA or DNA from dead organisms and we shall discuss the transfer of DNA from live
genetically modified microorganisms to other organisms. However, we shall not cover the
transfer of DNA from living GMO-plants to other plants in the environment since this topic
has already been covered in recent reports (e.g. Jørgensen, 1999; Jørgensen et al., 1999;
Rognli et al., 1999; Snow et al., 1999).
1.2 Dissemination of genes
The transfer of genes between bacteria has gained much interest since the first multi-drug
resistant bacteria appeared in the 60-ties and 70-ties as a consequence of the introduction of
new antibiotics for treatment of human infections and as growth enhancers in agriculture.
Since GMOs often contain antibiotic resistance genes, one of the main concerns has been if
dissemination of such genes from GMOs would result in disastrous, multi-drug resistant,
pathogenic bacteria similar to the situation in the 60-ties. In order for this to happen, the first
event would be the uptake or transfer of genetic elements to the recipient bacteria followed by
incorporation of the genes into the chromosome and stable propagation to the progeny.
Genes that are naturally transferred between organisms have been shown to be organized into
speciallised genetic elements designed for transfer. In their simplest form these elements are
just gene cassettes containing open reading frames encoding for example an antibiotic
resistance trait but if such cassettes are combined with other genetic elements they may
constitute more complex elements like transposons or plasmids. Gene cassettes and the more
complex elements, integrons, are described in chapter 2 and summerized in Table 1 below.
Transposons and self-transmissible plasmids (Table 1) have been described in detail in
various textbooks and will not be described in this report.
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GENETICALLY MODIFIED ORGANISMS
TABLE 1. Characteristics of different elements involved in transfer of genes
Element
Characteristics
Role in gene transfer
Gene cassette
Circular, nonreplicating DNA
elements containing only open
reading frames; may integrate
into integrons
May carry antibiotic resistance
genes
Integron
DNA element containing an
integrase, a promoter, and an
integration site for gene cassettes
Integron promoter expresses
gene cassette(s); forms clusters
of genes
Transposon
May contain integrons and other
genes. Can move from one DNA
segment to another within the
same cell
Can transfer genes between
genetic elements within the cell,
i.e. from chromosome to plasmid
or vice versa
Mobilizable plasmid
Circular, self-replicating
element; contains gene(s) that
allows the plasmid to use
conjugal apparatus provided by
self-transmissible plasmid
Transfer of genes between cells
Conjugative transposon
Integrated elements that can
excise to form a non-replicating
circular transfer intermediate;
carries genes needed for conjugal
transfer
Transfer of genes between cells;
may mobilize other elements
Self-transmissible plasmid
Circular, self-replicating
element; carries genes needed for
conjugal transfer
Transfer of genes between cells;
may mobilize other elements
The genetic elements outlined in Table 1 may be taken up by or transferred between bacteria
through various transfer mechanisms: transformation; transduction; and conjugation.
All the different transfer mechanisms have been shown to occur naturally in the environment
or in the human intestine. However, since few results exist on transfer of genes from GMOs
very little is known about possible routes of DNA transfer from GMOs to other organisms. In
this and the following chapters, we shall review the natural transfer mechanisms and we will
describe and discuss in detail the few results published on the transfer of DNA from GMOs to
other organisms.
TABLE 2. Environments where horizontal gene transfer has been documented
Terrestrial environment
Water environment
Other environments
Transformation
Soil
Marine sediments, rivers,
epilithon on river stones
Plants, insects, intestinal
flora in mice
Transduction
Soil, plant surfaces
Lakes, oceans rivers, sewage
in treatment facilities
Shellfish, intestinal flora in
mice
Conjugation
Soil, plant surfaces
Lakes, oceans, marine
sediment, rivers, epilithon on
river stones, sewage in
treatment facilities
Plants, insects, intestinal
flora in chickens, mice, and
humans
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TRANSFER OF DNA
1.2.1 Transformation
Transformation is a process by which a (bacterial) cell takes up naked DNA from the
surrounding medium and incorporates it. In order to be able to take up DNA via
transformation a bacterium must be in a state of competence. Within both Gram-positive and
Gram-negative bacteria it is part of the normal physiology to become competent for uptake of
DNA, though the exact conditions for competency varies. Both plasmids and chromosomal
DNA may be taken up by transformation and it is believed that transformation plays an
essential role in the transfer of DNA in nature.
The donor DNA for the transformation process may come from DNA released from live
bacteria or leaked from dead organisms. The accessibility, survival, persistence and
movement of naked DNA may vary depending on the surrounding environment. Several
biotic as well as abiotic factors influence the availability of donor DNA and the competence
of recipient cells. Parameters like soil composition, pH, temperature, nutrients, salts, UV/light
irradiation etc. have been shown to influence transformation. The transformation process is
described in more detail in chapter 4.
1.2.2 Transduction
Transduction is a DNA transfer mechanism where the transfer of bacterial genes from one
bacterium to another is mediated by bacterial viruses (bacteriophages). Transduction is
generally believed to be one of the most important gene transfer mechanisms in the generation
of diversity and bacterial evolution between closely related bacterial groups. It has been
shown that transduction occurs at high frequencies both in lake- and sea-water and it is
believed that this transfer mechanism is important in soil also. The recipient cells must
generally be in a state which is sensitive to infection by the transducing bacteriophage. As
with other viruses, bacteriophages require the presence of specialised cell-surface associated
receptors in order to infect a recipient cell. Two types of transduction are recognised,
generalised and specialised.
In generalised transduction any DNA fragment may be packaged into the bacteriophage head.
Some transducing bacteriophages contain only chromosomal or plasmid DNA and no
bacteriophage DNA whereas others contain both bacteriophage DNA and “foreign” DNA.
The geometry and the size of the bacteriophage head determine the amount of DNA that can
be packaged into the head and transferred by transduction.
In specialised transduction only a specific host chromosomal site is transduced. One or more
bacteriophage genes are replaced by host bacterial DNA through illegitimate recombination.
The transducing particles are produced only when chromosomally integrated prophage is
induced and switches to lytic growth.
1.2.3 Conjugation
In conjugation DNA transfer occurs from one bacterial cell to another through direct contact
between the cells. The DNA is transferred through a specialised conjugation apparatus
encoded for by various self-transmissible plasmids and conjugative transposons. The
conjugation apparatus, comprising the pili and proteins involved in transfer and replication of
DNA. It has been shown that conjugation may occur between closely related species, between
different genera and even between Gram-positive and Gram-negative bacteria.
Recently, it has been shown that self-transmissible plasmids are not the only conjugation
mediating elements. So-called conjugative transposons have been shown to play an important
role in the transfer of genetic material via conjugation between a wide variety of Grampositive bacteria, and similar conjugative transposons have also been observed in the Gram-3-
GENETICALLY MODIFIED ORGANISMS
negative bacterium Bacterioides. Conjugative transposons most often contain antibiotic
resistance genes and have been shown to have a very broad host range of transfer.
Some plasmids lack transfer genes and are not self-transmissible on their own. However,
those plasmids which contain so-called mobilisation and replicative origins may be
transferred utilising the transfer mechanisms provided in trans by a conjugative plasmid or
transposon. Similarly, the entire bacterial chromosome or parts of the chromosome may be
transferred via conjugation if a conjugative plasmid integrates into the chromosome.
However, as the bacterial chromosome is large relative to plasmids, transfer is often
interrupted resulting in the transfer of only a part of the chromosome.
1.3
References
Jørgensen, R.B., Andersen, B., Snow, A., and Hauser, T.B. (1999). Ecological risks of growing genetically
modified crops. Plant Biotechnol. 16 : 69-71
Jørgensen, R.B. (1999) Deliberate release of genetically modified plants. In: Genetically modified organisms in
Nordic habitats. (Nordic Council of Ministers, Copenhagen, 1999) (TemaNord 1999:542) p. 24-28
Miller, R.V. (1998). Bacterial gene swapping in Nature. Scient. Americ. 1: 47-51
Rognli, O.A., Tomiuk, J., Tufto, J., Nurminiemi, M., and Jørgensen, R.B. (1999). Gene flow from transgenic
crop plants to wild populations. NORDTEST-TR-409, 42 p.
Salyers, A.A. and Amábile-Cuevas, C.F. (1997). Why are antibiotic resistance genes so resistant to elimination.
Antimicrob. Ag. Chemother. 41: 2321-2325
Snow, A.A., Andersen, B., and Jørgensen, R.B. (1999). Costs of transgenic herbicide resistance introgressed
from Brassica napus into weedy B. rapa. Mol. Ecol., 8: 605-615
Winding, A., Kvaløy, K., Hendriksen, N.B., Gustaffson, K., Iverson, T-G., Helgason, E., and Kolstø, A-B.
(1998). Procedures for risk identification and assessment of genetically modified microorganisms. Nordtest
Report, no. NT Techn Report 381, ISSN0283-7234.
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TRANSFER OF DNA
2
SELECTABLE DNA MARKERS
by Peter Stougaard
2.1 Summary
Antibiotic resistant organisms have been shown in several investigations to be
naturally present in the environment, in animals, and in man even though no
antibiotics have been used. Resistance to antibiotics can be either natural (the
antibiotic target is absent) or acquired (caused by mutations or by uptake of
foreign resistance genes). Genes mediating acquired resistance have been used
as selective markers in development of genetically modified organisms (GMOs).
Antibiotic resistance genes found in nature are organised in gene cassettes and
may be included in integrons. Integrons may be transferred between bacteria
and higher cells at high frequencies.
However, antibiotic resistance determinants investigated so far have for obvious
reasons only been investigated in organisms which are culturable in the
laboratory. Since such culturable organisms constitute a minority of the total
microflora in the environment or in the human intestine (in some environments
1% or less, Amann et al. 1995), it is evident that a more profound knowledge of
the un-culturable organisms is needed in order to provide the missing data for
risk assessment. It has not been examined if the un-culturables constitute a
resistance sink similar to culturable organisms, or if un-culturable organisms
are able to interact in transfer of antibiotic resistance.
Furthermore, a more detailed study of the role of gene cassettes and integrons in
the dissemination of antibiotic resistance genes is needed. For example,
investigations are lacking on the mechanistic and genomic level of entrapment in
gene cassettes of resistance genes from GMOs. Also missing are determinations
of the dissemination frequencies of such genes via gene cassettes and integrons.
As the incidence of antibiotic resistant bacteria in the environment or in the
human intestine may be very high, dispersal of additional antibiotic resistance
genes from e.g. GMOs may not necessarily constitute a risk of change in the
microbial balance in the environment or in the dissemination of un-wanted
resistance determinants to human pathogenic bacteria. Thus, a detailed risk
assessment will comprise not only the un-answered questions outlined above but
also the nature of the antibiotic (whether in clinical use or not), frequencies of
horizontal transfer (to be discussed in the following chapters), and the
consequences of gene transfer.
2.2 Recombinant DNA and cells
GMOs, Genetically Modified Organisms, contain in addition to their normal, natural genetic
composition additional genetic information, either inherited as self-replicating, extrachrosomal, plasmid molecules or integrated into the host chromosome. The additional genetic
information comprises first of all the “genes of interest” encoding for example a
pharmaceutical protein or a food or industrial enzyme as well as sequence elements involved
in transcription and translation of “genes of interest”.
Furthermore, in most recombinant DNA constructions one or more selectable markers are
included in order to be able to select for the recombinant cells. The selectable markers are not
only required during the construction of the GMO, but may also be beneficial during large
scale fermentation or as tracking markers in deliberate release into the environment. The most
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GENETICALLY MODIFIED ORGANISMS
widespread selectable markers used are dominant, antibiotic resistance genes and genes
complementing auxothophic mutations in the host organisms.
Markers for direct visual tracking of recombinant GMOs in e.g. deliberate release
experiments have also been developed. Some markers require a chromogenic substrate
whereas other markers may be detected directly (e.g. flourescent proteins).
To assess the impact of DNA from GMOs on the environment or on human health, it is
necessary to evaluate the effect of the various additional genetic elements on the natural
microbial flora. The specific “genes of interest” may of course influence the balance between
microorganisms in the environment or the human gut, but as the diversity in the nature of
such genes is very large it is impossible to make an overall conclusion on environmental
impact or effect on human health. However, as antibiotic resistance genes are present in most
GMOs constructions the genetic organisation of antibiotic resistance genes and their
occurrence in natural environments is described.
2.3 Organisation of Antibiotic Resistance Genes
Since the introduction of antibiotics in the late 1940s it has been observed that antibiotic
resistance emerged in pathogenic bacteria and related bacteria. The first resistance
mechanisms observed was shown to be due to point mutations that altered the drug target. But
from the mid-1950s transmissible antibiotic resistance genes were reported from Japan (e.g.
Davies, 1997) and later in the 1980s and 1990s the molecular genetic basis for this transfer
ability of antibiotic resistance was elucidated (Baquero et al., 1998; Davies, 1994; Davies,
1997; Salyers & Amábile-Cuevas, 1997). However, the presence of antibiotic resistance in
microorganisms is not only caused by human use of antibiotics. A strain of Escherichia coli
collected and freeze-dried in 1946 was shown to harbour genes mediating resistance to
tetracycline and streptomycin although these antibiotics were used in clinic several years later
(Brock & Madigan, 1991). Other papers report similar results on the presence of antibiotic
resistance in bacterial strains collected from humans and animals that have not been treated
with antibiotics (Watanabe, 1963; Honoré & Cole, 1994).
On the other hand, several reports and reviews have been published that document the effect
of antibiotics on the presence and dissemination of antibiotic resistance genes (e.g. Baquero et
al, 1998; Davies, 1994; Davies, 1997). For example, quinolone-resistant E. coli appeared
shortly after the introduction of fluoroquinolone in a Spanish hospital (Baquero et al, 1998)
and streptomycin resistant and tetracycline-resistant bacteria appeared few years after the
introduction of the corresponding antibiotics in clinical use (reviewed by Davies, 1997).
Resistance to antibiotics can be either natural (the antibiotic target is absent) or acquired
(caused by mutations or by uptake of foreign resistance genes). For example, Mycobacterium
is naturally resistant to penicillins because they lack the peptidoglycan-layer which is the
target of penicillins, and Gram-negative bacteria are generally resistant to vancomycin
because they can not take up the antibiotic due to the small pores in their cell-envelope.
Acquired resistance may be caused by changed (mutated) antibiotic target (i.e. streptomycinresistant ribosomes or rifampicin-resistant RNA-polymerases), by increased metabolism or
inactivation of the antibiotic (i.e. penicillin- or chloramphenicol-resistance), and by increased
active efflux of the antibiotic (tetracycline resistance). The resistance mechanisms and the
origin and dissemination of antibiotic resistance determinants have been reviewed by e.g.
Davies (1994, 1997).
Whereas antibiotic resistance determinants responsible for natural resistance have not been
exploited as selective markers in genetic engineering experiments, genes mediating acquired
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TRANSFER OF DNA
resistance have been used thoroughly in biotechnology. Some of the most widespread
antibiotic resistance genes used as selection markers in genetic engineering originate from
plasmids known to be transferable between microorganisms. The antibiotic resistance genes
may even be located in special genetic compartments, gene cassettes, which themselves are
not transferable. However, if the gene cassettes are integrated into other genetic structures like
integrons, transposons, plasmids, or bacteriophages, the antibiotic resistance genes may be
transferred horizontally to other microorganisms.
2.3.1 Structure of gene cassettes
Gene cassettes form a diverse group of small mobile genetic elements found in a broad
spectrum of Gram-negative and Gram-positive bacteria. Usually, each cassette contains only a
single gene and a recombination site. At present, most of the genes found in gene cassettes are
antibiotic resistance genes although reports on cassettes containing other genes or on “empty”
cassettes have been published (Recchia & Hall, 1995). To date more that 40 different
antibiotic resistance gene cassettes have been observed (Recchia & Hall, 1995; Hall, 1997)
(Table 3).
TABLE 3. Compilation of gene cassettes found in Gram-negative and Gram-positive
bacteria
β-lactams
3 class A β-lactamases
1 class B β-lactamase
aminoglycosides
4 adenylyltransferases
8 acetyltransferases
chloramphenicol
trimethoprim
7 class D β-lactamases
1 chloramphenicol
exporter
5 class A dihydrofolate 3 class B dihydrofolate
reductases
reductases
3 acetyltransferases
streptothricin
1 acetyltransferase
antiseptics and
disinfectants
1 quaternary
ammonium compound
exporter
unidentified ORFs
5 ORFs
The cassettes vary considerably in total length and genotype due to the different sizes of the
various resistance genes in the cassettes. However, despite the diversity and functional
variation of the genes they contain, cassettes share common features (Recchia & Hall, 1995;
Hall, 1997). Each cassette contains in addition to the resistance gene, a recombination site
known as a 59-base element located down-stream of the resistance gene (Fig. 1).
59-base element
G TTRRRY
Gene
RYYYAAC
G TTRRRY
Cassette
Fig 1. Structure of a gene cassette. The core sites (GTTRRRY) at each end of the
integrated cassette is shown. The inverse core site (RYYYAAC) at the 5’ end of the 59base element is also shown. Dashed line indicates the central region of the 59-base
element.
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GENETICALLY MODIFIED ORGANISMS
DNA-sequence analysis of the known gene cassettes has shown that although the 59-base
element may vary in length and sequence, they share some common features. All gene
cassettes have been shown to harbor an inverse core site (RYYYAAC) at the end closest to
the 3’ end of the resistance cassette gene and a core site (GTTRRRY) positioned at the other
end. Most 59-base elements have a central axis of symmetry as the consensus regions are
imperfect inverted repeats, and all 59-base elements contain additional inverted sequences
positioned in the central 59-base element region.
Within the 59-base element family, groups of closely related elements can be identified. In
one group, 12-15 different resistance genes have been found, e.g. aadA genes, aadB, catB3,
and an open reading frame orfD (Recchia & Hall, 1995; Hall, 1997). Thus, it seems possible
that gene cassettes with a common 59-base element may function as a general “gene trap” for
integration of resistance genes. Though most gene cassettes normally contain a single gene
and a 59-base element, it has been observed that one cassette may contain more genes and one
59-base element. Such cassettes most probably have arisen from fusion between two or more
gene cassettes.
Though gene cassettes are not self-transmissible, they can be turned into mobile genetic
elements if they are integrated into transferable genetic elements through recombination at the
59-base element. While gene cassettes are most commonly found integrated at a specific site
in larger, mobile units, named integrons or at a non-specific, secondary site, they can exist as
covalently closed circular molecules (Collis & Hall, 1992).
2.3.2 Structure of integrons
In contrast to gene cassettes, which normally do not carry promoters, integrons harbor a
promoter located in or close to the 3’ end of the integrase gene. This promoter initiate’s
transcription of the gene cassette integrated in the integron and in the case of integration of
multiple gene cassettes, the promoter transcribes the genes as in an operon. The cassette most
proximal to the promoter is transcribed more frequently than the one more distal.
All known integrons are composed of three key elements necessary for the capture and
expression of open reading frames: 1) A gene coding for an integrase (intI), 2) a primary
recombination site (attI), and 3) a strong promoter. The integrase is responsible for the
integration of gene cassettes into the integron through recombination between the 59-base
element of the gene cassette and the integron attI site. Integrons may harbour one or more
gene cassettes and up to seven different antibiotic resistance gene cassettes have been
observed in one integron (Fig. 2). Integrated gene cassettes are normally oriented in the same
direction with respect to the integron promoter.
int
5’ conserved
res.gene 1 res.gene 2 res.gene 3 qacE∆1
3’
sulI
orf5
conserved
Fig 2. Structure of integron. int shows the position and direction of transcription of the
integrase. qac∆E1, sulI and orf5 shows the position of the 3’ conserved segment. In
between is shown the position of antibiotic resistance genes (res.gene) transcribed from
a promoter located in the 5’ conserved segment of the integron.
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TRANSFER OF DNA
Though several different integrons have been described, they share common features in the
integrase genes. A comparison of integrase sequences shows that despite the large variation in
total integron length and function, all integrons may be subdivided into three integron classes.
In one of the integron classes, class 1, all gene cassettes are integrated between the intI gene
and a sulphonamide resistance gene, sulI (Figure 2). A second class of integrons comprises
the transposon Tn7 and derivatives. In transposon Tn7 gene cassettes are integrated at a
unique site near the left end of the transposon, 3’ of a putative (but defective) intI gene,
intI2*. No sulphonamide resistance gene is observed at the other end of the integrated
cassette(s). A third class of integrons harbouring a third integrase, intI3, has also been
identified (Hall & Stokes, 1993; Sundström, 1998).
Though gene cassettes and integrons are defective for self-transposition, this defect may be
complemented through association with insertion sequence elements (IS-elements),
transposons or conjugative plasmids. In fact, it has been shown that the antibiotic resistance
genes identified in the broad host range, multi-resistance plasmids and in transposons isolated
in the 1950s and 1960s (e.g. pSa, NR1, R100, R46, R388, Tn7, Tn21, Tn1696) are organized
in cassettes and integrons. The transfer of antibiotic resistance genes via transposition,
transformation, transduction, or conjugation has been described above in chapter 1.
2.4 Antibiotic resistance in natural environments
After the introduction of antibiotics in the 1950s, whether chemically synthesized or from
natural sources, bacterial resistance to the drugs has emerged. For long time it was thought
that the use of antibiotics in hospitals or in agriculture was the major cause of the widespread
occurrence of bacterial multidrug resistance. There is little doubt, that this selection pressure
is important for the maintenance of multidrug resistant bacteria, but recently it has been
shown that human administered antibiotics are not the only selection pressures responsible for
maintenance of resistant bacteria.
In a study on leakage of sewage into the natural environment in the Lake District in England,
Jones et al. (1986a) observed that the incidence of antibiotic resistant bacteria was higher in
the lake water than in the sewage effluent and, quite unexpectedly, they found that the
frequency of antibiotic resistance was even higher in two remote upland tarns. Similar
observations were made in studies in which healthy humans or animals that had not received
antibiotic treatment showed high frequencies of antibiotic resistant intestinal bacteria (Datta,
1984; Levy, 1997; Davies, 1994).
Therefore, in order to be able to evaluate the potential impact of additional resistance genes
from GMOs on the natural bacterial flora it is necessary to investigate further the frequency
and nature of antibiotic resistances in the natural environment, being terrestrial or aquatic
environment or the human gut.
2.4.1 Naturally occurring antibiotic resistance
Presence of antibiotic resistance in the environment has been investigated over the past four
decades but with the occurrence of modern molecular methods it has become easy to
discriminate between natural and acquired resistance and furthermore, to detect if resistance is
plasmid or chromosomal encoded. Several studies on microorganisms in soil, water,
sediment, and sewage has been carried out, but due to the different methods employed it has
been difficult to compare the results from the different studies.
In soil, the occurrence of bacteria may exceed 1010 bacteria per gram dry matter. But as only
0.1 to 1% of the bacteria may be cultivated in the laboratory, the published data only describe
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GENETICALLY MODIFIED ORGANISMS
a minority of the microorganisms present in the environment. In a study by Mueller et al.
(1988) it was shown that naturally occurring Bradyrhizobium japonicum from soil with no
exogenously added antibiotics contained a high frequency of streptomycin-resistance
followed by multiple resistance against streptomycin and neomycin, streptomycin and
rifampicin, kanamycin and rifampicin, and kanamycin and nalidixic acid. In another study,
Brockman & Bezdick (1989) showed that Rhizobium leguminosarum showed resistance
against tetracycline, erythromycin, kanamycin, and neomycin. Similar results have been
reported by Sundin et al. (1994) who found that streptomycin-resistant Pseudomonas syringae
could be isolated from farmland never treated with streptomycin and that the streptomycinresistance gene was located on a transposon contained within a broad host range plasmid. In a
follow-up study Sundin et al. (1995) showed that other non-plant-pathogenic bacteria
contained the streptomycin resistance and that the frequency of occurrence was increased
upon treatment with streptomycin. In a recent paper, Schnabel & Jones (1999) presented
similar results on tetracycline resistance. Bacteria resistant to tetracycline could be isolated
from orchards not treated with tetracycline, and furthermore, the resistance genes were
located in integrons and/or on plasmid-borne transposons. In orchards treated with
tetracycline, bacteria containing tetracycline resistance genes were increased in numbers and
that resistance to other antibiotics (streptomycin and sulphonamide) was increased as well.
In aquatic habitats and sediments antibiotic resistance among bacteria has been reported as
well. As mentioned above, Jones (1986) and Jones et al. (1986a) have reported that antibiotic
resistant bacteria were observed in natural waters and sediment in the Lake District in
England. The occurence of resistant bacteria was higher in natural lake water than in sewage
effluent which discharged into the lake, and that the incidence of resistant bacteria was even
higher in two remote upland tarns. Similar results have been reported by Kobori et al. (1984)
who found antibiotic resistance and plasmids in bacteria isolated from Antarctic: 31% of the
isolates contained plasmids and approximately 10% were resistant to one or more antibiotics.
Investigations in Australia and Greece have documented very much the same results from
aquatic environments not influenced by exogenously added antibiotic or sewage, i.e. presence
of resistance to penicillin, ampicillin, and methicillin and to ampicillin, ticarcillin,
chloramphenicol, trimethoprim, kanamycin, and tetracycline, respectively (Boon, 1992;
Arvanitidou et al., 1997).
Thus, it is well documented that antibiotic resistance determinants are present in the aquatic
environment. However, human influence, either by deliberate release of antibiotics in fish
farming or by effluent sewage, has been shown to further increase the frequency of antibiotic
resistance. A comparison of resistance in bacteria from three fish-farming sites in Denmark
where antibiotics were used showed that 15% of the isolates were oxytetracycline resistant
and that 27% of the isolates were resistant to oxolinic acid. The resistance frequencies in nontreated waters were 6% and 16%, respectively (Spanggaard et al., 1993). Similar results on
human impact on the occurrence of antibiotic resistance determinants in aquatic environment
have been reported from a Spanish river. Acquired resistance to tetracycline and beta-lactams
(in Enterobacteriaceae) and to tetracycline and co-trimoxazole (in Aeromonas spp.) was
shown to be highest close to and immediatly down-stream from the discharge site of sewage
into the river (Goñi-Urriza et al., 2000). Despite the authors showed the presence of several
plasmids in the resistant isolates they were unable to demonstrate transfer of the antibiotic
resistance determinants.
Intestinal bacteria from healthy humans and animals have been shown in several
investigations to be resistant to antibiotics. Early investigations reviewed by e.g. Datta (1984),
Levy (1997), Davies (1994, 1997), showed that antibiotic resistant bacteria could be isolated
from individuals not treated with antibiotics. However, similar to the investigations of soil
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TRANSFER OF DNA
and aquatic environments it was observed that there was a positive correlation between the
use of antibiotics and high frequencies of resistance determinants.
However, in several of the early studies on bacteria from environmental, human and animal
samples, no attempts have been made to discriminate between the natural, intrinsic resistance
and the – often transferable – acquired resistance. As discussed by e.g. Jones et al. (1986b)
and Goñi-Urriza et al. (2000) other parameters make comparisons between different
investigations difficult. First of all, different isolation procedures (media, antibiotics and
concentration of antibiotics, incubation temperatures and time, inoculum sizes) may influence
the results. Furthermore, differences in the composition of the bacterial populations,
taxonomically determinations, and statistics complicate the comparison of data from different
investigations.
Two recent reports describe in more detail the prevalence of antibiotic resistant bacteria and
the spread of antibiotic resistance genes in the aquatic environment. The reader is referred to
these reports for further information (Petersen et al., 1997; Patel & Torsvik, 1999)
2.5
References
Amann, R., Ludwig, W., and Schleifer, K-H. (1995). Phylogenetic identification and in situ detection of
individual microbial cells without cultivation. Microb. Rev. 59: 143-169.
Arvanitidou, M., Tsakris, A., Constantinidis, T.C., and Katsouyannopoulos, V.C. (1997). Transferable antibiotic
resistance among salmonella strains isolated from surface waters. Water Res. 31: 1112-1116.
Baquero, F., Negri, M.C., Morosini, M.I., and Blazquez, J. (1998). Antibiotic-selective environments. Clin.
Infect Dis. 27: S5-11
Boon, P.I. (1992). Antibiotic resistance of aquatic bacteria and its implications for limnological research. Aust. J.
Marine Freshwat. Res. 43: 847-859.
Brock, T.D. and Madigan, M.T. (1994). Biology of Microorganisms. Prentice Hall International, Inc.
Brockman, F.J. and Bezdick, D.F. (1989). Diversity within serogroups of Rhizobium leguminosarum biovar
vicae in the paouse region of eastern Washington as indicated by plasmid profiles, Intrinsic antibiotic resistance
and topography. Appl. Environ. Microbiol. 55: 109-115.
Collis, C.M. and Hall, R.M. (1992). Site-specific deletion and rearrangement of integron insertion catalyzed by
the integron DNA integrase. J. Bacteriol. 174: 1574-1585.
Datta, N. (1984). Bacterial resistance to antibiotics, Ciba Found. Symp. 102: 204-218.
Davies, J. (1994). Microbial molecular diversity: Past and present. J. Ind. Microbiol. 13: 208-211.
Davies, J.E. (1997). Origins, acquisition and dissemination of antibiotic resistance determinants, Ciba. Found.
Symp. 207: 15-35.
Goñi-Urriza, M., Capdepuy, M., Arpin, C., Raymond, N., Caumette, P., and Quentin, C. (2000). Impact of an
urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp. Appl. Environ.
Microbiol. 66: 125-132.
Hall, R.M. (1997). Mobile gene cassettes and integrons: moving antibiotic resistance genes in Gram-negative
bacteria. Ciba Found. Symp. 207: 192-205.
Hall, R.M. and Stokes, H.W. (1993). Integrons: novel DNA elements which capture genes by site-specific
recombination. Genetica. 90: 115-132
Honoré, N. and Cole, S.T. (1994). Streptomycin resistance in Mycobacteria. Antimicrob. Agents Chemother. 38:
238-242.
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Jones, J.G. (1986). Antibiotic resistance in aquatic bacteria. J. Antimicrob. Chemother. 18: 149-154.
Jones, J.G., Gardener, S., Simon, B.M., and Pickup, R.W. (1986a). Antibiotic resistant bacteria in Windermere
and two remote upland tarns in the English Lake District. J. Appl. Bacteriol. 60: 443-453.
Jones, J.G., Gardener, S., Simon, B.M., and Pickup, R.W. (1986b). Factors affecting the measurement of
antibiotic resistance in bacteria isolated from lake water. J. Appl. Bacteriol. 60: 455-462.
Kobori, H., Sullivan, C.W., and Shizuya, H. (1984). Bacterial plasmids in Antarctic natural microbial
assemblages. Appl. Environ. Microbiol. 48: 515-518.
Lévesque, C., Piché, L., Larose, C., and Roy, P.H. (1995). PCR mapping of integrons reveals several novel
combinations of resistance genes. Antimicrob. Agents Chemother. 39: 185-191.
Levy, S.B. (1997). Antibiotic resistance: an ecological imbalance. Ciba Found. Symp. 207: 1-9.
Mueller, J.G., Skipper, H.D., Shipe, E.R., Grimes, L.W., and Wagner, S.C. (1988). Intrinsic antibiotic resistance
in Bradyrhizobium japonicum. Soil Biol. Biochem. 20: 879-882.
Patel, S. and Torsvik, V. (1999). Dissemination of antibiotic resistance in bacteria from the environment. Statens
Forurensningstilsyn, Oslo. Rapport 99:20. ISBN 82-7655-187-4. (in Norwegian)
Petersen, A., Olsen, J.E., and Dalsgaard, A. (1997). Forekomst af antibiotikaresistente bakterier i akvatiske
miljøer. Miljøprojekt nr. 361. Miljø- og Energiministeriet, Miljøstyrelsen. Copenhagen. ISBN 87-7810-800-4 (in
Danish)
Recchia , G.D. and Hall, R.M. (1995). Gene cassettes: a new class of mobile elements. Microbiology. 141: 30153027.
Rosser, S.J. and Young, H-K. (1999). Identification and characterization of class 1 integrons in bacteria from an
aquatic environment. J. Antimicrob. Chemother. 44: 11-18.
Rowe-Magnus, D.A. and Mazel, D. Resistance gene capture. (1999). Curr. Opin. Microbiol. 2: 483-488.
Salyers, A.A. and Amábile-Cuevas, C.F. (1997). Why are antibiotic resistance genes so resistant to eliminate?
Antimicrob. Ag. Chemoth. 41: 2321-2325.
Schnabel, E.L. and Jones, A.L. (1999). Distribution of tetracycline resistance genes and transposons among
phylloplane bacteria in Michigan apple orchards. Appl. Environ. Microbiol. 65: 4898-4907.
Spanggaard, B., Jørgensen, F., Gram, L., and Huss, H.H. (1993). Antibiotic resistance in bacteria isolated from
three freshwater fish farms and an unpolluted stream in Denmark. Aquaculture, 115: 195-207.
Sundin, G.W., Demezas, D.H., and Bender, C.L. (1994). Genetic and plasmid diversity with natural populations
of Pseudomonas syringae with various exposures to copper and streptomycin bacteriocides. Appl. Environ.
Microbiol. 60: 4421-4431
Sundin, G.W., Monks, D.E., and Bender, C.L. (1995). Distribution of the streptomycin-resistance transposon
Tn5393 among phylloplane and soil bacteria from managed agricultural habitats. Can. J. Microbiol. 41: 792799.
Sundström, L. (1998). The potential of integrons and connected programmed rearrangements for mediating
horizontal gene transfer. APMIS suppl. 106: 37-42.
Watanabe, T. (1963). Infective heredity of multiple drug resistance in bacteria. Bacteriol. Rev. 27: 87-115.
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3
CONTAINMENT SYSTEMS
by Peter Stougaard
3.1 Summary
Efficient containment systems based on broad universal principles (e.g.
membrane proteins, lytic proteins, and nucleases) have been developed and
shown to function in laboratory experiments. However, despite success in
laboratory experiments, 100% biological containment has never been achieved,
irrespectively of the killing function or the element controlling expression of the
killing factor. Even in the presence of the killing factor a sub-population of cells
always survive. The most efficient control circuits with double killing cassettes
and double control circuits result in less than 10-7-10-8 mutants per cell per
generation.
Biological containment systems have been shown to function outside the
laboratory. In experiments with rats it was shown that bacteria harbouring
containment systems disappeared faster than control bacteria. Similarly, in field
trials with recombinant Pseudomonas putida it was shown that bacteria with
containment systems were reduced in number by a factor of more than 1000
times when compared to non-contained control bacteria.
However, these field trials and experiments in rats were carried out with “first
generation” containment systems carrying only one killing gene and one control
circuit. Therefore, it is assumed that a second generation containment systems
with double killing cassettes and double control circuits which have proved to be
efficient in laboratory experiments may result in further reduction of viability in
animal tests or in field trials.
Furthermore, development of new control circuits regulated by relevant
environmental factors or by chemical or physical inducers may lead to even
more efficient biological containment systems.
3.2 Containment of organisms
When GMOs were developed there was public and scientific concern about these new
organisms and there was – and still is – a general wish for the development of containment
systems that prevent the proliferation of GMOs released into the environment, deliberately or
unintentionally. In physical containment systems the laboratory and the procedures for
handling of GMOs have been designed to minimise the escape or release of GMOs from the
contained laboratory. In order to further prevent escape and establishment of GMOs in the
environment biological containment systems were developed. The first biological
containment systems were based on disabled mutant GMO strains developed to survive only
under laboratory conditions. Other applications of disabled biological containment systems
are attenuated organisms as live vaccines to be used in medical and veterinary microbiology.
However, in the case of deliberate release of GMOs to the environment or in the use of GMOs
as food additives for humans or animals, physical containment systems or biological systems
with disabled strains are obviously not applicable. This report will not cover the analysis of
such physical or early biological containment systems but will in stead focus on new
biological containment systems designed for GMOs that are intended to be used as food
additives or to be released into the environment.
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GENETICALLY MODIFIED ORGANISMS
As more and more GMOs have been designed for deliberate release in the environment, there
has been an increasing demand for containment systems which effectively controls the
viability of GMOs without reducing their beneficial effects. One such solution is the
engineering of self-destructive microorganisms that express “suicide functions” when their
job is done, in the human gut or in the environment. This chapter will review different
containment systems based on suicide functions specially designed for the control of bacterial
GMOs in the environment, rather than the more classical attenuation methods used for e.g.
live vaccine delivery systems.
3.3 Suicide or killing functions
Most containment systems are combinations of two elements: 1) a suicide or killing function
and 2) a control element. Several molecules, even from the microorganism itself, are known to
be toxic to microbial cells and it would – in theory – be easy to find a proper killing agent.
However, a closer examination of microbial gene products thought to be toxic to the cells
reveals that many potential “killing functions” are merely inhibiting the growth of the
bacteria. The ideal killing agent should interfere with vital, central cellular functions, it should
preferably be active at low concentrations, and it should be stable in the cell.
The gef gene family meets most of these requirements. The gef gene products interact with
the cell membrane causing transmembrane potential collapse, termination of respiration,
influx to the cytoplasm of RNase from the periplasm, and ultimately, cell death. One of the
members of the gef gene family is the hok (host killing) gene from plasmid R1. Normally, the
hok gene is expressed constitutively but the cells are prevented from dying by another gene,
the sok gene (suppression of killing). The suppression of the Hok mediated killing is caused
by blocking of translation of the hok mRNA by the sok mRNA which is antisense with respect
to the hok mRNA (Gerdes et al., 1986a). The promoters of both the hok and the sok genes
have been modified in order to express the genes in a controlled fashion.
Other members of the gef gene family are the E. coli genes relF and gef which are
homologous to the hok gene from plasmid R1. All three genes, hok, relF, and gef, have been
shown to display similar killing effects, i.e. collapse of the membrane potential, fast drop in
oxygen uptake, and cell death. Whereas gef gene family systems are found in several Gramnegative bacteria (Bech, et al., 1985; Gerdes, et al., 1985; Gerdes, et al., 1986b; Gerdes, et al.,
1997; Poulsen, et al., 1991) its presence in Gram-positives has so far not been demonstrated.
However, a plasmid stabilising system has been identified in the Gram-positive bacterium
Enterococcus faecalis (Weaver & Tritle, 1994) which could be a postsegretional killing
system like the gef gene system.
As the central target of the gef gene family proteins is the cell membrane, it is most likely that
the killing effect is more universal, working in more if not all bacteria. Experiments with
various Pseudomonas species, Xanthomonas campestris, Serratia marcescens, and Bacillus
thuringensis indicate that this is the case (Gerdes, 1988; Pimeta, et al., 1992; Molin, et al.,
1993).
Another killing system is that of the relBE gene family. The relB and relE are co-transcribed
in an operon together with relF, the relBEF-operon (Bech et al., 1985). The RelE protein has
been shown to be a cytotoxin, lethal or inhibitory to host cells and the RelB protein is
believed to be an anti-toxin as it prevents the lethal action of the RelE protein (Gotfredsen &
Gerdes, 1998; Grønlund & Gerdes, 1999). The mode of action exerted by the RelE protein is
at present unknown, but it is believed that translation is inhibited (Gerdes, pers. comm.). The
toxic RelE protein are generally stable in the cell whereas the antitoxin, the RelB protein, are
degraded by the cellular proteases Lon and Clp (Gerdes, 2000). Data base analysis has
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TRANSFER OF DNA
revealed that homologues to the relB/relE system is found in all procaryotes, Gram-positives
and Gram-negatives as well as Archaea (Gerdes, 2000).
A universal killing system based on broad spectrum nucleases has also been developed.
Controlled expression of nucleases has been shown to cause regulated cell death in several
Gram-negative and Gram-positive bacteria. This system may have advantages over the Gefsystem since the target – nucleic acids – is universal and, thus, the genetic material itself is
degraded. Nuclease genes from Serratia marcescens and Staphylococcus aureus expressing
potent endonucleases have been isolated and engineered in expression systems in wich
expression can be switched on by the addition of exogenously added inducing agents or by
increasing the temperature (Molin et al., 1993; Arenholtz et al., 1994). In uninduced state, the
nucleases are not expressed and the cells grow normally but if induced, the expression of the
nuclease genes results in production of nuclease and ultimately in cell death. One drawback of
the system is that the nucleases are highly unstable when expressed as intracellular enzymes,
in contrast to the natural, extracellular forms which are highly stable.
Another nuclease system designed to suppress the lateral spread of cloned genes from GMOs
to indigenous bacteria has been developed by Díaz et al. (1994). It is based on the
endolytically activity of the bacterial toxin colicin E3, which has a target at the 3’ end of the
16S ribosomal RNA. The target is conserved in virtually all prokaryotic and many eukaryotic
organisms. Cleavage of the target sequence in rRNA separates the mRNA binding site from
the remainder of the 16S rRNA thereby inhibiting protein synthesis. Laboratory experiments
have shown that transfer of the E3 gene to recipient cells resulted in a reduction in viability in
the range of 4 to 5 orders of magnitude. However, as other nuclease based suicide systems,
the colicin E3 system requires expression of large numbers of nuclease molecules in order to
be effective (Díaz et at., 1994).
A fourth universal killing system is based on the streptavidin gene from Streptomyces avidinii
(Szafranski et al., 1997). The system targets the metabolism of one-carbon units at the
oxidation level of carbon dioxide by depleting an essential prosthetic group, D-biotin.
Expression of the streptavidin gene is controlled by a regulated promoter, and only under
certain conditions, streptavidin synthesis is switched on, resulting in biotin depletion. Binding
of biotin to streptavidin is almost irreversible and results in a direct inhibition of biotindependent carboxylases, decarboxylases, and transcarboxylases. Inactivation of these
enzymes blocks the first step of fatty acid metabolism, replenishment of the Krebs cycle, and
substrate uptake by some anaerobes. As biotin is required throughout the living world, the
streptavidin-based system should be as universal as the systems based on gef genes and
nucleases. This system was shown to work efficiently in Pseudomonas putida (see below).
Containment systems based on bacteriophage lysis proteins have been constructed and shown
to function in laboratory experiments. In one system, the lysis genes S, R, and Rz from
bacteriophage lambda were controlled by the inducible Ptac promoter (Kloos et al., 1994).
Addition of inducer to E. coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri carrying
this suicide system caused cell death and liberation of nucleic acids from the lysed cells. In a
similar system, the lysis gene E from bacteriophage φX174 caused lysis in E. coli and P.
stutzeri and simultanously degradation of the liberated nucleic acids (Kloos et al., 1994).
3.4 Controlled expression of killing functions
Besides the killing element, one of the key components in the design of an efficient
containment system lies in the regulation of the expression of the killing genes. Whether the
system is designed to control un-intentional leak from large scale fermentations or it is
engineered to regulate the survival of deliberately released GMOs, the system must ensure
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GENETICALLY MODIFIED ORGANISMS
normal metabolic processes under conditions where cell growth is wanted, i.e. expression of
the killing functions must be efficiently switched off. On the other hand, adequate amounts of
the killing functions must be synthesized when killing of the organism is desired.
Such regulated systems in which expression is tightly controlled are well-known in laboratory
experiments. Induction of killing genes by the chemical IPTG has been shown to function
effectively in laboratory experiments (Molin et al., 1987; Bej et al., 1988; Kloos et al., 1994;
Knudsen et al., 1995; Gotfredsen & Gerdes, 1998). In several of these systems, the killing
gene is under the control of the strong, inducible lac-promoter or derivatives thereof. Thus,
expression of the killing gene is regulated by the action of lac repressor and the inducer IPTG.
In the absence of IPTG, the promoter is inactive due to the action of the lac repressor, but
when IPTG is added, the promoter is active and the killing function is synthesized.
A similar regulated system based on temperature control of synthesis of the killing agent has
been also developed (Ahrenholtz et al., 1994; Gerdes et al., 1986b). The system is based on
killing element expression from the bacteriophage λ promoters, λpR and λpL, which in turn
are controlled by the temperature sensitive cI857 repressor. Thus, at low temperature no killing
element is produced whereas at high temperature, the killing factor is synthesized.
In laboratory experiments, systems based on IPTG inducible lac promoters or temperature
regulated λpL promoters may be satisfactory. If containment systems are used for the control
of un-intentional release from large-scale fermentors or to regulate the survival of deliberate
released GMOs, expensive chemicals like IPTG or the application of temperature increase is
hardly realistic. A more practical solution would be to design a control system where the
controlling elements are parts of the environment in which the containment system is intended
to function. For example, promoters from cold shock genes or from genes responding to plant
exudates could be used to design containment systems for control of GMO leakage from
fermentors or for regulation of survival of deliberately released GMOs on agricultural crops.
At the moment no such control systems have been constructed.
Promoters responding to various xenobiotics have also been used in the design of suicide
systems responding to changes in the environment. One of the best studied systems is based
on genes and promoters from the TOL plasmid from Pseudomonas putida. The strategy for
biological containment is to design bacteria that grow and metabolise 3-methylbenzoate
(3MB) but when their job is done and 3-MB is degraded the bacteria commit suicide due to
switching on the synthesis of Gef protein (Contreras et al., 1991; Jensen et al., 1993). The
containment system is a two component system comprising a suicide function and a control
element. The suicide function is composed of a lac promoter fused to the gef suicide gene.
The control system contains the E. coli lacI repressor gene under the control of of the Pm
promoter from the Pseudomonas putida meta-operon on the TOL plasmid. Expression of the
control system – lac repressor synthesis – is regulated by the presence of 3-MB and a Pm
activator, the XylS protein (from the xylS gene also contained in the meta-operon). Thus, in
the presence of 3-MB, XylS is activated and stimulates transcription from the Pm promoter
resulting in lac-repressor synthesis. This in turn prevents expression of the killing function.
However, if 3-MB is depleted, the XylS protein no longer stimulates transcription of the lacI
gene, and when no lac-repressor is synthesised the lac promoter becomes active resulting in
production of Gef killing function and cell death. This system has been shown to function
both in laboratory experiments and in field trials (see below).
All the above mentioned expression systems require external regulators, either temperature or
chemicals (IPTG or 3-MB). However, control circuits based on such regulators are not always
applicable. For example, deliberate release of GMOs on agricultural crops or use of GMOs as
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TRANSFER OF DNA
live vaccines require other control systems. One such system based on stochastic control has
been developed by Klemm and co-workers (1995). The system is based on a recombinational
switch which inverts a promoter containing DNA fragment with a frequency of approximately
2 x 10-3 per cell per hour. When such an invertible promoter is placed in front of the gef gene
in the “off mode” no killing function is synthesized. But with a frequency of 2 x 10-3, the
promoter is inverted to the “on mode”, Gef protein is synthesized and the cells are killed. The
system has been shown to be growth rate independent with a tendency of being more active in
slow growing cells (Klemm, et al., 1995; Molin, et al., 1993). However, the control system
has a serious drawback since it only works in E. coli and Salmonella, but not in other bacteria
e.g. Pseudomonas, Enterobacter, or Klebsiella.
3.5 Containment efficacy
Despite considerable successes in laboratory experiments, 100% biological containment has
never been achieved, irrespectably of the killing function or the control element. Even in the
presence of inducer a sub-population of cells always survive. The surviving fraction of cells is
in the range between 10-7 to 10-3 per cell per generation (Arenholtz et al., 1994; Contreras et
al. 1991; Molin et al., 1993). The surviving bacteria which no longer respond to the induction
of suicide may be mutated in the killing gene itself, in the expression system, or elsewhere.
Laboratory experiments has shown that mutations in the killing genes may explain the
majority of the mutations and that the mutation rate in the gef-like genes is in the range of
approximately 10-6 per cell per generation (Knudsen & Karlström, 1991).
One obvious way to reduce the problem with mutants is to duplicate the killing system. This
approach has been shown by Knudsen and co-workers (1995) to work with the relF gene from
E. coli. Two copies of promoter-relF cassettes placed in the same plasmid results in reduction
of mutation frequency, from 10-6 to approximately 10-8 per cell per generation, and it is
assumed that the frequency may be even lower if different killing systems are employed
(Knudsen et al., 1995).
Another second generation containment system with optimized killing frequencies has been
developed. Szafranski and co-workers (1997) have described a control system similar to the
3-MB/XylS/LacI system described above. In their system, synthesis of the lac repressor, LacI,
is also regulated by 3-MB and XylS. The lac promoter, controlled by the LacI repressor, is
fused to a gene encoding T7 RNA polymerase. Synthesis of the killing function, streptavidin
(see above), is expressed from the strong T7 gene 10 promoter, φ10. Thus, in the presence of
3-MB, the XylS activates synthesis of LacI which represses the synthesis of T7 RNA
polymerase from the lac promoter, and as no T7 RNA polymerase is present, no streptavidin
is produced from the φ10 promoter. If 3-MB is not present, XylS is not active, LacI is not
produced resulting in activation of the lac promoter. The lac promoter transcribe the T7 RNA
polymerase gene and the T7 RNA polymerase that facilitates streptavidin production. This
double control circuit is shown to work efficiently in Pseudomonas putida. Under favorable
laboratory conditions, clones escaping killing appeared at frequencies of only 10-7 to 10-8 per
cell per generation.
3.6 In vivo efficiency of biological containment systems
In contrast to the many laboratory experiments with containment systems, only a limited
amount of reports have been published on the performance of suicide systems in human or
animal models or in the environment.
Induced suicide caused by the gef gene family has been shown to function in germfree rats. In
experiments with germfree rats containing E. coli harbouring hok and gef genes controlled by
the stochastic inducible fimA promoter (Klemm et al. 1995) it was shown that hok and gef
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GENETICALLY MODIFIED ORGANISMS
induced suicide of E. coli did occur in the gut of germfree rats (Jacobsen et al. 1993¸ Jacobsen
et al. 1996). In the experiments, E. coli carrying hok and gef genes were decimated when
compared to control E. coli strains carrying plasmids without the hok or gef genes. Expressed
as T90, the time used for a 90% reduction of the bacterial viable cell number, the mean
elimination time was calculated to be 2.8 days for a hok strain compared to 5.3 days for the
reference strain and 2.5 days for a gef strain compared to 6.6 days for the reference strain
(Jacobsen et al. 1993¸ Jacobsen et al. 1996). This clearly indicates that the gef family suicide
functions works in germfree rats.
In experiments with two cassettes of the relF gene it was shown that RelF induced suicide of
E. coli was observed in seawater and in soil (Knudsen et al., 1995). The reduction in colony
forming units was more than 6 to 7 orders of magnitude both in field and in laboratory
experiments. The two suicide functions were shown to prevent transfer of plasmids in both
test tube and in rat intestine experiments. If plasmids harbouring the two relF genes were
transferred to other bacteria without the controlling lac repressor gene this would result in
RelF synthesis and lead to cell death. It was shown, both in test tubes and in germfree rats,
that the ratio of transconjugants to donor was less than 1 x 10-8 compared to a frequency of
approximately 4.6 x 10-5 for the control plasmid without relF genes.
Only one publication on field trials has been found. Molina et al. (1998) have shown that the
containment system based on the gef gene function and regulated by the 3-MB/XylS/LacI
control system works under field conditions. Recombinant Pseudomonas putida carrying the
Gef suicide and control functions described above and control strains without the gef gene
were inoculated into soil containing normal, indigenous microflora. Two sets of experiments
were carried out, one in autumn-winter and one in spring-summer. During the four autumnwinter months of experimentation the contained strain and the reference strain developed
differently. The reference strain was established in soil supplemented with and without 3-MB
(approx. 105 CFU per gram soil) whereas the contained strain was reduced in number (below
detection level if 3-MB was added and approx. 103 CFU per gram soil in the absence of 3MB). During the 4 month of the spring-summer experiment a less clear-cut result was
observed. Both the contained and the control strain was reduced in number, but the reduction
below detection level was first observed in the contained strain in the absence of 3-MB
(within the first 20 days). Although the difference was not so obvious in these field trials, this
is the first demonstration that containment systems actually work in outdoor environment.
3.7
References
Arenholtz, I., M.G. Lorenz, and W. Wackernagel (1994). A conditional suicide system in Escherichia coli based
on the intracellular degradation of DNA. Appl. Environ. Microbiol. 60: 3746-3751.
Bech, F.W., S.T. Jørgensen, B. Diderichsen, and O. Karlström (1985). Sequence of the relB transcription unit
from Escherichia coli and identification of the relB gene. EMBO J. 4: 1059-1066.
Bej, A.K., M.H. Perlin, and R.M. Atlas (1998). Model suicide vector for containment of genetically engineered
microorganisms. Appl. Environ. Microbiol. 54: 2472-2477.
Contreras, A., S. Molin, and J.L. Ramos (1991). Conditional suicide containment system for bacteria which
mineralize aromatics. Appl. Environ. Microbiol. 57: 1504-1508.
Díaz, E., M. Munthali, V. de Lorenzo, and K.N. Timmis (1994). Universal barrier to lateral spread of specific
genes among microorganisms. Mol. Microbiol. 13: 855-861.
Gerdes, K. (2000). Toxin-antitoxin modules may regulate the synthesis of macromolecules during nutritional
stress. J. Bacteriol. 182: 561-572.
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TRANSFER OF DNA
Gerdes, K., F.W. Bech, S.T. Jørgensen, A. Løbner-Olsen, P.B. Rasmussen. (1986a.) Mechanism of
postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the
relF gene product of E. coli relB operon. EMBO J. 5: 2023-2029.
Gerdes, K., A.P. Gultyaev, T. Franch, K. Pedersen, and N.D. Mikkelsen (1997). Antisense RNA-regulated
programmed cell death. Annu. Rev. Genet. 31: 1-31.
Gerdes, K., J.E.L. Larsen, and S. Molin. (1985). Stable inheritance of plasmid R1 requires two different loci. J.
Bacteriol. 161: 292-298.
Gerdes, K., P.B. Rasmussen, and S. Molin. (1986b). Unique type of plasmid maintenance: postsegretional killing
of plasmid-free cells. Proc. Natl. Acad. Sci. USA. 83: 3116-3120
Gotfredsen, M. and K. Gerdes (1998). The Escherichia coli relBE genes belong to a new toxin-antitoxin gene
family. Mol. Microbiol. 29: 1065-1076.
Grønlund, H. and K. Gerdes (1999). Toxin-Antitoxin systems homologous with relBE of Escherichia coli
plasmid P307 are ubiquitous in prokaryotes. J. Mol. Biol. 285: 1401-1415.
Jacobsen, B., Schlundt, J, and Fischer, G. (1993). Study of a conditional suicide system for biological
containment of bacteria in germ-free rats. Microbiol. Ecol. Health Disease. 6: 109-118.
Jacobsen, B., Schlundt, J, and Fischer, G. (1996). The use of germfree rats for the study of fate and effect of
genetically modified microorganisms. Microecol. Therapy 24: 59-63.
Jensen, L.B., J.L. Ramos, Z. Kaneva, and S. Molin. (1993). A substrate-dependent biological containment
system for Pesudomonas putida based on the Escherichia coli gef gene. Appl. Environ. Microbiol. 59: 37133717.
Klemm, P., L.B. Jensen, and S. Molin (1995). A stochastic killing system for biological containment of
Escherichia coli. Appl. Environ. Microbiol. 61: 481-486.
Kloos, D.-U., M. Strätz, A. Güttler, R.J. Steffan, and K.N. Timmis (1994). Inducible cell lysis system for the
study of natural transformation and environmental fate of DNA released by cell death. J. Bacteriol. 176: 73527361.
Knudsen, S.M. and O.H. Karlström (1991). Development of efficient suicide mechanisms for biological
containment of bacteria. Appl. Environ. Microbiol. 57: 85-92.
Knudsen, S., P. Saadbye, L.H. Hansen, A. Collier, B.L. Jacobsen, J. Schlundt, and O. Karlström (1995).
Development and testing of improved suicide functions for biological containment of bacteria. Appl. Environ.
Microbiol. 61: 985-991.
Molin, S., L. Boe, L.B. Jensen, C.S. Kristensen, M. Givskov, J.L. Ramos, and A.K. Bej (1993). Suicidal genetic
elements and their use in biological containment of bacteria. Annu. Rev. Microbiol. 47: 139-166.
Molin, S., P. Klemm, L.K. Poulsen, H. Biehl, K. Gerdes, and P. Andersson, Conditional suicide system for
containment of bacteria and plasmids. Bio/Technology, 5: 1315-1318.
Molina, L., C. Ramos, M.C. Ronchel, S. Molin, and J.L. Ramos (1998). Construction of an efficient biological
contained Pseudomonas putida strain and its survival in outdoor assays. Appl. Environ. Microbiol. 64: 20722078.
Pimeta, A.D.L., Y.B. Rosato, and S. Astolfi-Filho (1992). Effect of parB on plasmid stability and gene
expression in Xanthomonas campestris. Lett. Appl. Microbiol. 14: 233-237.
Poulsen, L.K., A. Refn, S. Molin, and P. Andersson (1991). The gef gene from Escherichia coli is regulated at
the level of transtation. Mol. Microbiol. 5: 1639-1648.
Szafranski, P., C.M. Mello, T. Sana, C.L. Smith, D.L. Kaplan, and C.R. Cantor (1997). A new approach for
containment of microorganisms: Dual control of streptavidin expression by antisense RNA and the T7
transcription system. Proc. Natl. Acad. Sci. USA. 94: 1059-1063.
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GENETICALLY MODIFIED ORGANISMS
Weaver, K.E. and D.J. Tritle (1994) Identification and characterization of an Enterococcus faecalis plasmid
pAD1-encoded stability determinant which produces small RNA molecules necessary for its function. Plasmid
32: 168-181.
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4
ENVIRONMENTAL ASPECTS OF DNA TRANSFER
by Flemming Jørgensen
4.1 Summary
Microbial uptake and incorporation into the genome of naked DNA from the
environment constitutes an important issue for safe use of genetically modified
organisms. Natural competence for uptake of naked DNA can appear in both
Gram-positive and Gram-negative bacteria. The conditions required for the
bacterial competence are variable; the uptake often involves a pili type-IV
apparatus, in a few cases this result in a high frequency of DNA uptake. Naked
DNA has been found to survive for some time in soil despite high levels of DNase
activity. DNA can be protected by binding to minerals, and has by laboratory
procedures allowed the isolation of bacterial transformants. In experiments with
release of genetically modified material no DNA transfer events have been
observed or reported. It is conceivable that such events could occur at frequencies
below the present detection level. Horizontal gene transfer between non-related
species is probably an important element in bacterial evolution, and examples of
such development within the last 100 million years have been found by genome
sequence projects.
4.2 Introduction
The engineering of GMOs and their use for industrial purposes has been debated both in
public and scientific communities. The prime topics in this debate have been the safety of
GMOs and the environmental consequences of their release. Modern biotechnology has
allowed genetic material to be moved and combined in ways hitherto not possible. Breaking
natural biological barriers by moving species from one continent to another has in many cases
been documented to cause problems. Containment, the prevention of recombinant DNA from
being released or from entering and establishing in natural life forms, has consequently been a
regulatory requirement for GMO use.
Bacteria are the hub in a model describing the dissemination of genetic material (Heineman,
1991). This model proposal is presented in Fig. 3. Transformation, conjugation and
transduction are the known methods for exchange of genetic material among bacteria.
Examples of gene transfer from bacteria to organisms that belong to one of the four other
kingdoms have also been documented. In this way bacteria become the center of DNA flow,
and the uptake of naked DNA by natural transformation becomes a very important process in
dispersal and in evolution.
Natural transformation is the process by which bacteria take up DNA present in the
environment. The uptake mechanism is produced like any other cellular structure in the
bacterium. The DNA that has been taken up may become integrated in the bacterium resulting
in stable transformation. Natural transformation has only been found in bacteria, where it was
first discovered more than 50 years ago. Once DNA has become incorporated into the host
chromosome the genes encoded by the inserted DNA may become active and provide the
bacterium with new capabilities.
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GENETICALLY MODIFIED ORGANISMS
anim als
fungi
archae
plants
G ram negative
bacteria
GMOs
G ram positive
bacteria
DNA
natural
sources
Fig 3. Dissemination of genetic information as proposed by Heineman (1991).
4.3 Natural competence
Bacterial transformation, meaning the uptake and subsequent expression of naked DNA, can
be achieved in a number of ways. In the laboratory, chemical and electrical treatments are
routinely used to make bacterial cells competent for transformation, but also particle
bombardment have been employed as a mean of transformation. Besides these artificial
methods, many bacteria have been found to exhibit natural competence, where the ability to
take up naked DNA from the environment is genetically encoded and regulated.
In 1994 Lorenz and Wackernagel listed 42 prokaryotic species capable of natural
transformation, and many of these bacteria only expressed competence transiently under
specific conditions. The conditions needed to induce the ability to take up naked DNA are
probably to be discovered in many more bacteria. Recently, competence gene homologues
have been identified in bacteria previously regarded as non-competent (Håvarstein &
Morrison, 1998; Håvarstein, 1998). Genome sequencing have revealed a complete set of late
competence genes in Lactococcus lactis IL1403 (Bolotin et al., 1999).
In short, the transformation pathway generally consists of the following steps (Lorenz &
Wackernagel, 1994; Dubnau, 1999): 1) Double stranded DNA binds to a limited number of
specific receptors present on the cell surface. However, some Gram-negative cells are also
able to bind and take up single-stranded DNA. Depending on the bacterial species in question,
these receptors bind DNA either non-specifically, or they require a short specific DNA
sequence for binding. 2) The bound DNA is cleaved at random positions. Depending on the
bacterial species in question, either a single-stranded or double-stranded cleavage of the
double stranded DNA takes place. 3) One strand of the DNA molecule is transported across
the membrane into the bacterial cell, the molecular end generated by DNA cleavage being
used as the starting point. The other strand of the DNA molecule is being degraded outside
the cell. 4) Transported, single-stranded DNA associates with cytoplasmic proteins and
integrates into the chromosome by way of homologous recombination. Plasmids are
reconstituted by way of overlapping fragments.
Natural transformation is sensitive to DNases, a characteristic that experimentally is used to
distinguish this event from conjugation and transduction.
Cell-contact transformation is a special form of natural transformation that shows
resemblance with conjugation, but it has not been well characterized. The process needs cell
to cell contact, but unlike conjugation it is partially DNase sensitive. Furthermore, the process
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TRANSFER OF DNA
is bi-directional, both plasmid and chromosomal DNA can be transferred, and it works with
dead donor cell material (Yin & Stotzky, 1997).
DNA uptake mechanisms of selected bacterial families in which the natural competence has
been examined will be described. Genes responsible for the regulation of natural competence
and the expression of cellular transformation components will be describes in these separate
sections.
4.3.1 DNA uptake by Streptococcus
In Streptococcus pneumoniae a 17 amino acid peptide pheromone induces competence
(Håvarstein et al., 1995). This competence-inducing peptide (CSP) is encoded by a signal
peptide containing form of the comC gene. The comA and the comB gene products make up
the CSP-specific secretion system allowing a slow accumulation of CSP in the surrounding
medium. Competence is induced, when the concentration of CSP in the medium reaches 2-10
ng/ml. A histidine kinase receptor encoded by the comD gene is activated and phosphorylates
the cognate kinase receptor (comE), which is thereby turned into a DNA binding activator of
transcription (Ween et al., 1999).
Competence is developed in early- to mid-phase growth, and depending on the specific strain
it lasts for 40-60 minutes. The DNA uptake is non-specific, but the coordinated competence
development may favor exchange of genetic material between related organisms. A single
stranded nick initiates DNA uptake that involves a number of more and less characterized
proteins. For a more detailed description of streptococcal competence the reader is referred to
recent reviews and articles on the subject (Lunsford 1998; Håvarstein 1998; Whatmore et al.,
1999).
4.3.2 DNA uptake by Bacillus
In Bacillus subtilis development of natural competence takes place between late exponential
and early stationary growth phase. The competence is expressed in a sub-population
consisting of 1-10% of the bacterial cells. Cell density and nutrition regulate induction of
competence in a complex way combining sporulation and motility signals with the synthesis
of degradative enzymes and secondary metabolites (Grossman, 1995; Solomon & Grossman,
1996). At least 40 genes have been identified of which half are involved in regulation of
competence development, while the rest make up the cellular apparatus for DNA uptake.
Cell density is reflected by the accumulation of two extracellular pheromone peptides, the
competence stimulating factor (CSF) and the ComX protein, that both stimulate transcription
of srfA. The ComS protein is required for development of competence and consists of 46
amino acids, which are separately encoded within the larger srfA gene product. The srf operon
consists of four genes (srfABCD); together these enzymes catalyze the synthesis of surfactin,
an antibiotic lipopeptide. CSF is a penta-peptide that is transported into the cell by the Spo0K
oligopeptide permease. Inside the bacterial cell low concentrations of CSF stimulate
competence development, whereas high CSF concentrations stimulate sporulation and inhibit
competence development (Lazazzera et al., 1997). ComX is a modified 5-10 amino acid
peptide that has not yet been fully characterized. A two-component histidine kinase system
consisting of ComP and ComA is responsible for the activation of ComX that in return
stimulates the srfA promoter
ComK is a transcription regulatory protein, that binds to DNA sequences upstream of its own
and other competence product encoding gene promoters. This binding triggers the expression
of late competence genes involved in DNA interaction, processing and acquisition DNA (van
Sinderen et al., 1995). ComK also activates transcription of genes for DNA repair and
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GENETICALLY MODIFIED ORGANISMS
recombination. In Bacillus subtilis ComK serves as a molecular switch committing the cell to
transformation and repressing other developmental possibilities. Hence, it is not surprising
that ComK is exquisitely regulated.
During exponential growth ComK is trapped by MecA, an adapter protein also recognizing
the ClpC protease, resulting in degradation of ComK (Turgay et al., 1998; Persuh et al.,
1999). A high cell density leads to increased levels of the ComS protein, that also binds to
MecA for ClpC mediated degradation. In doing so ComS reduces ComK degradation, the
presence of free ComK rapidly increases due to autocatalytic transcription, and the cell is
destined to become competent for transformation (Hahn et al., 1996; Turgay et al., 1997; Liu
& Zuber, 1998; Ogura et al., 1999).
Genes responsible for binding, processing and transport of DNA are organized into four comK
regulated transcriptional units (comC, comE, comF, comG). A recent review by Dubnau
(1997) describes that in Bacillus subtilis transformation shows no DNA specificity and
involves a double stranded DNA cleavage event. The comC and comG operon encoded
proteins appears to be responsible for the DNA binding apparatus. The comG gene product
shows resemblance to type-IV pili. The comE and comF operon encoded proteins are assumed
to make up the DNA transport machinery, with ComE serving as the link between the DNA
processing and transport apparatus.
4.3.3 DNA uptake by Neisseria
Natural competence is constitutively expressed in gonococci. Transformation depends on the
presence of a specific DNA sequence, which in the case of Neisseria gonorrhoeae has been
identified as GCCGTCTGAA (Goodman & Scocca, 1988; Goodman & Scocca, 1991). The
recognition sequence is distributed throughout the genome, and it ensures that exchange of
genetic information preferentially takes place between related bacteria. Strains that are
mutated in the type-IV pili are also transformation negative suggesting that bacterial
competence is linked with the type-IV pili (Fussenegger et al., 1997).
Natural transformation of N. gonorrhoeae takes place with equal frequency when single- and
double-stranded DNA is introduced (Stein, 1991). The DNA is probably concentrated on the
cell surface by electrostatic interaction with positively charged Opa protein (Hill, 2000).
Extracellular DNA is converted into a DNase-resistant form by interaction with a type-IV pili
structure, where PilC, PilT and ComP are required. The PilC protein is present at the tip of the
type-IV pili structure, and PilC-depleted mutants are defective in both transformation and in
adherence to epithelial cells (Rudel et al., 1995). A strain with PilT mutation produces typeIV pili that looks and functions normally, except that cells are defective in natural
transformation and twitching motility (Wolfgang et al., 1998). Lack of ComP also results in a
transformation negative strain with otherwise normal type-IV pili functions (Wolfgang et al.,
1999). The transforming DNA apparently uses the type-IV pili and its outer membrane
penetration channel as the point of entry in the uptake process. When DNA has reached the
periplasm, transport across the inner membrane is mediated by Tcp and ComL which are both
peptidoglycan associated and together with ComA form a structure located in the inner
membrane (Fussenegger et al., 1997). Finally, the incoming single-stranded DNA is
integrated into the chromosome via RecA-dependent recombination. The products of the
RecBCD pathway have also been found to contribute to the integration process (Mehr &
Seifert, 1998; Chaussee et al., 1999).
4.3.4 DNA uptake by Acinetobacter
Acinetobacter is a group of Gram-negative bacteria that are widely distributed in soil and
water ecosystems. In Acinetobacter sp. strain BD4, formerly classified as Acinetobacter
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TRANSFER OF DNA
calcoaceticus DB4, a high level of natural competence is induced by dilution of lag phase
culture into fresh medium. Competence gradually decreases during exponential growth, and
there is no sequence specificity for the DNA taken up. Only one out of approximately 750
DNA fragments resulted in bacterial transformation, but as many as 65 DNA fragments were
taken up by each bacterial cell (Palmen & Hellingwerf, 1997).
Knowledge concerning the genetics and mechanics of natural transformation in Acinetobacter
is still limited. A type-IV pilus biogenesis factor like protein, ComC, has been described
(Link et al., 1998). Three other identified proteins (ComE, ComF, ComP) with pilin like Nterminal sequences, are suggested to form or be part of a rudimentary pilus structure, that is
responsible for DNA import (Busch et al., 1999; Porstendörfer et al., 1997).
4.4 DNA transfer and bacterial evolution
The existence and importance of horizontal gene transfer between non-related species has
been well debated (Lawrence, 1999; Syvanen, 1994 & 1999). Valuable information about
microbial evolution is obtained from genome sequence projects (Doolittle, 1997). Analysis of
phylogenetic relationships is based on the assumption, that genetic deviation between related
species has resulted from gradual changes, and that an early version of an ancestor has
existed. However, several examples of transfer between distantly related bacteria, provide a
more likely explanation for the genetic diversity.
The ‘selfish operon’ model emerged from the analysis of cofactor B12 synthesis and use in
Salmonella enterica (Lawrence & Roth, 1996; Lawrence 1997). Many bacterial genes that
code for enzymes, which contribute to reactions in a specific pathway, are clustered in
cotranscribed groups known as operons. The operon organization allows an entire metabolic
pathway to be acquired by horizontal gene transfer and selection will advance such an event.
This is presented as the evolving force behind the ‘selfish operon’ model.
Horizontal transfer of genetic material might generate DNA sequences that deviate in GC
content or codon usage from the surrounding genome. The complete E. coli genome sequence
has been analyzed for these indications of horizontal gene transfer (Lawrence & Ochman,
1998). From a total of 4288 genes, 755 (17.6%) genes detected in the chromosome seemed to
originate from horizontal gene transfer. Because the acquired genes are expected to undergo a
non-equilibrium conversion towards the DNA composition of E. coli, a process termed
amelioration, that is based on nucleotide substitution rates, the evolutionary age could be
proposed. E. coli and S. enterica diverged 100 million years ago, and since then horizontal
gene transfer into the E. coli genome has averaged 16 kb/million years.
Operational genes are more prone to horizontal transfer than informational genes (Rivera et
al., 1998; Jain et al., 1999). The informational genes are involved in transcription, translation
and related processes, while the operational genes involve housekeeping metabolic pathways.
Operational genes are more readily organized into adequate operons that allow for
competitive exploration of new ecological niches. The complexity of the genetic system only
allows partly transfer of material, unlikely to function in combination with the informational
genes in the receiving organism and does not provide any selective value (Jain et al., 1999).
It is not known to what extent natural transformation has contributed to the observed
horizontal gene transfer during bacterial evolution. A genetically regulated low frequency of
natural cell transformation is consistent with the idea that promiscuous uptake of foreign
DNA serves an essential role in global evolution. Although a very low level of horizontal
transfer by way of natural transformation may be optimal for the time scale in which
evolution operates, mutations in the control circuits regulating natural transformation could be
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GENETICALLY MODIFIED ORGANISMS
important exceptions. If bacterial mutants with high DNA uptake frequencies emerge
regularly in nature, the risk of horizontal DNA transfer may be underestimated.
Both a transfer barrier and an establishment barrier have to be crossed in a gene transfer
event. The mismatch repair system was found to be the most important barrier for DNA
transfer between E. coli and S. typhimurium (Matic et al., 1995), but it played only a minor
role for Bacillus, where instead heteroduplex formation was inferred to limit interspecies
DNA exchange (Majewski & Cohan, 1998).
The structure of bacterial populations ranges from being clonal to being panmictic (Maynard
Smith et al., 1993). Salmonella enterica and E. coli are examples of clonal bacterial
populations, where recombination events are too rare to destroy linkage disequilibrium
between the analyzed loci. A sexual or panmictic population structure is found for N.
gonorrhoeae, and frequent recombination is generally observed in bacterial species with
natural competence.
Evidence for several events of horizontal gene transfer within the genus Neisseria have been
reported (Bowler et al., 1994; Vázquez et al., 1995; Zhou et al., 1997, Smith et al., 1999). For
at least two classes of antimicrobial agents, the ß-lactams and the sulfonamides, intergeneic
recombination have led to the emergence of antibiotic resistances in the pathogenic bacteria
Streptococcus pneumoniae, Neisseria meningitidis and Neisseria gonorrhoeae (Maiden,
1998).
4.5 Persistence of naked and recombinant DNA
The risk associated with the use and release of recombinant DNA is influenced by
environmental DNA turnover characteristics. Naked DNA is the input for natural
transformation, and in this section the persistence of naked DNA in soil and water, will be
discussed. The naked DNA source may be from pure preparations, or be released from dead,
decaying cells, or it may be excreted from cells living in the environment. Previously, naked
DNA was thought to have very limited survival due to high DNase levels in natural systems,
but that view has changed. Adsorption is able to protect DNA against degradation, and naked
DNA is consequently able to persist in the environment.
4.5.1 Fate of naked DNA in soil
The fate of naked DNA in soil has been reviewed meticulously (Lorenz & Wackernagel,
1994; Paget & Simonet, 1994; Trevors, 1996). Adsorption to soil particles protects naked
DNA against degradation by the DNases naturally present in soil. The protective binding
depends on the soil type used; clay soils have a high binding capacity, whereas sand only
binds limited amounts of naked DNA. Typically 10-20 µg DNA is adsorbed per gram clay
soil, which is sufficient to bind the genomic DNA from 109 bacteria, corresponding to the
number frequently found in one gram of soil. Shorter DNA fragments are preferentially
adsorbed by most soil types (Ogram et al., 1994). Although protected against the DNases
present in soil, bound DNA retains its ability to transform competent cells (Khanna &
Stotzky, 1992; Romanowski et al., 1992; Gallori et al., 1994). DNA bound by humic acids
have similarly been found to be protected against degradation and retained ability to
transform competent cells (Crecchio & Stotzky, 1998).
Both plasmid and chromosomal DNA of bacterial origin have been found to persist for at
least 10 days in soil when using a transformation assay for detection. Persistent DNA could be
observed for at least 30 days, when PCR was used as the detection method (Romanowski et
al., 1992; Recorbet et al., 1993; Gallori et al., 1994; England et al., 1997). Plant DNA from
genetically engineered tobacco was found to persist in soil under natural conditions for
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TRANSFER OF DNA
several months. The genetic marker used could be recovered after one year, but soil samples
taken 3 years after harvest of the tobacco plants tested negative (Paget et al., 1998).
Blum et al. (1997) used radioactive labeled phage DNA (molecular size 41.5 kb) to study the
fate of naked DNA in 3 different non-sterile soil types from agricultural fields. Depending on
the soil type used, 40-80% of the added DNA became bound within 1-3 hours. Close to 60%
of the adsorbed DNA could afterwards be recovered in a special extraction buffer. Nonadsorbed DNA was rapidly degraded within 24 hours, while adsorbed DNA persisted for at
least 5 days. Southern blot analyses of DNA extracted from soil showed that DNases also
affected bound DNA. After 5 days DNA fragments with a maximal size of 2 kb were still
present in the soil microcosms used. Growing prokaryotes are the probable producers of soil
DNases, because addition of rifampicin and chloramphenicol, but not of cycloheximide, could
prevent a burst in DNase activity associated with soil rehydration.
Adsorption to mineral material protected DNA against degradation by an extracellular
endonuclease from Serratia marcescens, a bacterium readily isolated from soil.
Approximately a 100-fold more endonuclease was required for the inactivation of the
transforming activity obtained with bound DNA compared to non-bound (Ahrenholtz et al.,
1994).
The understanding of DNA binding to clay particles is still in its infancy (Paget & Simonet,
1994). DNA bound on montmorillonite, but not on kaolinite, could be amplified by PCR
(Alvarez et al., 1998), an observation that suggested important differences between clay
minerals. Cycles of air-drying and wetting reduced the ability of clay bound DNA to
transform competent cells, but the reason for this effect is not understood (Pietramellara et al.,
1997).
4.5.2 Fate of naked DNA in aquatic systems
Naked DNA has a half-life of several hours in natural freshwater and in marine waters. The
degradation rate is faster in wastewater, where plasmid DNA is destroyed with a half-life of
10-20 min (Lorenz & Wackernagel, 1994; Alvarez et al., 1996).
Measurements of the naked DNA concentrations in aquatic samples often give results in the
1-10 µg/l range, and molecular sizes up to 35 kb have been reported (Lorenz & Wackernagel,
1994).
Bacterioplankton is considered the dominant source of extracellular DNA. Dead cells,
excretion from live cells, phage lysis and protozoan grazing all contribute to the release of
naked bacterial DNA. The apparent discrepancy between the rapid turnover rate and the
measured concentration of naked DNA may be a result of protective binding, but the ability of
dead cells and detritus to bind and protect naked DNA in aquatic systems is unknown.
Experiments with microcosms have demonstrated, that naked DNA in natural aquatic systems
can be used to transform competent bacteria (Paul et al., 1991; Frischer et al., 1994). Natural
transformation of plasmid DNA into indigenous marine bacteria was accompanied by plasmid
alteration, presumably a reflection of the single strand DNA uptake mechanism and
associated recombination (Williams et al., 1997).
4.5.3 Persistence of recombinant DNA
Plasmid DNA has been reported to retain up to 0.6% of the original transformation activity
after autoclavation (121°C for 15 min) in the presence of 0.5-2.0 M NaCl (Masters et al.,
1998). Thus, standard procedures to prevent recombinant DNA release may have to be
revised.
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GENETICALLY MODIFIED ORGANISMS
Genetically engineered E. coli seeded into nonsterile soil at 107 cells/g dropped below
detection limit (102 colony CFU/g) after 15 days, while the number of chromosomal targets
remained at 105/g after 40 days (Recorbet et al., 1993).
Transgenic plant material and DNA persist for several months under field conditions (Widmer
et al., 1997). The decay rate depended on the type of plant litter; for tobacco leaves buried in
soil only 0.06% of the marker DNA was detected after 77 days, while for tomato stems and
leaves 2.7% persisted after 84 days.
A system with inducible cell lysis has been developed to mimic the release of naked DNA
from dead cells (Kloos et al., 1994) The lysis-released DNA was found to behave like
purified DNA in transformation assays.
4.6 Natural transformation in the environment
Bacteria can be found in nearly all habitats and horizontal gene transfer has been revealed in
soil, aquatic systems and human bacterial communities. Determination of gene transfer
frequency under field conditions is limited by the available methodology. Low frequency
detection is often difficult in experiments with bacterial populations that do not allow the use
of selective markers (Clerc & Simonet, 1998).
4.6.1 Bacterial DNA uptake in soil
Horizontal gene transfer in soil has been the subject of recent reviews, that have focused on
the possible transfer from transgenic plants to soil microorganisms (Nielsen, 1998; Nielsen et
al., 1998; Wackernagel et al., 1998; Bertolla & Simonet, 1999). Most experimental work
concerning natural transformation of bacteria has been performed using laboratory in vitro
systems or microcosms, and experimental data for natural soil situations is limited.
Pseudomonas stutzeri JM300 discriminates against foreign DNA both during uptake, and by a
restriction system that operates after uptake (Lorenz et al., 1998). Once single-stranded DNA
has been taken up, the double-stranded DNA generated by homologous recombination
appears to be degraded by DNA restriction enzymes. Paget and Simonet (1997) used a
chromosomally integrated tetracycline gene, and found that DNA adsorbed to sterile soil
could transform P. stutzeri with frequencies up to 10-8 transformants per viable cell.
Sikorski et al. (1998) used complementation of a his-1 mutation to study natural
transformation of P. stutzeri. Plasmid transformation was around 2 orders of magnitude more
efficient, when selection was for complementation of the his-1 mutation as compared to
plasmid encoded streptomycin resistance selection. Selection for streptomycin resistance
displayed a two-hit mechanism, suggesting that two linear plasmids had to be taken up and
recombined in order to obtain a resistant transformant. A one hit mechanism was observed,
when histidine prototroph transformants were selected. The majority of recombination events
between plasmid DNA and the chromosome resulted in plasmid free his+ cells. The his+
transformants could be isolated from a non-sterile soil microcosm. DNA (plasmid or
chromosomal) capable of complementing the his-1 mutation was loaded onto the soil
microcosm, and non-adsorbed DNA was washed out. Afterwards, naturally competent P.
stutzeri cells (his-1) were added, and the transformation efficiency was determined. Using
plasmid DNA the maximal transformation frequency obtained was 6x10-6. When plasmid
DNA was incubated in the non-sterile soil for 3 days prior to the addition of competent P.
stutzeri cells, 3% of the initial transformation activity remained.
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TRANSFER OF DNA
Lee and Stotzky (1999) have reported transformation of Bacillus subtilis present in a soil
system. They observed a transformation frequencies ranging from 2x10-6 to 7x10-8 in sterile
soil when using chromosomal genes encoding amino acid synthesis. Transformation
frequencies were less in water saturated soil than in non-saturated soil (33 kPa water tension).
Transformants were also detected in non-sterile soil, but indigenous bacteria and fungi, that
were resistant to the antibiotics used, hampered enumeration.
Naturally competent cells of Acinetobacter sp. BD413 are transformed at high frequencies,
and the DNA uptake does not discriminate between heterologous and homologous DNA.
Chromosomal DNA containing a kanamycin resistance gene, nptII, was used by Nielsen et al.
(1997) for transformation of Acinetobacter sp BD413 in both sterile and non-sterile soil
microcosms. Bacterial cells added to the soil microcosm remained competent for up to 1 day
but DNA lost its transforming ability after a few hours in the soil microcosm. Transformation
frequencies depended on the soil type used, and for the sterile soil microcosms values in the
10-4 to 10-7 range were obtained. In non-sterile soil the transformation frequencies were lower.
Addition of nutrient was found to induce development of natural resistance in nontransformable soil Acinetobacter sp BD413 cells. (Nielsen et al., 1997). Soil moisture affected
transformation frequencies; 35% soil moisture gave the lowest frequency and it increased in
more dry soil.
De Vries and Wackernagel (1998) have developed a highly sensitive marker-rescue system
for the detection of nptII gene (kanamycin resistence). Cells of Acinetobacter sp BD413
containing a plasmid with a 10 bp deleted nptII gene was used in a natural transformation
assay in selection of kanamycin resistant transformants. Uptake of DNA with an intact nptII
gene, a marker frequently found in transgenic plants, resulted in transformants with a
functional kanamycin resistant gene. In general, the marker-rescue transformation system
yielded 1-2 transformants per 104 nptII gene copies. This sensitivity is 500-1000 fold less than
what can be detected by PCR. Material from 8 different transgenic plants all resulted in
isolation of transformants. The detection limit of the nptII gene system corresponded to ½ mg
of fresh leaf material from a transgenic tomato.
Gebhard and Smalla (1998) also used a plasmid encoding the nptII gene but with a 317 bp
deletion. Horizontal DNA transfer from transgenic sugarbeet to Acinetobacter sp BD413 was
detected. Homogenates of sugar beet leaves gave a transformation frequency of 1.5x10-10,
while purified sugar beet DNA gave a frequency of 5x10-9.
Restoration of the nptII gene was more efficient in both non-sterile and sterile soil
microcosms, when a functional nptII gene was integrated in homologous chromosomal DNA
and allowed to transform Acinetobacter sp BD413 cells. Heterologous chromosomal DNA
from Pseudomonas fluorescens and Burkholderia cepacia with functional intact nptII genes
were 4- to 16-fold less effective (Nielsen et al., 2000). In non-sterile soil the homologous
lysate gave a transformation frequency of 1x10-6. On average the cell lysates retained 31% of
their transforming activity after 1 hour in the non-sterile soil.
Schlüter et al. (1995) could not detect signs of horizontal gene transfer from a transgenic
potato to Erwinia chrysanthemi, a bacterial pathogen causing soft rot. The transgenic potato
contained a chromosomally integrated pBR322 replicon, and the gene encoding resistance to
ampicillium was used in selection of E. chrysanthemi transformants. Control experiments
with increasing resemblance to the field conditions were used and a hypothetical transfer
frequency of 2x10-17 was calculated for the horizontal gene transfer event. The authors
estimated that a potato field of 43.000 m2 was equivalent to the calculated frequency.
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GENETICALLY MODIFIED ORGANISMS
Gebhard and Smalla (1999) looked for evidence of horizontal gene transfer in a transgenic
sugar beet field. Using PCR and with the nptII gene as target they could not document any
horizontal transfer from transgenic sugar beet to indigenous soil bacteria. However, directly
extracted DNA from non-culturable soil bacteria was not analysed, because it was impossible
to prevent transgenic plant material from contaminating the DNA extraction process.
4.6.2 DNA uptake from water
Determination of natural transformation frequencies in aquatic systems is limited. Only a few
organisms and environments have so far been investigated (Goodman et al., 1994; Stotzky,
1997; Day et al., 1998).
Paul et al. (1991) used a high-frequency-of-transfer Vibrio strain and a small non-conjugative
plasmid, pQSR50, to study natural plasmid transfer in marine waters. Marine water
microcosms yielded transformation frequencies of 10-6 to 10-10 transformants per recipient
Vibrio cell for naked plasmid in multimeric form. Transformation in marine sediment was not
observed.
The ability to take up pQSR50 by natural competence was found in 3 out of 30 culturable
marine isolates, and 15 out of 105 water sample isolates could take up and express
homologous chromosomal DNA (Frischer et al., 1994). Based on actual concentrations of
DNA and bacteria found in estuarine waters, a hypothetical rate of 10-5 transformants per liter
per day was estimated using the pQSR50 data. For the Florida Tampa Bay area this number
may be converted to 1010 transformation events per year, a number that demonstrates the
genetic power of ecosystem scale processes.
Williams et al. (1996) demonstrated natural transformation of Acinetobacter sp. BD413 in
river epilithon. Transformation rates of 10-4 were obtained in unenclosed experiments in the
river Taff. Selection was for prototrophy, where a his- recipient strain and a crude lysat of a
donor strain (his+) were placed on a filter securely attached onto a sterilized rock in the
riverbed. Using plasmid DNA a 10-7 transfer rate was obtained. No transformation could be
detected below 10°C in the tested river during winter.
Surprisingly, E. coli was found to be naturally competent in freshwater (Bauer et al., 1996).
Transformation frequencies in river water, spring water and bottled mineral water were in the
10-2 to 10-5 range. Measurements were performed using standard laboratory strains (JM109
and HB101) together with a laboratory plasmid (pUC18) coding for resistance to ampicillin.
The tested water samples contained 0.3-11 mM Ca2+ and the observed number of ampicillinresistant transformants was correlated with this Ca2+ concentration.
4.6.3 DNA uptake from food
Bauer et al. (1999) reported transformation of E. coli in 12 tested food products. A maximal
transformation rate of 10-7 was observed in milk and orange juice. In the transformation assay
laboratory E. coli strains and pUC18 plasmid DNA were used. Bacterial cells grown in milk
or juice before addition of plasmid DNA could be transformed, and transformation took place
both at 0°C storage conditions and at 37°C. The transformation frequencies did not show a
correlation to the Ca2+ content of the tested foodstuffs.
Bräutigam et al. (1997) found natural transformation of Bacillus subtilis, when bacterial cells
and chromosomal DNA were added to milk and chocolate milk. In the assay a mutant strain
requiring tryptophan was transformed using chromosomal DNA from the ancestral
prototrophic strain. In general transformation frequencies between 1 and 2 orders of
magnitude higher than for sterile tap water were observed for the tested milk products. The
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TRANSFER OF DNA
highest transformation frequency, 3x10-3, was obtained for chocolate milk with 1.5% fat.
Bacillus subtilis cells growing in chocolate milk also developed natural competence.
Zenz et al. (1998) concluded that chocolate milk for unknown reasons was a suitable medium
for B. subtilis transformation. A neutral protease with potential in cheese production, npr,
encoded by the B. subtilis plasmid was used in transformation experiments. B. subtilis cells
growing in chocolate milk with 0.3% fat were found to yield 5 times more transformants than
cells growing in chocolate milk with 1.8% fat. The maximum frequency obtained for cells
growing in chocolate milk were 102 transformants per 0.01 µg DNA.
Heller et al. (1995) evaluated the possibility for horizontal gene transfer events in yogurt.
They found low transformation efficiency for Streptococcus thermophilus using
electroporation.
4.6.4 Miscellaneous data on transformation
Survival and transforming ability of plasmid DNA added to human saliva were studied by
Mercer et al. (1999). The plasmid DNA was rapidly degraded when incubated with fresh
saliva, but Streptococcus gordonii transformants were obtained in decreasing numbers for a
period of 10 min. The authors also found evidence that factors present in human saliva,
promoted development of competence in the oral bacterium.
Adam et al. (1999) examined the transmission by transformation of genetic material from
Saccharomyces cerevisiae to E. coli. Lysates prepared from a yeast strain with
chromosomally integrated pUC plasmids were used to transform artificially prepared, highly
competent E. coli cells. Ampicillin resistant transformants were obtained at a low frequency;
one µg of DNA yielded approximately 50 transformants. This suggests that transfer of
chromosomal yeast genes by transformation into E. coli under natural conditions must be a
rare event.
The plant pathogen Ralstonia solanacearum was confirmed to develop natural competence
(Bertolla et al., 1997). However, a requirement for at least 50 bp of homologous DNA made
integration of DNA from other species an inefficient event.
4.7 Conclusions
Natural transformation is only known to develop in bacteria involving cooperation between
multiple gene products. The ability to take up naked DNA is a general feature common in
different bacterial genera. Specific regulation and mode of the competence features have most
likely evolved as a consequence of need for specific adaptation. A low frequency of
horizontal gene transfer via natural transformation has been documented, and both natural and
recombinant DNA can be shuffled between bacteria even between unrelated genera. However,
the knowledge of these shuffling rates is insufficient. One reason is that rare events are hard
to detect, and the methods available need to be improved. Few naturally occurring bacteria
can be cultivated and studied (for soil the number is less than 1%). The impact on genetic
shuffling by this high number of non-culturable bacteria should be investigated.
Recombinant DNA released into the environment must be expected to display a very low
level of horizontal gene transfer. Such rare transfer event should be assured not to generate
superior or otherwise harmful bacteria. Even autoclaved DNA has been reported to retain
transformability under specific conditions. Procedures for safe disposal of genetic material
may need to be re-evaluated and the efficiency tested by transformation experiments.
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GENETICALLY MODIFIED ORGANISMS
Natural transformation has been detected in foodstuffs, using laboratory strains. Natural
transformation in food by indigenous bacteria should be investigated.
4.8
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Lawrence, J.G. (1997). Selfish operons and the speciation by gene transfer. Trends Microbiol. 5: 355-359.
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Lawrence, J.G. and Roth, J.R. (1996). Selfish operons: horizontal transfer may drive the evolution of gene
clusters. Genetics 143: 1843-1860.
Lazazzera, B.A., Solomon, J.M. and Grossman, A.D. (1997). An exported peptide functions intracellularly to
contribute to cell density signaling in B. subtilis. Cell 89: 917-925.
Lee, G.H. and Stotzky, G. (1999). Transformation and survival of donor, recipient, and transformants of Bacillus
subtilis in vitro and in soil. Soil Biol. Biochem. 31: 1499-1508.
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Lorenz, M.G., Meyer, B., Wittstock, M., Graupner, S. and Wackernagel, W. (1998). Selective DNA uptake and
DNA restriction as barriers to horizontal gene exchange by natural genetic transformation in Pseudomonas
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London.
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Maiden, M.C.J. (1998). Horizontal genetic exchange, evolution, and spread of antibiotic resistance in bacteria.
Clin. Infec. Dis. 27 (Suppl 1): S12-20.
Majewski, J. and Cohan, F.M.. (1998). The effect of mismatch repair and heteroduplex formation on sexual
isolaton in bacillus. Genetics 148: 13-18.
Masters, C.I., Miles, C.A. and Mackey, B.M. (1998). Survival and biological activity of heat damaged DNA.
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Matic, I., Rayssiguier, C. and Radman, M. (1995). Interspecies gene exchange in bacteria: the role of SOS and
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Maynard Smith, J., Smith, N.H., O’Rourke, M. and Spratt, B.G. (1993). How clonal are bacteria? Proc. Natl.
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Mehr, I.J. and Seifert, S. (1998). Differential roles of homologous recombination pathways in Neisseria
gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair. Mol. Microbiol. 30: 697-710.
Mercer, D.K., Scott, K.P., Bruce-Johnston, W.A., Glover, L.A. and Flint, H.J. (1999). Fate of free DNA and
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Environ. Microbiol. 65: 6-10.
Nielsen, K.M., Bones, A.M. and van Elsas, J.D. (1997). Induced natural transformation of Acinetobacter
calcoaceticus in soil microcosms. Appl. Environ. Microbiol. 63: 3972-3977.
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TRANSFER OF DNA
Nielsen, K.M., van Weerelt, M.D.M., Berg, T.N., Bones, A.M., Hagler, A.N. and van Elsas, J.D. (1997). Natural
transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms. Appl.
Environ. Microbiol. 63: 1945-1952.
Nielsen, K.M. (1998). Barriers to horizontal gene transfer by natural transformation in soil bacteria. APMIS 106:
77-84.
Nielsen, K.M., Bones, A.M., Smalla, K. and van Elsas, J.D. (1998). Horizontal gene transfer from transgenic
plants to terrestrial bacteria - a rare event ? FEMS Microbiol. Rev. 22: 79-103.
Nielsen, K.M., Smalla, K.and van Elsas, J.D. (2000). Natural transformation of Acinetobacter sp. strain BD413
with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in soil microcosms.
Appl. Environ. Microbiol. 66: 206-212.
Ogram, A.V., Mathot, M.L., Harsh, J.B., Boyle, J. and Pettigrew Jr., C.A. (1994). Effects of DNA polymer
length on its adsorption to soils. Appl. Environ. Microbiol. 60: 393-396.
Ogura, M., Liu, L., LaCelle, M., Nakano, M.M. and Zuber, P. (1999). Mutational analysis of ComS: evidence for
the interaction of ComS and MecA in the regulation of competence development in Bacillus subtilis. Mol.
Microbiol. 32: 799-812.
Paget, E. and Simonet, P. (1994). On the track of natural transformation in soil. FEMS Microbiol. Ecol. 15: 109118.
Paget, E. and Simonet, P. (1997). Development of engineered genomic DNA to monitor the natural
transformation of Pseudomonas stutzeri in soil-like microcosms. Can. J. Microbiol. 43: 78-84.
Paget, E., Lebrun, M., Freyssinet, G. and Simonet, P. (1998). The fate of recombinant plant DNA in soil. Eur. J.
Soil Biol. 34: 81-88.
Palmen, R. and Hellingwerf , K.J. (1997). Uptake and processing of DNA by Acinetobacter calcoaceticus - a
review. Gene 192: 179-190.
Paul, J.H., Frischer, M.E. and Thurmond, J.M. (1991). Gene transfer in marine water column and sediment
microcosms by natural plasmid transformation. Appl. Environ. Microbiol. 57: 1509-1515.
Persuh, M., Turgay, K., Mandic-Mulec, I. and Dubnau, D. (1999). The N-terminal and C-terminal domains of
MecA recognize different partners in the competence molecular switch. Mol. Microbiol. 33: 886-894.
Pietramellara G., Dal Canto, L., Vettori, C., Gallori, E. and Nannipieri, P. (1997). Effects of air-drying and
wetting cycles on the transforming ability of DNA bound on clay minerals. Soil Biol. Biochem. 29: 55-61.
Porstendörfer, D., Drotschmann, U. and Averhoff, B. (1997). A novel competence gene, comP, is essential for
natural transformation of Acinetobacter sp. strain BD413. Appl. Environ. Microbiol. 63: 4150-4157.
Recorbet, G., Picard, C., Normand, P. and Simonet, P. (1993). Kinetics of the persistence of chromosomal DNA
from genetically engineered Escherichia coli introduced into soil. Appl. Environ. Microbiol. 59: 4289-4294.
Rivera, M.C., Jain, R., Moore, J.E. and Lake, J.A. (1998). Genomic evidence for two functionally distinct gene
classes. Proc. Natl. Acad. Sci. USA 95: 6239-6244.
Romanowski, G., Lorenz, M.G., Sayler, G. and Wackernagel, W. (1992). Persistence of free plasmid DNA in
soil monitored by various methods, including a transformation assay. Appl. Environ. Microbiol. 58: 3012-3019.
Rudel, T,, Facius, D., Barten, R., Scheuerpflug, I., Nonnenmacher, E. and Meyer, T.F. (1995). Role of pili and
the phase-variable PilC protein in natural competence for transformation of Neisseria gonorrhoeae. Proc. Natl.
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Schlüter, K., Fütterer, J. and Potrykus, I. (1995). “Horizontal” gene transfer from a transgenic potato line to a
bacterial pathogen (Erwinia chrysanthemi) occurs - if at all - at an extremely low frequency. Bio/Technology 13:
1094-1098.
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Sikorski, J., Graupner, S., Lorenz, M.G. and Wackernagel W. (1998). Natural transformation of Pseudomonas
stutzeri in a non-sterile soil. Microbiology 144: 569-576.
van Sinderen, D., Luttinger, A., Kong, L., Dubnau, D., Venema, G. and Hamoen, L. (1995). comK encodes the
competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis.
Mol. Microbiol. 15: 455-462.
Smith, N.H., Holmes, E.C., Donovan, G.M, Carpenter, G.A. and Spratt, B.G. (1999). Networks and groups
within the genus Neisseria: analysis of argF, recA, rho, and 16S rRNA sequences from human Neisseria species.
Mol. Biol. Evol. 16: 773-783.
Solomon, J.M. and Grossman, A.D. (1996). Who’s competent and when: regulation of natural competence in
bacteria. Trends Genet. 12: 150-155.
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Turgay, K., Hamoen, L.W., Venema, G. and Dubnau,D. (1997). Biochemical characterization of a molecular
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Vázquez, J.A., Berrón, S., O’Rourke, M., Carpenter, G., Feil, E., Smith, N.H. and Spratt, B.G. (1995).
Interspecies recombination in nature: a meningococcus that has acquired a gonococcal PIB porin. Mol.
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de Vries, J. and Wackernagel, W. (1998). Detection of nptII (kanamycin resistance) genes in genomes of
transgenic plants by marker-rescue transformation. Mol. Gen. Genet. 257: 606-613.
Wackernagel, W., Sikorski, J., Blum, S., Lorenz, M.G. and Graupner, S. (1998). Natural genetic transformation
of bacteria in soil. In Horizontal gene transfer. M. Syvanen and C. Kado (eds), pp. 168-178. Chapman and Hall,
London.
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regulator of natural competence in Streptococcus pneumoniae. Mol. Microbiol. 33: 817-827.
Widmer, F., Seidler, R.J., Donegan, K.K. and Reed, G.L. (1997). Quantification of transgenic plant marker gene
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Environ. Microbiol. 62: 2994-2998.
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transformation to populations of marine bacteria. Molec. Mar. Biol. Biotechnol. 6: 238-247.
Wolfgang, M., Lauer, P., Park, H.S., Brossay, L., Hébert, J. and Koomey, M. (1998). PilT mutations lead to
simultaneous defects in competence for natural transformation and twitching motility in pilated Neisseria
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gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural
transformation. Mol. Microbiol. 31: 1345-1357.
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TRANSFER OF DNA
Yin, X. and Stotzky, G. (1997). Gene transfer among bacteria in natural environments. Advances in Applied
Microbiology 45, 153-212.
Zenz, K.I., Neve, H., Geis, A. and Heller, K.J. (1998). Bacillus subtilis develops competence for uptake of
plasmid DNA when growing in milk products. System. Appl. Microbiol. 21: 28-32.
Zhou, J., Bowler, L.D. and Spratt, B.G. (1997). Interspecies recombination, and phylogenetic distortions, within
the glutamine synthetase and shikimate dehydrogenase genes of Neisseria meningitidis and commensal Neisseria
species. Mol. Microbiol. 23: 799-812.
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GENETICALLY MODIFIED ORGANISMS
5
TRANSFER OF DNA FROM LIVE GMO
by Lars Hagsholm Pedersen
5.1 Summary
Genetic information, including genetically engineered information can flow
between different living bacterial species by conjugation and conjugative
transposition of plasmids and conjugative transposons, respectively. The
frequencies by which conjugation and transpositions occur under laboratory and
natural conditions are very variable and highly dependent on the experimental
conditions, i.e. the biotic and abiotic factors. In situ expression and hybridization
techniques are and will be valuable tools for future studies of bacterial DNA
transfer processes in man made environments. Inter kingdom transfer of the Ti
plasmid or parts of the (engineered) Ti plasmid to organisms other than plants is
possible under optimized conditions. These organisms include yeast and
filamentous fungi.
5.2 Conjugative plasmid transfer and DNA transposition
The transfer of DNA from live bacteria to other live bacteria is possible by conjugation and
transposition, respectively, and is believed to be the principal way by which live bacteria
exchange genetic information. Conjugation and conjugative transposition requires that donor
and recipient cells are in close proximity. Also, the appropriate transfer, mobilization and
origin of transfer (oriT) functions have to be present for the transfer of plasmid DNA or
transposomal DNA (Lorentz and Wackernagel, 1996; Kroer et al., 1996; Heinemann, 1998;
Yin & Stotzsky, 1997; Davison, 1999).
The transfer mechanisms within genera and species of Gram-negative and Gram-positive
bacteria are different. In Gram-negative bacteria special structures, pili, form a tunnel which
allows transfer of single stranded DNA molecule between bacteria, whereas other
mechanisms such as pheromone induced conjugation exist in Gram-positive bacteria. Nonconjugative plasmids may be mobilized by the transacting functions of self-transmissible
plasmids or by co-integrative (conductive) transfer (Yin & Stotzsky, 1997). Transferred
plasmid DNA can either replicate as a free extra-chromosomal element if a functional
replicon is present or replication factors are supplied by the host. The plasmid DNA may also
become integrated on the host chromosome e.g. as the F factor. A chromosomally integrated
plasmid may later be able to transfer itself and host-chromosomal information to a new host.
Conjugative transposons can either integrate on (other) plasmids or in the chromosome of the
new host (Salyers et al., 1998; Hall, 1998).
A special case of inter kingdom conjugation is the horizontal transfer of the T-DNA element
of the Agrobacterium tumefaciens Ti-plasmid or the Agrobacterium rhizogenes Ri-plasmid to
plant cells (Kado et al., 1998; Fründt et al., 1998).
5.3 Which bacteria can transfer and receive genetic information
Many known genera of both Gram-negative and Gram-positive bacteria can receive new
genetic information by conjugative processes (e.g. see Table 2 in Davison, 1999). The intra
and inter species spread of plasmids is dependent on the host range, the incompatibility with
other plasmids in the recipients and of the restriction system compatibility of the host (Kroer
et al., 1996; Yin & Stotzky, 1997). In theory all genes can be transferred, including
recombinant, and thereby provide recipients with new transient or permanent functions e.g.
antibiotic and heavy metal resistance beneficial to the organism (Kroer et al., 1996; Yin &
Stotsky, 1997; Davison, 1999).
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TRANSFER OF DNA
5.4 Factors of importance for the transfer process
Numerous laboratory investigations, artificial microcosm experiments and field tests have
shown or indicated that conjugative transfer take place or can take place in nature. There are
many physical, chemical, topological and biological constraints to the transfer processes
(Kroer et al., 1996; Yin & Stotsky, 1997; Christensen et al., 1998; Dahlberg et al, 1998;
Davison, 1999). Major biological obstacles to the transfer process are physiological state,
plasmid incompatibility and DNA restriction (Yin & Stotzsky, 1997), and the spatial structure
may also be important (Christensen et al., 1998; Hausner & Wuertz, 1999). Given that the
recipient and the donor bacteria are competent e.g. metabolically active, their numbers
adequate and the abiotic factors allows for it, then conjugational transfer can take place (Kroer
et al., 1996; Yin & Stotsky, 1997; Davison, 1999).
Reports have shown or indicated that intra and inter species conjugative DNA transfer
between different bacterial genera can be observed in nearly any kind of environment (Yin &
Stotsky, 1997; See Table 2, in Davison (1999)). Tracking of resistance genes has often been
the preferred method of detecting DNA transfer. For a list and description of the examined
environments, biotic and abiotic factors refer to Kroer et al. (1996); Yin & Stotzsky (1997);
Davison (1999); Newby et al. (2000); Thomas et al. (2000).
Direct visualization of horizontal gene transfer have been performed by in situ hybridization
and gene expression studies using the bio-reporter molecule green fluorescent protein (GFP).
In situ studies have shown that horizontal gene transfer by conjugation take place in artificial
biofilms (Christensen et al., 1998), in seawater environments (Dahlberg et al., 1998) and in
activated sludge (Geisenberger et al., 1999). Hausner and Wuetz (1999) provided evidence for
high rates of conjugative transfer (10-2 to 10-3 h-1) in artificial biofilms. In situ techniques may
turn out to be significant tools for direct measurement of conjugational transfer to bacteria,
which are non-culturable, and for investigating transfer in various man-made environments
(e.g. food processing machines).
5.5 Conjugational transfer rates
The frequencies by which conjugative events take place in laboratory systems or under
natural conditions are highly variable. The values range from below the detection limit
(approximately 10-10) to about 10-1 transconjugants per recipient under optimized laboratory
conditions but are highly dependent on the individual experiments (Kroer et al., 1996; Yin &
Stotzsky, 1997; Christensen et al., 1998; Hausner & Wuertz, 1999; Geisenberger et al., 1999;
Newby et al., 2000). Recently, it was demonstrated that conjugational gene transfer in the gut
occurred at rates higher than or comparable to forced filter mating frequencies (Netherwood et
al., 1999).
5.6 Molecular phylogenetic evidence for transfer
The experimental evidence for intra and interspecies transfer in nature is also supported by
phylogenetic analysis of aligned DNA sequence data. By using molecular phylogenetic
analysis techniques evidence for horizontal transfer of resistance genes between different
bacterial species (Salyers et al., 1998; Lawrence & Roth, 1998; Wiener et al. 1998) and
between bacterial species and plants have been presented (Fründt et al., 1998).
5.7 Transfer of DNA from Ti and Ri plasmids
It is well known that T-DNA can be transferred from A. tumefaciens and A. rhizogenes to
plants (Kado, 1997; Fründt et al., 1997; Nam & Gelvin, 1997). Under laboratory conditions
the (genetically engineered) T-DNA region of Ti-plasmids has been transferred from A.
tumefaciens to Esherichia coli and Streptomyces lividans, and to the fungi Saccharomyces
pombe, Saccharomyces cerevisiae (Heineman, 1991,1998). Recently, A. tumefaciens has been
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GENETICALLY MODIFIED ORGANISMS
shown to increase the efficiency of transformation of filamentous fungi by a conjugative
process (de Groot et al., 1998; Gouka et al., 1998). Whether such processes also take place
under natural conditions is unknown.
Whether recombinant DNA containing T-DNA can be transferred from plants by
transformation of wild type A. tumefaciens strains or A. rhizogenes remains to be shown
(Nielsen et al. 1998; Bertolla & Simonet, 1999). Gebhard and Smalla (1998), who indicated
the possibility of this event under laboratory conditions transformed Acetinobacter with
chromosomal DNA purified from transgenic sugarbeets.
5.8 Conclusions
Under laboratory, simulated natural, and natural conditions, plasmids and conjugative
transposons can be transferred from one bacterium to bacteria from other genera, but only at
low frequencies, which are strongly dependent on several biotic and abiotic factors.
It is possible to obtain significant rates (i.e. rates equal to or higher than the general detection
limit of 10-10 to 10-9) of horizontal transfer under optimized laboratory and microcosm
conditions, but the rates are variable and highly dependent on the experimental setup. In situ
expression and hybridization techniques are and will be valuable tools for future studies of
bacterial DNA transfer processes.
Laboratory experiments have shown that inter kingdom transfer of the Ti-plasmid or parts of
the (engineered) Ti-plasmid to organisms other than plants is possible under optimized
conditions. These organisms include yeast and filamentous fungi. The transfer rate(s) of
information from plants to agrobacteria under natural conditions is very low under laboratory
conditions and remains to be determined under natural conditions.
5.9
References
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putative process for gene transfers between transgenic plants and micro organisms. Res. Microbiol. 150: 375384.
Christensen, B. B., Sternberg, C., Andersen, J. B., Eberl, L., Møller, S., Givskov, M., and Molin, S. (1998).
Establishment of new genetic traits in a microbial biofilm community. AEM 64 (6), 2247-2255.
Dahlberg, C., BergströmM., and Hermansson, M. (1998). In situ detection of high levels of horizontal plasmid
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Davison, J. (1999). Genetic exchange between bacteria in the environment. Plasmid 42, 73-91.
Fründt, C., Meyer, A. D., Ichikawa, T., and Meins, F. (1998). Evidence for the ancient transfer of Ri-plasid TDNA genes between bacteria and plants. Syvanen, M. and Kado, C. I. Horizontal gene transfer (8), 94-106..
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Gebhard, F. Smalla K. (1998). Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet
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bacteria. Syvanen, M. and Kado, C. I. Horizontal gene transfer. 53-62.. London, Chapman and Hall.
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mellem bakterier, Miljøprojekt nr. 324. Miljø- og Energiministeriet and Miljøstyrelsen. 1-85 København.
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natural bacterial populations. Tomiuk, J., Wöhrman, K., and Shoemaker, N. B. Transgenic organisms Biological and social implications. 45-57.. Basel, Birkhäuser Verlag.
Nam, J. and Gelwin, S. B. (1998). Arabidopsis ecotypes resistant to crown gall tumorigenesis. Syvanen, M. and
Kado, C. I. Horizontal gene transfer. (7), 75-93. London, Chapman and Hall.
Netherwood, T., Bowden, R., Harrison, P., O'Donnell, A. G., Parker, D. S., and Gilbert, H. J. (1999). Gene
transfer in the gastro intestinal tract. AEM 65 (11), 5139-5141.
Newby, D. T., Josephson, K. L., and Pepper, I. L. (2000). Detection and characterization of plasmid pJP4
transfer to indigenous soil bacteria. AEM 66 (1), 290-296.
Nielsen, K. M., Bones, A. M., Smalla, K., and van Elsas, J. D. (1998). Horizontal gene transfer from transgenic
plants to terrestrial bacteria - a rare event? FEMS Microbiology reviews 22, 79-103.
Salyers, A. A., Coper, A. J., and Shoemaker, N. B. Lateral broad host range transfer in nature.how and how
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Thomas, D. J., Morgan, J. A., Whipps, J. M., and Saunders, J. R. (2000). Plasmid transfer between the Bacillus
thurengensis subspecies kurstaki and tenebrionis in laboratory culture and soil and in lepidopteran and
coleopteran larvea. AEM 66 (1), 118-124.
Wackernagel, W., Sikorski, J., Blum, S., Lorenz, M. G., and Graupner, S. (1998). Natural genetic transformation
of bacteria in soil. Syvanen, M. and Kado, C. I. Horizontal gene transfer 168-191. London, Chapman and Hall
Wiener, P., Egan, S., Huddleston, A. S., and Wellington, E. M. (1998). Evidence for transfer of antibioticresistance genes in soil populations of streptomyces. Molecular Ecology 7 (9), 1205-1216.
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Microbiology 45, 153-212.
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GENETICALLY MODIFIED ORGANISMS
6
DNA TRANSFER FROM FUNGI TO NON-FUNGAL HOSTS
by Lars Hagsholm Pedersen
6.1 Summary
Fungi constitute a diverse group of organisms of which some contain plasmids
and transposable elements. Under optimized laboratory conditions plasmids have
been transferred between different fungal species, and there is molecular
phylogenetic evidence of ancient transfer of both transposons and plasmids. It is
indicated that unstable transfer of genetic information from plant material to
fungi may take place. Rates of horizontal transfer of DNA between fungi and
between other organisms and fungi under natural conditions are largely
unknown. Further investigations are necessary to estimate the fate and rate of
horizontal DNA transfer under natural or simulated natural conditions.
6.2 Exchange of genetic material between fungi
The fungal kingdom is composed of a diverse group of different uni- or multi-cellular
eukaryotic organisms, often with very complicated lifecycles (Griffin, 1994). Some fungi can
be considered as large organisms due to the large spatial distribution of their mycelia, which
offers the possibility of multiple contact points for putative anastomosis events of hyphae
belonging to different species. Some fungal genera have complicated sexual life cycles
(Staben., 1995), whereas other genera have no known sexual life cycles (Adams et al., 1995).
A unique feature of some fungi is the coenocytic organization, i.e. the presence of numerous
nuclei in the cytoplasm (e.g. cells of mycelia growth points contain many nuclei).
In a laboratory experiment it was demonstrated that a chromosome could be transferred from
one biotype of Colletotrichum. gloeosporiodes to another biotype (He et al., 1998). Whether
such a transfer could happen under natural circumstances between different fungi is not
known.
Like bacteria some fungi contain small or large extra-chromosomal elements e.g. plasmids.
Both linear and circular plasmids have been found in a diverse range of unicellular and
multicellular fungal genera (Griffiths, 1995; Kempken, 1995; Poplawski et al., 1997).
Kempken (1995) demonstrated that a linear mitochondrial plasmid could be horizontally
transferred from Ascobolus immersus to Poduspora anserina. Another example of “natural
horizontal inter genera” transfer between fungi is found in the host-parasite system Absidia
glauca – Parasitella parasitica where auxotrophic mutants of the zygomycete fungus Absidia
glauca became prototrophic upon transfer of genetic information from the myco-parasitic
fungus Parasitella parasitica (Wöstemeyer et al., 1998). In addition it was shown that an
autonomously replicating plasmid could be transferred from Parasitella parasitica to Absidia
glauca. In the laboratory it has been demonstrated that different species of Saccharomyces
can mate and form new hybrids (Marinoni et al, 1999). More distant species formed few
hybrids and their stability was low. Whether mating between distantly related yeast is found
in nature remains to be shown.
6.3 Transposons
Fungi also contain different types of transposons, which do not appear to contain resistance
genes (See Table in Kempken & Kück (1998)). There is molecular phylogenetic evidence,
which suggests that fungal transposons may have been and are involved in horizontal transfer
of information between fungi but this area of research is rather new and more experimental
evidence is needed.
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TRANSFER OF DNA
6.4 Transfer of genetic information to fungi from other organisms
Transfer of hygromycin B resistance from transgenic Brassica datura and Vicia plants to the
filamentous phytopathogenic fungus Aspergillus niger by transformation was investigated in
laboratory experiments (Hoffman et al., 1996). Transgenic hygromycin B resistant plants
were transformed by Agrobacterium tumefaciens or by direct gene transfer. Afterwards, plant
material was inoculated with A. niger. Hygromycin resistant A. niger colonies, of which some
contained the hygromycin resistance gene, hph, were isolated indicating that DNA transfer
had taken place. However the hygromycin resistance was only transiently expressed, and the
mechanism of transfer was unknown. As mentioned in chapter 5 it has been shown that under
optimized laboratory conditions A. tumefaciens can transfer (part) of its Ti plasmid to both
yeast and filamentous fungi (Heinemann, 1998; de Groot et al., 1998; Gouka et al., 1999).
6.5 Conclusions
There is direct and molecular phylogenetic evidence for horizontal transfer of plasmid and
transposon DNA between fungi. There is also laboratory evidence for transfer of DNA from
plants and A. tumefaciens to fungal hosts. The information concerning transfer rates are
lacking.
More research is needed with respect to investigations concerning fungal uptake (by
transformation and "conjugation") of foreign DNA from plants, bacteria or other fungi under
natural or simulated microcosm conditions.
6.6
References
Adams, T.H. Asexual sporulation of lower fungi. Gow, N. A. R. and Gadd, G. M. (1995). The growing fungus.
367-382. London, Chapman & Hall.
Gouka, R. J., Gerk, C., Hooykaas, P. J., Bundock, P., Musters, W., Vrrips, C. T., and de Groot, M. J.
(1999).Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologues
recombination. Nat.Biotechnology 17(6), 598-601.
Griffin, D.H. (1994). Fungal physiology. Wiley-Liss, 1 - 457, New York,
Griffiths, A. J. (1995). Natural plasmids of filamentous fungi. Microbiol.Rev. 59(4), 673-685.
de Groot, M. J., Bundock, P., Hooykaas, P. J., and Beijersbergen, A. G. (1998).Agrobacterium tumefaciensmediated transformation of filamentous fungi. Nat.Biotechnology 19(9), 839-842.
He, C., Rusu, A. G. Poplawski A. M., Irwin, J. A., and Manners, J. M. (1998). Transfer of a supernumerary
chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides.
Genetics 150(4), 1459-1466.
Heinemann, J. A. (1998). Looking sideways of the origin of replicons. Syvanen, M. and Kado, C. I. Horizontal
gene transfer 11-23. London, Chapman and Hall.
Hoffman T, Golz C, and Scheider O. (1996). Preliminary findings of DNA transfer from transgenic plants to a
wild-type strain of Aspergillus niger. Schmidt ER and Hankeln Th. Transgenic organisms and biosafety. 77-85.
Berlin, Springer Verlag.
Kempken, F. Horizontal transfer of a mitochondrial plasmid. Mol.Gen.Genet. 248(1), 89-94. 1995.
Kempken, F. and Kück, U. (1998). Transposons in filamentous fungi - facts and perspectives. Bioessays 20(8),
652-659.
Marinoni, G., Manuel, M., Petersen, R. F., Hvidtfeldt, J., Sulo, P., and Piskur, J. (1999). Horizontal transfer of
genetic material among Saccharomyces yeasts. J.Bacteriol. 181(20), 6488-6496.
Poplawski, A. M., He, C., Irwin, J. A., and Manners, J. M. (1997). Transfer of an autonomously replicating
vector between vegetatively incompatible biotypes of Colletotrichum gloeosporioides. Curr.Genet. 32(1), 66-72.
- 43 -
GENETICALLY MODIFIED ORGANISMS
Staben, C. (1995). Sexual reproduction in higher fungi. Gow, N. A. R. and Gadd, G. M. The growing fungus.
383-402. London, Chapman & Hall.
Wöstemeyer, J., Wöstemeyer, A., Burmester, A., and Czempinski, K. (1998). Horizontal genetransfer in the
host-parasite system Absidia glauca-Parasitella parasitica. Syvanen, M. and Kado, C. I. Horizontal gene transfer.
118-128. London, Chapman and Hall.
- 44 -
TRANSFER OF DNA
7
DNA TRANSFER FROM FOOD AND NON-BACTERIA
by Lars Hagsholm Pedersen
7.1 Summary
Evidence of natural horizontal transfer of (parts) of high molecular weight DNA
from foods of animal or plant origin to eukaryotic cells and bacteria is sparse.
The rate of transfer of recombinant eukaryotic DNA to bacteria may be very low.
The rate of transfer of recombinant eukaryotic DNA from food to eukaryotic
epithelial cells may be very low or non-detectable. The significance of such an
uptake process, if occurring at all, is not known and needs to be verified
experimentally.
7.2 Genetically modified foods
There is an increasing interest in the possibility of DNA transfer from recombinant food and
feed to other organisms as genetically modified plant food and feed such as maize and
soybean has become available (Berkowitz, 1990; Acuff et al., 1991; Moseley, 1999).
Only genetically engineered foods of plant origin are currently commercially available. These
include tomato, soybean, maize, sugar beet and rape (Schauzu, 1998). To our knowledge no
bacterial, fungal, fish and animal foods containing recombinant DNA are currently
commercially available, albeit such foods have been constructed (Teuber, 1996).
There are many potential sources of recombinant DNA present in foods. Recombinant
chromosomal DNA may be present in primary foods such as tomato and maize or may be
present in complex foods together with other basic ingredients (soybean in burgers, rapeseed
oil in other ingredients). Although not released to the market recombinant DNA present in
microorganisms (e.g. bacteria) with GRAS status may also become a source of DNA in the
near future, e.g. in certain dairy products. Recombinant DNA may also be present in trace
amounts of commercially available secondary foods, i.e. food additives or food grade
enzymes produced by engineered organisms (fungi, bacteria), although such residual DNA is
not allowed (Teuber, 1996).
The literature concerning PCR-detection and other nucleic acid detection protocols of
recombinant organism DNA in food is not covered here, although such techniques are
important with respect to detection of recombinant DNA fragments in e.g. complex foods and
intestinal contents (MacCormick et al. 1998; Hubner et al., 1999)
7.3 Uptake of DNA from recombinant foods
There are references which address the potential risks associated with digestion of genetically
modified material (Berkowitz, 1990; Mitten, 1996). They argue that, in theory large un-nicked
fragments of chromosomal (or plasmid) DNA are unlikely to reach the intestine because of
the acid environment in the stomach combined with the nucleolytic activities of the intestine.
Thus, the probability of a specific piece of recombinant DNA derived from a transgenic
organism (genome) being taken up by epithelial cells and be stabile integrated into the
chromosome suggested to be very low (non-measurable). Furthermore, the way by which
recombinant food is or may be processed industrially combined with the matrix properties of
the food may influence the integrity of the recombinant DNA.
7.4 Evidence for horizontal transfer of DNA
It is well known that conjugation of plasmids and transformation of bacteria can take place in
the gut (Yin & Stotzky, 1997; see chapter 4 and references herein for further details). Whether
microflora of the salivary system or the gut microflora can be transformed by high molecular
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GENETICALLY MODIFIED ORGANISMS
weight or partially degraded DNA (e.g. recombinant plant DNA) under natural conditions is
more speculative, but the transformation frequencies may be comparable to the ones
calculated for uptake of plant DNA by bacteria in soil systems (see Chapter 4).
Laboratory experiments showed that segments of plasmid DNA feed to mice can be found
transiently in epithelial cells, leukocytes, spleen and liver cells of the animals by PCR
techniques some time after ingestion (Schubbert et al., 1997 & 1998, see chapter 8 for more
details). The authors claimed that the foreign DNA was integrated into the host chromosome,
but their evidence was weak and not convincing. The amount of DNA (50 µg) ingested by the
mice corresponds to approximately 1013 plasmid genomes, which is a huge amount of genome
equivalents. To illustrate this we calculated the amount of tomatoes, which correspond to this
number of gene equivalents. A plant cell is set to 25 x 25 x 25 µm equivalent to 1.6 x 10-14
m3. The diameter of the tomato is set to 10 cm equivalent to 5 x 10-4 m3). Thus one tomato
contains 3.1 x 1010 cells - and genomes. Therefore the mouse should ingest 300 tomatoes
(equivalent to approximately 25 kg ) in order to obtain 1013 genomes. If the mouse should
ingest a bacterial culture containing the plasmid (assuming an OD450 of 50) this would
correspond to ingesting 400 ml of bacterial culture. If these numbers should be normalized to
a human being (70 kg) these figures should be multiplied by 2500.
The information which can be obtained from such an experiment, is that under optimized
conditions naked plasmid DNA can be taken up by epithelial cells and transferred to other
organs. Whether such plasmid-uptake processes is taking place under natural conditions, i.e.
in the presence of complicated fragmented food matrices and bacteria is not known and needs
to be verified experimentally. Whether the salivary system or the gut microflora or human
epithelial cells can take up high molecular weight or partially degraded DNA (e.g.
recombinant plant DNA) under natural conditions, i.e. by intake of "natural doses" of genetic
material (e.g. 4 transgenic tomatoes and 6 potatoes) is more speculative and remains to be
shown.
7.5 Conclusions
Evidence of (transient) natural horizontal transfer of fragments of high molecular weight
DNA from foods of animal or plant origin to eukaryotic cells and bacteria is sparse.
The rate of transfer of recombinant eukaryotic DNA (e.g. a bacterial kanamycin resistance
gene used as selective marker in plants) to bacteria may be very low and comparable to the
rate calculated for transformation of bacteria in soils (<10-17).
The rate of transfer of recombinant eukaryotic DNA present in food to eukaryotic epithelial
cells may be very low or non-detectable. The significance of such an uptake process, if
occurring at all, is not known and needs to be verified experimentally.
7.6
References
Acuff, G. R., Albanese, R. A., Batt, C. A., Berndt, D. L., Byers, F. M., Dale, B. E., Denton, J. H., Fuchs, R. L.,
Gastel, B., and Heidelbaugh, N. D. (1991). Implications of biotechnology, risk assessment, and communications
for the safety of foods of animal origin Mechanisms and consequences of horizontal gene transfer in natural
bacterial populations. J.Am.Vet.Med.Assoc. 1991 (12), 1714-1721.
Berkowitz, D. B. (1990). The food safety of transgenic animals. Biotechnology 8, 819-825.
Bertolla, F. and Simonet, P. (1999). Horizontal gene transfer in the environment: natural transformation as a
putative process for gene transfers between transgenicc plants and microorganisms. Res.Microbiol. 150, 375384.
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TRANSFER OF DNA
Hubner, P., Studer, E., and Luthy, J. (1999). Quantitation of modified organisms in food. Nat. Biotechnology. 17,
1137 - 1138.
MacCormick, C.A., Griffin, H.G., Underwood, H.M., Gasson, M.J. (1998) Common DNA sequences with
potential for detection of genetically manipulated organisms in food. J. Appl. Microbiol. 84, 969-980.
Mitten, D., Redenbaugh, K., and Lindemann, J. (1996). Evaluation of potential gene transfer from transgenic
plants. Schmidt, E. R. and Hankeln, Th. Transgenic organisms and biosafety. (9), 95-100. Berlin, Springer
Verlag.
Moseley , B.E.B. (1999). The safety and social acceptance of novel foods. Int.J.Food Microbiol. 50, 25-31.
Schauzu, M. (1998). Lebensmittel aus transgenen pflanzen - zulassungsverfahren und sicherheitsbewertung.
Gordian 98(11), 167-170.
Schubbert, R., Renz, D., Schmitz, B., and Doerfler, W. (1997). Foreign (M13) DNA ingested by mice reaches
periferal leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse
DNA. PNAS 94, 961-966.
Schubbert, R., Hohlweg, U., Renz, D., and Doerfler, W. (1998). On the fate of orally ingested foreign DNA in
mice: chromosomal association and placental transmission to the fetus. Mol. Gen. Genet. 259:569 - 576
Syvanen, M. and Kado, C. I. (1997) Horizontal gene transfer. Chapman and Hall, London.
Teuber, M. (1996). Genetically modified food and its safety assessment. Tomiuk, J., Wöhrman, K., and Sentker,
A. Transgenic organisms - Biological and social implications. 181-195. Basel, Birkhäuser Verlag.
Yin, X. and Stotzky, G. (2000). Gene transfer among bacteria in natural environments. Advances in Applied
Microbiology 45, 153-212.
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GENETICALLY MODIFIED ORGANISMS
8
DNA UPTAKE AND TRANSFER IN EUKARYOTES
by Mads Grønvald Johnsen
8.1 Summary
Even when a large quantity of naked DNA is orally ingested, it appears unlikely
that eukaryotic tissue cells will be transformed. When recombinant DNA is fed to
mammals in the more mobile form of plasmids, no amplification by present
microflora has been observed or reported in conducted experiments. In the field
of vaccine technology naked DNA is presently being tested for its ability to
provide immunity against specific antigens. It has been observed that CpG motifs
can result in proinflammatory responses. The engineering of vaccine bacteria that
are invasive to man may challenge new safety issues. The risk assessments to be
developed in connection with this kind of vaccine productions will most likely
apply to the evaluation of other recombinant DNA procedures as well.
Knowledge is needed on the transfer frequency of DNA elements to “nonculturables”. Only few experiments resembling the in vivo transformation
conditions have been conducted. The considerations concerning the amplification
of recombinant DNA by microflora may well be examined by studies of the
transmission and maintains of DNA elements that are well characterized and
already known to be part of the food and microflora ingested by man.
8.2 Exposure to and ingestion of naked DNA.
If naked DNA is defined as purified preparations suspended in buffer solutions, recombinant
DNA is hardly ever introduced to the environment as truly naked DNA. However, DNA
liberated from either live or dead microorganisms is more likely to come in contact with the
surrounding environment including mammals. DNA will, when released into the environment
or ingested, to a large extend be broken into smaller fragments (Schubbert at al., 1994). DNA
is expected to become, or already be, in complexes with agents originating from the host or
the environment. Development of techniques for transformation has been an important
research tool and the formulation of DNA in complexes has been intensively studied (Lehr,
1996). It has been observed that proper formulation of DNA enhances the uptake by
mammalian tissue cells (Meyer et al., 1995; Schughart et al., 1999). Consequently, evaluation
of DNA uptake should include DNA bound in complex with molecules likely to be present in
the environment. Recombinant DNA mixed with different mammalian food will perform
differently compared to naked DNA administrated directly to the recipient. To our knowledge
no studies have been conducted of the relationship between persistence of recombinant DNA
in combination with ingestion of different food. Results reported from transformation studies,
suggested, that DNA mixed with greasy agents will be both more persistent and may even
transform mammalian tissue cells (Alton et al., 1999).
Several experiments have been conducted with food-ingested recombinant DNA by Schubbert
et al.; 1994; 1997; 1998. These are the most extensive studies on the fate of naked DNA,
administrated orally to mammals that have been reported to date. It is reported that fragments
of recombinant DNA can be traced in the different organs of mice, and potential transmission
to placental fetus is monitored. Not only are these the most extensive reports to date, but they
are also, the first ever to suggest chromosomal integration by orally administrated DNA. As
the results of these studies are somewhat surprising, the experimental procedures deserve a
more profound description and evaluation.
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TRANSFER OF DNA
Four different molecular detection methods have been applied. 1) Detection by polymerase
chain reaction (PCR), 2) fluorescent in situ hybridization, 3) southern hybridization and 4) DNA
cloning procedures were conducted with different organ samples and preparations. From the
results that were obtained with these techniques and with reference to appropriate control
group of mice it appear evident that recombinant DNA fragments can be detected in the blood
of mice and fetuses when orally ingested. Apparently, some DNA fragments have survived
the passage through the gastrointestinal tract (GI) and crossed the mucosal stomach
epithelium. The daily dose of 50 µg pr. mouse (approx. 22g) correspond to an intake of 159
mg naked DNA for a person weighing 70 kg. A daily dose containing this many identical
molecules is unlikely ever to be ingested by humans. However, it appears evident if a large
amount of DNA is ingested that, transfer of DNA fragments to the blood of mammals may
result.
For the verification of potential chromosomal insertion a DNA cloning technique was
implied. Obtained clones contained fragments of the administrated DNA, M13mp18 (Roche
Molecular Biochemicals), in combination with mouse homologous DNA. However, this
experiment did not include cloning from the group of control mice. The Lambda cloning
vector DASHII was prepared for insertion of DNA fragments that were digested with the
restriction endonuclease BamHI. The recognition site of this restriction endonuclease is
present once in every administrated plasmid molecule. The PCR analyses indicated that
administrated plasmid DNA, M13mp18, was broken into fragments when passing through the
GI. Fragments of administrated plasmid DNA only containing one vector complementing site
have to combine with a chromosomal fragment also containing a vector complementing site
in order to become integrated in the Lambda cloning vector. The ligation step in the cloning
procedure used in this study will enforce recombination between administrated plasmid
fragments and chromosomal fragments as presented in Fig. 4. The clones that were isolated
are not likely to represent integration events, but are more likely to be a result of the cloning
procedure. The frequency of recombination during ligation could have been evaluated by
cloning chromosomal DNA isolated from the blood of animals treated with plasmid.
Alternatively, a cloning procedure using an endonuclease restriction site not present in the
administrated plasmid could have reduced the frequency of forced combination during
ligation.
Both the Lambda cloning vector system and the M13mp18 recombinant DNA pass an
airborne bacteriophage developmental stage when propagated (Sambrook et al., 1989). For
very limited cross contamination to take place in a laboratory processing both systems would
be almost inevitable. The cloning system will even allow a few insertions contain
chromosomal DNA originating from the E. coli host used for the amplification. One such
clone was also obtained in these experiments, emphasizing the ease with which low frequency
alterations can be obtained. One clone, ‘c’, was obtained that included highly homologous
mouse DNA suggesting the occurrence of a true integration. DNA sequence information on
the inserted mouse DNA was obtained, which would have allowed verification of insertion by
PCR analysis of mouse chromosomal DNA. No such verification attempts were reported. The
conclusion on chromosomal association of recombinant DNA from these experiments needs
extensive verification in order to prove likely. The authors appear to be aware of this problem
(Schubbert et al., 1998) and are challenging the question by proclaiming an investigation to
come of the possible insert detection in offspring generations of mice.
The cloning procedure is a specific amplification of a single event and represents the
occurrence in a single cell out of the entire organ tissue. For a recombination to have any
profound effect in a mammal, it would have to act on the gametes. If mice were transformed
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GENETICALLY MODIFIED ORGANISMS
simply by orally administrating DNA it would be a sensation in risk assessment, but also
provide a new application in the field of pharmaceutical applications.
Fig 4. Lambda DASHII recombinant DNA insertion. Black bars present the Lambda vector
fragments. Hatched bars represent mouse chromosomal DNA. Recombinant M13mp18 DNA is
presented as dotted bars. The fragments at the top can combine and be inserted at the vector sites,
this represents the most likely event of how administrated plasmid DNA was cloned in
combination with mouse chromosomal DNA. The chromosomal endonuclease restriction fragment
at the bottom represents the event where recombinant plasmid DNA was inserted while the mouse
was alive.
8.3 DNA uptake and transfer by mammalian microflora.
Uptake of recombinant DNA by the microflora of mammalians is a likely event as
microorganisms have been documented to have multiple functions for uptake and transfer of
DNA (Davison, 1999). This may have implications for the host as the recombinant DNA can
be multiplied to large quantities and may alter the composition of the microflora in an
unfavorable way (Netherwood et al., 1999a). However, an evaluation of this scenario is
presently difficult since 60-80% of mammalian microflora consists of non-culturables (Suau
et al., 1999). The ability of these bacteria to take-up, process, or maintain recombinant DNA
has to our knowledge never been examined in humans.
In the study of Klijn et al. (1995) genetically marked lactococcal bacteria were ingested by
humans. In this study viable bacteria from the feces were retrieved for three days after
ingestion. With a delay of one day a corresponding decline of specific DNA was detected.
This result suggested that no specific DNA was transferred to non-culturables or even
transiently amplified. In rats, the ability of live recombinant lactococcal cells to colonize and
exchange DNA with the microflora is assessed (Brockman et al., 1996; Jacobsen et al., 1996).
In these studies a plasmid with highly promiscuous host replicating ability was used, but only
resulted in the detection of a single horizontal transfer. The frequency of culturable bacteria
that had obtained this plasmid DNA was found to be very low, despite the high potential
transfer ability.
The ability of recombinant bacteria to exchange mobile DNA with the corresponding non
recombinant strain has been documented to take place at a frequency as high as 3 out of every
100 examined cells (Netherwood et al., 1999b). This frequency was reached in experiments
with chicks, but could suggest that traditional procedures underestimate in vivo transfer rates.
If invasive bacteria of the microflora easily take up recombinant DNA elements, the DNA
element could be presented inside the mammalian cell.
8.4 Naked DNA as vaccine delivery systems.
Presently, transient transformation of human tissue and experimental animals is being
attempted. Vaccine research, that is exploiting recombinant DNA as delivery system for
antigens, is faced with the difficulty of maintaining gene response (Luo & Saltzman, 2000).
Even when a high concentration of DNA containing eukaryotic expression elements, is
directed to a specific tissue, only limited response is obtained (Luo & Saltzman, 2000).
Vaccine plasmid constructions include well defined recombinant E. coli replication and
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TRANSFER OF DNA
selection elements have been developed (Fig 5). The plasmids that are transiently present in
the mammal cell are expected to express a specific antigen and thus provoke the immune
system. This approach may provide many new solutions to the development of functional
vaccines. One of the most appealing applications is to introduce antigens with the need for
specific glycosylation or other host cell factors needed for the proper conformation of
antigenic form. At present, this could prove an alternative to the production in cultivated
human cell systems, which is difficult as well as expensive. For these same reasons, and the
far more simple preservation, the DNA based vaccines may substitute products that are
presently produced as proteins.
While performing vaccination procedures on mice it has been observed that alterations of the
vector backbone had different immune activating effects in combination with the antigen
introduction. This observation has led to the identification of specific DNA sequences, CpG,
which stimulate proinflammatory responses (Lipford et al., 1998). It is reported that plasmid
DNA of both Gram-positive and Gram-negative bacteria provide this effect and may be part
of the human infection response. The effects are presently being studied for the beneficial
applications, but may also be found to have harmful effects, such as contributing to toxic
chock or sensitize other immune responses. When introduced in man, recombinant DNA
constructions replicated by bacterial hosts may unintentionally induce different degree of
immune responses. However, at present it takes large amounts of DNA to obtain response in
animals and these responses may not be extrapolated to man.
Manufactures of vaccines are currently looking for easier production systems to provide
effective long-lasting immunity and the use of live vaccines has been subjected to some
examination (Dertzbaugh, 1998). With the aid of DNA recombination techniques, invasive
bacteria may be attenuated (Grillot-Courvalin et al., 1999) or bacteria that are normally
ingested with food may be engineered for the delivery of vaccine products (Pouwels et al.,
1998). In humans, Salmonella strains harboring genetically growth restriction mutations have
been tested (Tacket et al., 1992.). Similarly, Salmonella strains have been shown to function
effectively, as containers for the delivery of recombinant DNA vaccines in vitro (Lowe et al.,
1999.).
CMV
ISS
antigen
ampR
ISS
A
ISS
ColE1
Fig 5. Generalized vaccine vector map. The protein expression unit includes an enhanced
eukaryotic promoter (CMV), the antigen encoding gene (antigen) and a transcriptional termination
provided by poly-adenylation (A). The prokaryotic replication unit or vector backbone. This
comprises an origin of replication (ColE1), a selection marker (ampicillin resistance gene, ampR)
and immunostimulatory sequences rich in CpG (ISS).
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GENETICALLY MODIFIED ORGANISMS
Recombinant DNA engineering for the production of vaccines is yet an experimental fields
that is in progress. Once experimental work reaches the production stage, an extensive line of
risk assessment have to be explored. Much of the experience from accessing risks in
connection with the recombinant vaccine technology will most likely also apply to the
evaluation of the hazards, that man may experience when exposed to other forms of
recombinant DNA.
8.5
References
Alton,E.W., Stern,M., Farley,R., Jaffe,A., Chadwick,S.L., Phillips,J., Davies,J., Smith,S.N., Browning,J.,
Davies,M.G., Hodson,M.E., Durham,S.R., Li,D., Jeffery,P.K., Scallan,M., Balfour,R., Eastman,S.J., Cheng,S.H.,
Smith,A.E., Meeker,D., and Geddes,D.M. (1999) Cationic lipid-mediated CFTR gene transfer to the lungs and
nose of patients with cystic fibrosis: a double-blind placebo-controlled trial Lancet 353: 947-954.
Brockmann,E., Jacobsen,B.L., Hertel,C., Ludwig,W., and Schleifer,K.H. (1996) Monitoring of Genetically
Modified Lactococcus lactis in Gnotobiotic and Conventional Rats by Using Antibiotic Resistance Markers and
Specific Probe or Primer Based Methods System.Appl.Microbiol. 19: 203-212.
Davison,J. (1999) Genetic exchange between bacteria in the environment Plasmid 42: 73-91.
Dertzbaugh,M.T. (1998) Genetically engineered vaccines: an overview Plasmid 39: 100-113.
Grillot-Courvalin,C., Goussard,S., and Courvalin,P. (1999) Bacteria as gene delivery vectors for mammalian
cells Curr.Opin.Biotechnol. 10: 477-481.
Jacobsen,B.L., Brockmann,E., Hertel,C., Ludwig,W., and Schleifer,K.H. (1996) monitoring of gene transfer
from genetically marked lactococcus lactis in the gastrointestinal tract of gnotobiotic and conventional rats. In
Germfree Life and its Ramifications. Hashimoto,K.e.a. (ed). Shiozawa: XII ISG Publishing Committtee, pp. 101104.
Klijn,N, Weerkamp,A.H., and de Vos,W.M. (1995) Genetic marking of Lactococcus lactis shows its survival in
the human gastrointestinal tract Appl.Environ.Microbiol. 61: 2771-2774.
Lehr,CM. (1996) From sticky stuff to sweet receptors-achievements, limits and novel approaches to bioadhesion
Eur.J.Drug.Metab.Pharmacokinet 21: 139-48.
Lipford,G.B., Heeg,K., and Wagner,H. (1998) Bacterial DNA as immune cell activator Trends Microbiol. 6:
496-500.
Lowe,D.C., Savidge,T.C., Pickard,D., Eckmann,L., Kagnoff,M.F., Dougan,G., and Chatfield,S.N. (1999)
Characterization of candidate live oral Salmonella typhi vaccine strains harboring defined mutations in aroA,
aroC, and htrA Infect.Immun. 67: 700-707.
Luo,D., Saltzman,W. (2000) Synthetic DNA delivery systems Nat.Biotechnol. 18: 33-37.
Meyer,K., Thompson,M., Levy MY, Barron LG, and Szoka FC Jr. (1995) Intratracheal gene delivery to the
mouse airway: characterization of plasmid DNA expression and pharmacokinetics Hum.Gene Ther. 2: 450-460.
Netherwood,T., Bowden,R., Harrison,P., O'Donnell,A.G., Parker,D.S., and Gilbert,H.J. (1999) Gene transfer in
the gastrointestinal tract Appl.Environ.Microbiol. 65: 5139-5141.
Netherwood,T., Gilbert,H.J., Parker,D.S., and O'Donnell,A.G. (1999) Probiotics shown to change bacterial
community structure in the avian gastrointestinal tract Appl.Environ.Microbiol. 65: 5134-5138.
Pouwels,P.H., Leer,R.J., Shaw,M., Heijne den Bak-Glashouwer,M.J., Tielen,F.D., Smit,E., Martinez,B., Jore,J.,
and Conway,P.L. (1998) Lactic acid bacteria as antigen delivery vehicles for oral immunization purposes
Int.J.Food Microbiol. 41: 155-167.
Sambrook,J., Fritsch,E.F., and Maniatis,T. (1989) Molecular cloning, a laboratory manual Sambrook,J.,
Fritsch,E.F., and Maniatis,T. (eds). New York: Cold Spring Harbor Laboratory Press.
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TRANSFER OF DNA
Schubbert,R., Lettmann,C., and Doerfler,W. (1994) Ingested foreign (phage M13) DNA survives transiently in
the gastrointestinal tract and enters the bloodstream of mice Mol.Gen.Genet. 242: 495-504.
Schubbert, R., Renz,D., Schmitz,B., and Doerfler,W. (1997) Foreign (M13) DNA ingested by mice reaches
peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse
DNA Proc.Natl.Acad.Sci.U.S.A 94: 961-966.
Schubbert,R., Hohlweg,U., Renz,D., and Doerfler,W. (1998) On the fate of orally ingested foreign DNA in mice:
chromosomal association and placental transmission to the fetus Mol.Gen.Genet. 259: 569-576.
Schughart,K., Bischoff,R., Rasmussen,U.B., Hadji,D.A., Perraud,F., Accart,N., Boussif,O., Silvestre,N.,
Cordier,Y., Pavirani,A., and Kolbe,H.V. (1999) Solvoplex: a new type of synthetic vector for intrapulmonary
gene delivery Hum.Gene Ther. 10: 2891-2905.
Suau,A., Bonnet,R., Sutren,M., Godon,J.J., Gibson,G.R., Collins,M.D., and Dore,J. (1999) Direct analysis of
genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human
gut Appl.Environ.Microbiol. 65: 4799-4807.
Tacket,C.O., Hone,D.M., Curtiss,R., III, Kelly,S.M., Losonsky,G., Guers,L., Harris,A.M., Edelman,R., and
Levine,M.M. (1992) Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta
crp Salmonella typhi strains in adult volunteers Infect.Immun. 60: 536-541.
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9
DNA TRANSFER BY BACTERIOPHAGES
by Holger Riemann
9.1 Summary
The frequent presence of bacteriophages in the environment is exemplified, and
the importance of general physical and biological factors that influence
transduction in the environment are briefly illustrated. Examples of specific
transduction are presented and it is pointed out that in situ transduction is only
moderately examined. The examples include aquatic systems, soils, plants, animal
systems, the human intestine as well as comestibles and most of the studies are
based on the transfer of resistance markers.
9.2 Introduction
In general bacteriophages are common in the environment. They represent relatively stable
nucleic acids (being protected by a protein coat) which are more compact and thus more
diffusible than naked DNA. Bacteriophages can be found in the environment either as part of
an actively replicating population or they can, under the right conditions, be stored as an
inactive stable pool. By changes in the environment the phages can be activated and following productive infection of their respective hosts - form part of an actively replicating
pool of infectious agents.
This chapter discusses the potential for unintended DNA transfer via phages present in the
environment. The naturally occurring phages and the transduction of genes from bacterial
GMOs is essential, as the outlet of genetically modified phages is of no significant
importance. As all infectable bacteria, in principle, can act as hosts for various phage types,
reference is made to this report’s other sections on bacterial DNA transfer, if a thorough
analysis on the environmental load of bacterial GMOs is desired.
The different mechanisms for spreading of genetic information via phage transduction are
wellknown and extensively described in the literature. In the process of phage propagation,
phages transfer nucleic acids synthesized in one bacterium to another bacterium. If a phage
infecting a new host contains genetic material from the previous host rather than its own
DNA, the extra genetic information may be transmitted to the new host, resulting in
transduction. For a more detailed interpretation of life cycle and transduction types, reference
is made to the literature on the subject.
9.3 Phages in the environment: Presence and transduction
Phages are very common in the environment and some estimates suggest that 90-100% of all
natural bacterial isolates are lysogenic for at least one phage and that many contains several
phages (Yin & Stotzky, 1997). In spite of this, comparable in situ data from natural
environments are not available and information on the total phage load has only been
monitored for a single aquatic system (Jiang & Paul, 1998). Transduction has been shown to
be a significant mediator of gene transfer within natural ecosystems as diverse as soils, plant
surfaces, aquatic systems, and animals. The phage mediated gene transfer in natural
environments is influenced by several biological and physiochemical factors acting as barriers
and modulating the degree of transduction.
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TRANSFER OF DNA
9.3.1 Biological barriers to transduction
Barriers exist to the broad distribution of genes by transduction - the most significant ones
being of biological nature. For most phages, the requirement for a specific receptor for phage
entry limits the host range to a few bacterial species, although phages with host ranges that
extend to many species have been reported. These promiscuous phages have been shown to
mediate interspecific as well as intergeneric transduction suggesting this to be happening in
the environment too (Yin & Stotzky, 1997).
Also, the restriction-modification systems employed by bacteria to defend themselves against
infecting phages represents barriers to the free distribution of foreign genetic information in
the bacterial population. However, the DNA is seldom completely destroyed by these systems
and many phages have developed specific mechanisms to avoid destruction. Furthermore,
environmental factors, such as elevated temperature and UV radiation, can influence the
efficiency of the restriction-modification system allowing DNA to escape from degradation
(Yin & Stotzky, 1997; Gonzales et al, 1999).
Lysogeny is normally accompanied by immunity to superinfection with one or more related
phages thus representing a barrier to transduction. In some cases, nevertheless, immunity only
rescues the bacteria from lysis and actually increases the frequency of transduction (Yin &
Stotzky, 1997). The widespread distribution of lysogens may also be important in maintaining
the potential for transduction in natural environment. Phages released from host cells have a
finite lifetime and lysogens may replenish the pool of transducing particles by spontaneous
induction of prophages.
9.3.2 Physiochemical barriers to transduction
The exact influence of physical and chemical factors on the survival of phages in the
environment and the subsequent infection of bacteria is not well documented. It is generally
known, that solar radiation may cause the induction of prophages to the lytic stage and more
specifically that sunlight can inactivate, for example, fecal bacteriophages from sewagepolluted seawater (Sinton et al., 1999). Other factors potentially influencing transduction in
the environment includes the surface-character of particles, the concentration of cations, the
ratio of solid to liquid, and pH. These parameters have been investigated to some degree but
their relative significance needs to be further established through the development of new
microenvironments in the laboratory (Yin & Stotzky, 1997).
It should be mentioned, though, that clay minerals seems to be very important for naturally
occurring transduction as they prolong the life-span of free phage particles and acts as
concentrating surfaces for bacteria and phages as well as for proteins and other nutrients. The
mere presence of clay particles in any environment probably increases the risk of transduction
although the high "concentration" in soils i.e. the high solid:liquid ratio, may actually reduce
the risk of transduction by limiting the movement of bacteria and phages.
9.3.3 Transduction in situ.
In spite of the known presence of phages and their hosts almost anywhere in the environment
in situ transduction has only been moderately studied. The below examples include aquatic
systems, soils, plants, animal systems, the human intestine and feces as well as comestibles
and most are based on the traceable transfer of resistance markers.
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9.3.4 Phage transduction: Plants
Plant surfaces (i.e. phytosphere) support many metabolically active bacterial species and are
good sites to look for transduction. Most phage studies on phytosphere bacteria have been
limited to only sugarbeet and bean seedlings grown in sterile soil (Kidambi et al., 1994;
O´Sullivan et al.,1990). As for most other systems the current material does not allow for
direct comparison. Nevertheless, it reveals that transduction does indeed occur on leaf
surfaces and that migration of the involved microorganisms does take place between nearby
plants. A new model system for field-grown sugarbeet sets the standard for the development
of new terrestrial plant systems for in situ transduction of genetic information. The system
was designed for the study of in situ population dynamics of bacterial viruses and showed
temporal changes in several phage populations as well as changes in the relative distribution
of the specific phages making up these populations (Ashelford. et al., 1999). It should be
noticed, though, that the data has been collected during a few growing seasons only and that it
takes quite a number of years to properly assess trends. Nevertheless, data on natural
fluctuations in the specific transduction potential of microcommunities can only be generated
through the use of field trials - exemplifying the insufficiency of standardized laboratory
setups for certain purposes.
9.3.5 Phage transduction: soils
Most terrestrial studies have focused on specific phage-host interactions in simple soil
systems i.e. sterile or non-sterile microcosms and rely on the resistance to antibiotics as
markers of transduction (Yin & Stotzky, 1997). Again, the results do not allow for direct
comparison but only shows that transduction takes place in soil environments and that some
transductants can survive for at least a month (Zeph et al., 1988). To asses the risk associated
with the spread of genetically engineered bacterial DNA through transduction in soils, new
standardized systems obviously have to be developed.
Phage transduction: aquatic systems
Almost all studies on transduction in natural environments were initially performed in
freshwater habitats like river water, lake water, and wastewater treatment facilities (Yin &
Stotzky, 1997; Saye et al., 1987 & 1990). More recently, studies have been performed on
totally indigenous phage-host systems from both marine and freshwater environments (Jiang
& Paul, 1998; Ripp et al., 1994). Needless to say transduction does occur and there has even
been an attempt to quantify the rate of transduction in a specific marine microbial community
using a mathematical model (Jiang & Paul, 1998). The rate was estimated to about 1.3 X 1014
events per year and the fact that this calculation involves a variety of assumptions is of little
importance as no comparable aquatic results exists from studies on other systems. Again, the
results from different transduction studies do not fit into a comparable standard.
Nevertheless, data obtained from these studies do show that environmentally endemic
bacteriophages are formidable transducers of naturally occurring microbial communities, and
that transduction may be an important mechanism for gene transfer in aquatic environments.
The recent isolation and characterization of natural phage-host systems from both marine and
freshwater environments suggests the possibility of developing model systems to test
transduction in any specific aquatic environment (Ripp et al., 1994; Jiang et al., 1998; Jiang
& Paul, 1998). Such modelsystems, built on indigenous phages and bacteria, will enable
realistic assessments of the unwanted spread of genetic information by transduction from
introduced genetically modified bacteria in natural aquatic environments.
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TRANSFER OF DNA
9.3.6 Phage transduction: Animals
Transduction as a possible phenomenon involving bacterial communities inside animals has
been reported for systems as diverse as chick embryos, gills of oysters and mice kidneys or
intestines (Yin & Stotzky, 1997). Although resistance to antibiotics as erythromycin,
tetracycline, and chloramphenicol are often used as markers, the results do not fit into a
comparable standard and the data can only pinpoint the possibility of transduction in animal
systems. A more extensive search for literature on the subject might reveal some extra
information but the field is not very well substantiated. Especially there is a need for
development of more standardized modelsystems, i.e. the use of specific gut models.
9.3.7 Phage transduction: The human intestine
The occurrence of phages in feral and domestic animal wastes as well as in human feces
shows that probably all mammalian species harbors phages (Calci et al., 1998). The great
majority of the investigated animal species i.e. cattle, chickens, dogs, ducks, geese, goats,
pigs, horses, seagulls, sheep, and humans excreted phages at very low levels but nonetheless
this implicates an enormous potential for the spread of genetic information to the environment
through wastewater treatment plants. Phages have been detected by various methods from
urban sewage (Puig et al., 1999; Muniesa & Jofre, 1998) and the human origin of specific
phages has also been established (Tartera et al., 1989).
No direct evidence of tranduction in the human intestine exists but bacteriophage-mediated
transfer has been suggested to explain the distribution of a phage-encoded toxin among
different lineages of pathogenic enteric bacteria (Kapur et al., 1992). Similarly, other phages
code for virulence factors that are expressed upon lysogenization of pathogenic enteric
bacteria (Figueroa-Bossi & Bossi, 1999; Schmidt et al., 1999; Waldor & Mekalanos, 1996).
Furthermore transduction of enteric pathogens isolated from sewage and physiological stool
has been shown using natural phages isolated from sewage and from pathogenic enteric
bacteria (Hertwig et al., 1999; Schmidt et al., 1999) suggesting the possibility of transduction
in the human intestine to. Also, transduction of enteric bacteria isolated from the stool
microflora of healthy individuals has been shown using an antibiotic resistance marker
(Schmidt et al., 1999).
9.3.8 Phage transduction: Comestibles
Scarce information as to phage presence in comestibles can be found although it is known that
phages are widely spread in e.g. dairy environments (Heller et al., 1995). As genetically
engineered starter cultures are proposed for the improvement of novel foods, the phage load
as well as the susceptibility of these cultures to infection should be investigated to reveal any
safety problems before the release of new products. This is especially vital to the use of dairy
starter cultures having potential phage-problems.
Data concerning the significance of transduction for the spread of genetic information among
bacteria in comestibles are extremely scarce. An example of real in situ transduction has been
published though, illustrating that transduction does occur in human foods (Heller et al.,
1995). The mere fact that the in situ system was yoghurt, a product of the (potentially) phageinfested dairy industry, clearly demonstrates that the field needs more attention.
9.4 Conclusions
It can be concluded, that transduction is commonly occurring in natural environments. This
form of DNA transfer might be the most significant exchange method in specific
microenvironments where the phage biology will be important for both DNA stability and
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GENETICALLY MODIFIED ORGANISMS
DNA transmission. This is especially true for highly structured environments with a high
solid:liquid ratio such as soils, where cell to cell contact is rare and for harsh environments
where naked DNA will not survive for long.
There is an evident lack of transduction studies focusing on exchange of DNA between
introduced and naturally occurring bacteria in the environment. Apart from an obvious in situ
approach, future studies should be based on the availability of new and well-characterized
natural microenvironments and the possibilities they offer for optimized laboratory
experiments. In terms of safety and standardization, these physically contained systems of
phages and bacteria are more advantageous than proper release experiments. The physical
containment permits studies with very little or no biological containment, which is indeed
relevant for most naturally occurring transduction systems. Also, the controlled laboratory
setup allows for a high degree of standardization, thus increasing the data applicability as well
as the feasibility of proper quantification of transfer frequencies.
Apart from the significance of phages for transduction of toxic characteristics between natural
unmanipulated pathogens present in the human intestines, data for the illustration of the health
relevance of transducing phages is basically non-existent. It is thus conclusive that the phagemediated exchange of DNA between bacterial GMOs and the bacterial flora in human
population and in food, respectively, is a non-substantiated area.
9.5
References
Alisky, J., Iczkowski, K., Rapoport, A. & Troitsky, N. (1998). Bacteriophages show promise as antimicrobial
agent. J. Infect. 36, 5-15.
Ashelford, K.E., Day, M..J., Bailey, M.J., Lilley, A.K. & Fry, J.C. (1999). In situ population dynamics of
bacterial viruses in a terrestrial environment. Appl. Environ. Microbiol. 65, 169-174.
Calci, K.R., Burkhardt, W.3rd., Watkins, W.D. & Rippey, S.R. 1998. Occurrence of male-specific bacteriophage
in feral and domestic animal wastes, human feces, and human-associated wastewaters. Appl. Environ. Microbiol.
12, 5027-5029.
Figueroa-Bossi, N. & Bossi, L. 1999. Inducible prophages contribute to Salmonella virulence in mice. Mol.
Microbiol. 33, 167-176.
Gonzales, P., Zigler, J.S., Epstein, D.L. & Borrás, T. (1999). Increasing DNA transfer efficiency by temporary
inactivation of host restriction. BioTechniques 26, no. 5.
Heller, K.J., Geis, A. & Neve, H. (1995). Behaviour of genetically modified microorganisms in yoghurt. System.
Appl. Microbiol. 18, 504-509.
Hertwig, S., Popp, A., Freytag, B., Lurz, R. & Appel, B. (1999). Generalized transduction of small yersinia
enterocolitica plasmid. Appl. Environ. Microbiol. 65, 3862-3866.
Jiang, S.C., Kellogg, C.A. & Paul, J.H. (1998). Characterization of marine temperate phage-host systems
isolated from Mamala Bay, Oahu, Hawaii. Appl. Environ. Microbiol. 64, 535-542.
Jiang, S.C. & Paul, J.H. (1998). Gene transfer by transduction in the marine environ. Appl. Environ. Microbiol.
64, 2780-2787.
Kapur, V., Nelson, K., Schlievert, P.M. Selander, R.K. & Musser, J.M. (1992). Molecular population genetic
evidence of horizontal spread of two alleles of the pyrogenic exotoxin C gene (speC) among pathogenic clones
of Streptococcus pyogenes. Infect. Immun. 60, 3513-3517.
Kidambi, S.P., Ripp, S. & Miller, R.V. (1994). Evidence for phage-mediated gene transfer among Pseudomonas
aeruginosa strains on the phylloplane. Appl. Environ. microbiol. 60, 496-500.
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TRANSFER OF DNA
Muniesa, M. & Jofre, J. (1998). Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7
and that carry the shiga toxin 2 gene. Appl. Environ. Microbiol. 64, 2443-2448.
O´Sullivan, M., Stephens P. M., & O´Gara, F. (1990). Interactions between the soil-borne bacteriophageFo-1
and Pseudomonas spp. on sugarbeet roots. FEMS Microbiol. Lett.. 68: 329-334.
Puig, A., Queralt, N. Jofre, J. & Araujo, R. (1999). Diversity of bacteroides fragilis strains in their capacity to
recover phages from human and animal wastes and from fecally polluted wastewater. Appl. Environ. Microbiol.
65, 1772-1776.
Ripp, S., Ogunseitan, O.A. & Miller, R.V. (1994). Transduction of a freshwater microbial community by a new
Pseudomonas aeruginosa generalized transducing phage, UT1. Mol. Ecol. 3, 121-126.
Saye, D.J., Ogunseitan, O., Sayler, G.S. & Miller, R.V. (1987). Potential for transduction of plasmids in a natural
freshwater environment: Effect of plasmid donor concentration and a natural microbial community on
transduction in Pseudomonas aeruginosa. Appl. Environ. Microbiol. 53, 987-995.
Saye, D.J., Ogunseitan, O.A., Sayler, G.S. & Miller, R.V. (1990). Transduction of linked chromosomal genes
between Pseudomonas aeruginosa strains during incubation in situ in a freshwater habitat. Appl. Environ.
Microbiol. 56, 140-145.
Schmidt, H., Bielaszewska, M. & Harch, H. 1999. Transduction of enteric Escherichia coli isolates with a
derivative of shiga toxin 2-encoding bacteriophage Φ3538 isolated from Escherichia coli O157:H7. Appl.
Environ. Microbiol. 65, 3855-3861.
Sinton, L.W., Finlay, R.K. & Lynch, P.A (1999). Sunlight inactivation of fecal bacteriophages and bacteria in
sewage-polluted seawater. Appl. Environ. Microbiol. 8, 3605-3613
Tartera, C., Lucena, F. & Jofre, J. (1989). Human origin of Bacteroides fragilis bacteriophages present in the
environment. Appl. Environ. Microbiol. 55, 2696-2701.
Waldor, M.K. & Mekalanos, J.J. (1996). Lysogenic conversion by a pilamentous phage encoding cholera toxin.
Science 272, 1910-1914.
Yin, X. & Stotzky, G. Gene transfer among bacteria in natural environments. In: Advances in Applied
Microbiology 45, 153-212. Academic Press 1997
Zeph, L. R., Onaga, M. A., & Stotzky, G. (1988). Transduction of Echerichia coli by bacteriophage P1 in soil.
Appl. Environ. Microbiol. 54: 1731-1737.
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GENETICALLY MODIFIED ORGANISMS
10 DNA TRANSFER BY EUKARYOTIC VIRUSES
by Holger Riemann
10.1 Summary
Genetically modified viruses influencing the health of human beings originates from multiple
sources of which the most important are the use of viral gene therapy and live viral vaccines.
A comprehensive description of the undesirable effects connected with the production and use
of recombinant viral vectors for gene therapy as well as for live viral vaccines is not within
the scope of this report. This section will concentrate on aspects directly related to viral gene
therapy only. Viral systems for gene therapy are introduced and the concept of biological
containment is presented. Various concerns connected to the unintended effects of viral
vectors e.g. the creation of replication competent viruses, the shedding of viruses from
infected individuals, the risk of germline transmission, as well as more general concerns are
discussed. Finally, the prime concern is concluded to be the lack of standardized assays for
detection of potentially replication competent viruses.
10.2 Viral gene therapy
Several requirements must be fulfilled for the successful use of gene therapy: The
understanding of the underlying biology of the disease, the construction of recombinant DNA
constructs to correct the abnormality and finally the use of a highly efficient delivery system
for the transfer of the therapeutic genes into the cells of the patient. The development of viral
gene delivery vehicles that are both safe and efficient has become the greatest challenge for
the successful use of gene therapy. Although gene therapy is potentially a powerful clinical
approach, it still needs an unequivocal clinical success. For a recent review on gene therapy
see Smith (1999).
10.3 Viral therapy systems
It is often stressed, by people strongly biased by their own inventions or investments, that
some viral systems are obviously superior to other systems - both in terms of efficiency and
safety - but few generalizations can be made as to vector selection for any particular
application. Each disease has its own specific requirements such as target tissue and amount
of gene-product required and similarly each vector has characteristics that may or may not be
desirable for a particular application. Furthermore, objective studies comparing the relative
merits of different delivery systems are rarely carried out, making comparisons difficult
(Giannoukakis et al., 1999, Hanazono et al., 1999, Lozier et al., 1997). The viral vectors used
for gene transfer include retroviruses, adenoviruses, adeno-associated viruses, herpes simplex
virus and flavivirus. The vectors most commonly used by companies involved in gene therapy
research i.e. retroviruses, adenoviruses and adeno-associated viruses (Commander, 2000.) are
briefly described in terms of type of genome, the capacity for carrying genes and major
advantage and disadvantage.
Retroviral systems
Retroviruses are RNA-viruses, which can integrate their transgenes into the genome of the
host cell. They have a god capacity for carrying genes (approximately 9-12 Kb) and appear to
avoid detection by the host immune system. The major drawback for retroviral vectors is the
risk of adverse event caused by their permanent integration into the genome of the host cell.
Two viral subsets of the family Retroviridae has been developed as gene-delivery vehicles i.e.
members of the subfamilies oncovirinae and lentivirinae.
Members of the oncovirinae, mainly murine viruses, were the first retroviruses to be
domesticated as vectors for gene-delivery. For a recent review see (Vile & Russell, 1995).
The major disadvantage of the commonly used onco-retroviral vectors is their inability to
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transduce non-dividing cells as these viruses can only integrate their genome as the cell
divides.
Lentiviruses combine all the advantages of onco-retroviral vectors with the added ability to
deliver therapeutic genes to non-dividing cells. For recent reviews see Amado & Chen (1999),
Kilmatcheva et al. (1999). However, exceptions to this ability to transduce non-proliferative
tissues have been reported, suggesting that lentiviral vectors cannot infect all non-dividing
cells (Emerman, 2000). The major disadvantage of first and second-generation lentivirus
vectors is connected to the fact that they are constructed on the basis of HIV-genomes and it
is still an open question whether or not HIV-derived vectors should be used for treatments
outside AIDS. Hence, a number of non-primate lenti-viruses have been considered as
potential gene therapy vectors. The most advanced are the equine infectious anemia virus
(EIAV), and the feline immune deficiency virus (FIV), neither of which is associated with any
human disease despite prevalent natural exposure. These lentiviruses do not infect human
cells but the gene determining the host range can be substituted with another gene allowing
the viruses to infect and deliver genes to human tissue. However, the consequences of their
possible recombination with HIV-1 in infected individuals as well as of their introduction of
novel sequences into the viral pool infecting humans should be carefully considered. It is
important to emphasize that no data presently exist regarding the relative safety of primate
versus nonprimate lentivirus vectors (Bukovsky et al., 1999; Johnston et al., 1999).
Adenoviral systems
Adenoviruses are DNA viruses, which do not integrate their DNA into the host cell genome,
but reside in the nucleus as an extra-chromosomal element. These vectors are useful for shortterm high level in vivo expression of a transgene and infect dividing and as well as nondividing cells. They have a moderate to very high capacity for carrying therapeutic genes (430 Kb) and can be used for transient in vivo and ex vivo gene therapy. The major drawback of
most adenoviral vectors is their known ability to cause serious inflammatory side effects. First
and second-generation adenoviral vectors still include viral proteins, which are produced
within the transduced cell and are recognized by the immune system. This normally leads to
clearance in a relatively short time but in some applications the immune response can lead to
serious inflammation (Fox, 1999; Iwakuma et al., 1999). Researchers are addressing this by
developing gutless vectors in which almost all the viral genes are deleted allowing longerterm expression of the transgene in the host cell. These high-capacity vectors have the added
ability to transduce cells with more than 30 Kb of nonviral DNA (Kochanek, 1999). For a
recent review see Benihoud et al. (1999).
Adeno-associated viral systems
Adeno-associated viruses (AAV) are also DNA viruses but have far fewer genes than
adenoviruses and need a helper virus (adenovirus) in order to replicate. These viruses can
integrate their DNA into the host genome for long-term expression of the transgene and
appears to avoid the host immune responses. As for retroviruses, the integration of the viral
DNA into the genome of the target cells presents safety-problem to be considered. AAV have
only a small capacity to carry therapeutic genes, and systems for their production are still at
the developmental stage. However, AAV have great promise for both in vivo and ex vivo
gene therapy in the future as AAV, unlike adenovirus, are not known to cause disease in
humans and do not contain genes that would allow them to induce an immune response. Trials
are underway using AAV in treating cystic fibrosis ref (Commander, 2000). For a recent
"review" see Linden. & Woo (1999).
10.4 Viral containment
Each specific group of viruses has its own limitations for use in human gene therapy as well
as its own problems concerning safety. Nevertheless, all viruses intended for use as genedelivery vehicles has, at least to some degree, been designed following the most general
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principles of biological containment i.e. the principle of trans-complementation of replication
deficient viral vectors.
Biologically contained viruses for gene therapy has been made by dissection of the viral
genomes into crippled and physically separated sub-genomes i.e. helper and vector genomes.
The ideal vector genome contains only viral cis-acting sequences necessary for the vector to
infect the target cell and for transfer of the therapeutic gene. The ideal helper genome contains
only viral trans-acting elements necessary for vector packaging such as sequences coding for
the structural proteins and the enzymes required to generate vector particles. For most systems
the challenge has been to design the genomes as to avoid the creation of replicationcompetent viruses. The approach has been to develop system with almost no sequence
homology between helper and vector genomes thus minimizing the risk of homologous
recombinational events leading to the generation of non-defective viruses. Many viral vectors
are generally created in a transient tranfection system i.e. the simultaneous introduction of
helper and vector genomes into the producer cells. As this procedure provides an unnecessary
opportunity for the crippled genomes to engage in numerous recombinatorial events, the
development of stable and well-characterized packaging cells, supplying viral trans-factors
expressed from separate packaging genomes, has greatly increased the safety.
The safety of onco-retroviral systems is well documented as relatively safe retroviral systems
with almost no homology between vector and helper genomes have existed for at least ten
years. For a comprehensive review of the principles of constructing safe retroviral systems
and a thorough description of retroviral vectors and packaging cells see Riemann (1990). For
recent reviews see Vile & Russell (1995) or Kim et al. (1998). Few improvements have been
added to the safety of these systems (Ismail et al., 2000), probably because of the fact that
onco-retroviral vectors cannot transduce non-dividing cells. The relatively recent successful
use of lentiviral systems with the ability to transduce many non-dividing tissues has, however,
reactivated the interest in the development of safer retroviral gene-delivery systems. Most
lentiviral approaches copy the strategy from the safest onco-viral systems apart from the use
of packaging cells (Déglon et al., 2000). Often transient transfection systems are applied, as
the commonly used envelope gene (trans-factor) encoding the G glycoprotein of the vesicular
stomatitis virus (VSV-G) is toxic to the producer-cells. This problem has been solved by
placing VSV-G into a helper genome under the control of an inducible promoter thus
allowing the design of packaging cells (Kafri et al., 1999).
The biological containment is historically well established for adenoviruses also, and recent
modifications of the adenovirus backbone have led to the development of helper-dependent
vectors completely devoid of all viral protein-coding sequences (Morsy & Caskey, 1999).
These vectors can be grown to high titers but often first-generation helper genomes are used
making it difficult to separate helper virus from the gutted vector (Robbins et al., 1998). No
well characterized packaging system free of wild type virus is available for other large DNA
viruses as a recenly introduced first-generation packaging cell line for Epstein-Barr virusderived vectors still suffers from unwanted recombination between helper and vector
genomes (Delecluse et al., 1999).
As for AAV, this virus is inherently biologically contained i.e. it needs trans-factors from
adenoviruses to replicate and like other safe viral gene-delivery vectors it is further contained
by the exchange of viral sequences for the transgene and by the removal of genes essential for
replication.
Phage-based systems has been introduced as an alternative to more conventional genedelivery systems but their apparent virtues has to be proven in practice (Kassner et al., 1999;
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Larocca et al., 1999; Poul & Marks, 1999). Nevertheless, the feasibility of applying phagemediated transduction to screen multivalent phage-libraries for new targeting ligands cannot
be questioned (Becerril et al., 1999; Ivanenkov et al., 1999).
In general, the viral systems with the highest degree of containment are the least effective and
even these systems can not be expected to be fully contained. In fact no eucaryotic viral genedelivery system can be expected to be totally non-leaky as the helper and vector genomes
contain essential viral trans- and cis-functions which, by some homologous or nonhomologous process including non-controllable viral elements such as endogenous viruses or
super-infecting viruses, can generate replication competent virus.
However, for practical purposes it somehow has to be decided which viral systems have a
satisfying level of biological containment for any particular application. Although this choice
obviously has to be made on a case-to-case basis some kind of standardized assays for the
degree of leakiness would be a great asset. At present viral systems are assayed for safety in
many different ways which do not allow direct comparison and which do not readily provide
quantitative guidelines (Déglon et al., 2000; Giannoukakis et al., 1999; Kafri et al., 1999).
The containment level of viral systems is therefore expressed in probabilistic terms rather
than in actual performance terms.
10.5 Unintended presence of therapeutic viruses.
For viral gene therapy, transfer of genetic information is the aim. The efficiency of these
gene-delivery vehicles is well established and it does not make any sense to examine if DNA
transfer can take palce. Rather, the question is whether or not it is possible to control this
transfer time- and space-wise. The unavoidable leakiness of the biological containment and
the non-existing physical containment calls for a thorough characterization of the problems
connected with the practical handling of these infectious particles. For a recent review on
general approaches to safety evaluation see Pilling (1999).
Two major problems connected with virus-mediated DNA transfer in relation to gene therapy
are a) the unintended creation of infectious viral particles with the ability to introduce
unwanted changes in the infected individual and b) the uncontrollable shedding of viral
particles from patients in therapeutic treatment. Another major concern is related to the risk of
adverse events caused by the permanent integration of the viral DNA i.e. inadvertent
activation/deactivation of genes causing cancer and the risk of germ line transmission.
10.5.1 Replication competent viruses
As pointed out in previous sections, the inadvertent creation of replication-competent helpergenomes cannot be excluded for any vector system, and the degree should be quantitated in
some way to enable a reasonable risk assessment. Development of specific helper detection
assays for viral vectors will be necessary prior to testing in humans but, unfortunately, present
data for the undesirable formation of infectious helper particles are by no means comparable.
The leakiness of most biologically contained viral systems, i.e. the generation of replication
competent viruses, is described by means of in vitro systems and in vivo model systems. The
detection principles are mainly based on the use of simple or nested PCR and the rescue of
defective reporter genomes but histological staining, in situ hybridization, in situ PCR, and
seroconversion has also been applied (Déglon et al., 2000; Dewey et al., 1999; Iwakuma et
al., 1999; Kafri et al., 1999; Kim et al., 1998).
At present, standardized assays for the detection of potentially replication-competent viruses
generated in various defective viral systems does not exist. It will be critical to the safety of
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novel gene therapy vectors to develop rapid and sensitive assays for the detection of such
recombinants in vector preparations. Development of fast in vitro bioassays (biosensors)
based on the use of indicator cells i.e. cells reacting to the presence of helper virus displaying
either immediate cytophatic effects or expression of inducible reporter genes, would be a vast
improvement to the present situation. In order to secure that the detection conditions can be
standardized, each specific assay should be designed in a concerted action among several
groups specialized in the use of a particular viral vector.
Shedding of therapeutic virus
Any in situ therapeutic approach involving large amounts of infectious particles - defective
therapeutic as well replication-competent viruses - gives reason to concerns as to the degree
of shedding. Shedding of virus particles from patient through all sorts of body fluids i.e.
saliva, nasal secretion (snot), semen, feces, urine, and blood but also shedding via skin-debris
and aerosols from breathing should be followed and quantitated. This is especially important
in connection with the intentional use of replication-competent viruses as seen for certain
applications of adenoviral vectors in cancer therapy (Robbins et al., 1998).
The methods most commonly applied are different versions of PCR as seen for detection of
human viruses in urban sewage, municipal swimmingpools, estuaries, shellfish and elsewhere
in the environment. Also, the registration of clinical signs of viremia or the simple registration
of seroconversion as practiced when following shedding from wild life animals can be used
(Bofill-Mas et al., 2000; Castignolles et al., 1998; Papapetropoulou et al., 1998; Pina, 1998;
Tessaro et al., 1999). Present in vitro models, as described for the detection of non-defective
helper viruses, are useful only when shedding load is high again stressing the need for new
biosensors with improved sensitivity.
In situ model systems exists and can be used for the capture of viral vectors shedded from
patients in treatment. The captured viruses can be identified by means of PCR and
immunological methods such as ELISA or, alternatively, by histopathological analyses to
detect viral genomes, transgene expression, chronic inflammation and related tissue-damage
(Dewey et al., 1999; Gambhir et al., 1999). The selected animal species should be susceptible
to infection with the wild-type virus related to the vector and at the same time should allow
specific activation of the promoters controlling expression of the transgenes as to reveal all
possible inflammatory responses. In addition, immune-competent models alone are not
sufficient as only immune-deficient models can be predictive of the problems connected with
infection of immune-deficient individuals in which replicating vectors are not eliminated.
Moreover, these animal systems should be exploited as shedding models too; especially for
the specific analyses of the long-term shedding from immune-deficient individuals.
Recombinant manipulations narrowing the tissue and host-range of the infectious particles
limits the spread (Douglas et al., 1999; Girod et al., 1999), but this kind of biological
containment is clearly not enough and, furthermore, is impossible to implement for the
majority of viral gene-transfer systems. This lack of specificity is most obvious for the
lentiviral systems using VSV-G as part of the packaging construct as VSV-G displays an
inherent affinity to lipid membranes as such.
Although physical containment in the strict sense of the word cannot be attained when dealing
with human patients, some sort of physical restrainment always has to be considered in order
to control the shedding of virus. If possible, some sort of ex vivo approach should be applied
i.e. in vitro infection of patient cells grown in culture with subsequent screening to rule out
the presence of potential replication competent viruses before reimplantation of the
manipulated cells. Quarantine, of course, should always be part of the short-time routine
physical measures as should be the use of gloves and protective clothing for anybody in close
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contact with patients. Recent safety studies has pointed out that even long-term quarantine
might be relevant for certain gene therapy compounds as the viral vector in question, although
designed to be only transiently present, could be detected for several months (Dewey et al.,
1999).
Integration and germline transmission
Two major concerns are related to the permanent integration of the viral DNA i.e. the risk of
insertional mutagenesis causing cancer and the risk of germ line transmission.
Insertional mutagenesis, caused by the random integration of viral DNA, can induce
phenotypic changes to the cells and tissues of infected individuals. Although these changes
are rarely registered, adverse event caused by incorporation of the viral DNA into the host cell
genome is an important issue for gene therapy procedures as the ability of viruses to induce
human cancer is well documented (Eckhart, 1998; Mueller, 1999) the best known being
retroviruses (Blattner, 1999). For this reason, the extent of vector integration should clearly be
investigated especially when it is unintentended but also when it is intended and inherent to
the viral system.
As no guidance is currently available from regulatory authorities as to the precise
circumstances or methods to describe the extent of vector integration, the choice is at the
discretion of the investigators. While PCR-methods can easily be used for the mere detection
of viral vectors, methods involving the use of PCR to distinguish between integrated and
unintegrated viral vectors has to be developed on a case to case basis. This, for instance, could
be done for the retroviral self-inactivating vectors (SIN-vectors) taking advantage of the
known specific differences between the free vector and the integrated proviral DNA.
Ideally, the viral vectors employed should not allow inadvertent gene activation by
introduction into the host genome and, of course, this is especially important for inherently
integrating vectors i.e. vectors based on retroviruses and AAV. The ancient principle of the
onco-retroviral self-inactivating vectors (SIN-vectors), which has recently been copied into
the lentiviral system (Iwakuma et al., 1999), is the only successful attempt to create such
vectors. The SIN vectors are designed as to eliminate the potentially undesirable effects of the
viral promoter and enhancer sequences of the viral long terminal repeat (LTR) and the
resultant integrated provirus has no functional LTR´s. Nevertheless, other enhancer and
promoter elements of the integrated transgene might still influence the activity of cellular
genes adjacent to the provirus.
The development of viral systems for in situ viral gene therapy, as opposed to ex vivo gene
therapy, has raised concerns over the possibility of germ line transfer of the viral vector.
Although the risk of a deleterious outcome as a result of inadvertent germ line transmission
has been estimated to be in the range of one in a billion chances, regulatory authorities have
confirmed that concerns with germline spread is likely to persist until sufficient data has been
generated to allow the risk to be refined (Pilling, 1999).
Biodistribution assays has indicates that viral vectors are widely distributed throughout the
body following in situ administration and are frequently detected in the gonads (Pilling, 1999)
but, as for other safety assessment situations, there are no consensus as to what investigative
methods should be applied. Methods involving the use of in situ PCR to describe the
biodistribution and localization to germ cells should be improved and standardized as many
scientists have considerable problems getting reproducible results. A more direct approach
could be the use of non-advanced PCR procedures to detect the transduced vectors in semen
samples from mature treated male animals, although this crude and simple methodology has
obvious limitations.
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The development of tissue-specific vectors with novel tropism determinants and the ablation
of endogenous tropism (Douglas et al., 1999; Ivanenkov et al., 1999; Poul & Marks, 1999)
represents a promising solution to the problem of germ line transmission. This approach can
be expected to greatly diminish the problem, but the risk cannot be totally removed unless the
use of in vivo gene therapy, especially based on retroviruses and AAV, is completely
prohibited.
10.5.2 General concerns.
General concerns connected to the unintended presence of virally introduced genetic
information relate to the strategy of the therapeutic treatment and the accidental spread of
virus to other human beings. Whether or not the treatment is based on in vivo or ex vivo
infections, transient or permanent presence of the therapeutic principle, guided or unspecific
infection or on terminally differentiated cells or growing cells any unintended outcome of the
therapeutic treatment gives reason to concern.
In connection with a transient strategy, for instance, any permanent proof of the therapeutic
principle in the patient - as caused by a persistent infection or an unintended incorporation will be problematic. For patients treated in accordance with a permanent strategy any signs of
unspecific incorporation outside target tissue, destructive incorporation, incorporation into
germ line cells, or persistent infection outside the target tissue will represent problematic
complications.
The above-mentioned concerns, of course, to an even higher degree apply to untreated
humans. In that case, any detection of viral vectors are unacceptable, as there are no
therapeutic benefits connected with viral presence. In particular, the problem of unintended
viremic infection is not only a problem for the patient in treatment or the accidentally infected
person as infected immune-deficient individuals will represent some sort of a reservoir
generating and shedding infectious particles. In order to minimize this potential spread of
recombinant virus some sort of physical containment should be applied to protect vulnerable
groups. Especially immune-deficient individuals such as newly born infants, individuals
infected with HIV, and patients displaying symptoms of AIDS should not be exposed to the
uncontrollable shedding of viral particles from patients in therapeutic treatment. This means
that clinical tests, if not performed on a hospital specialized in this field only, should not be
performed in close physical proximity to e.g. maternity wards and nor should patients
receiving gene therapy and displaying obvious symptoms of viremea, be allowed to leave the
quarantine area.
Statistically significant data exemplifying above general problems are not easily available to
the scientific and general community. Although serious adverse events in connection with
gene therapy are reported to the regulatory authorities it will always be difficult to find an
appropriate balance between public disclosure and confidentiality, especially when
considering ongoing trials. Adverse events in the form of unexplained deaths, whether
ultimately proven to be related to the treatment or not, always creates public arousal as well as
immediate reactions on the stockmarkets (Commander, 2000).
Patient evaluation and the design of future viral gene therapy approaches must address not
only the efficiency of the therapeutic treatment but also any aspect of safety including the risk
of adverse events in the form of unexplained deaths, long-term active inflammation, tissue
damage and persistent expression of the transgene. This means that, whenever possible, the
safety of viral systems for gene therapy should be tested using in situ model systems build on
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relevant species in which the biological response to therapy most closely mimics the expected
human response. Healthy human volunteers have a mission to, but they cannot replace the use
of immune-deficient animal models.
10.6 Environmental risk assessment
A comprehensive description of the undesirable environmental effects connected with the
production and use of recombinant viral vectors for e.g. creation of transgenic animals, human
gene therapy, and live viral vaccines is not within the scope of this report. This will only by a
brief comment on the subject, and reference is made to "An Orphan in science: Environmental
Risks of Genetically Engineered Vaccines" (Traavik, 1999).
In theory, the potential for undesirable viral DNA transfer in the environment is enormous.
The presence of indigenous viruses in the environment is wellknown and recombinant live
viral vaccines have been known to establish themselves in wildlife host populations of e.g.
bank vole (Traavik, 1999). Virus mobilizable GMO-DNA in the environment originates from
multiple sources such as e.g. viral gene therapy, live viral vaccines, transgenic animals, the
production of therapeutics through the use of mammalian cells containing viral elements, and
from DNA vaccines containing viral sequences (e.g. viral promoters).
The prime concern regarding most viruses used as vaccines, e.g. vaccinia, is not their
recombinant nature. Rather, the prime concern is connected to the mere fact of their
introduction as possible pathogens e.g. the use of HIV-based vectors for vaccines and for gene
therapy. However, the consequences of recombinations among viruses within infected
animals as well as of the introduction of novel sequences into the viral pool infecting humans
and wildlife animals should be carefully considered. Presently, it is important to emphazise
that no standardized or comparable data regarding the environmental effects connected to the
production and use of recombinant viral vectors apparantly exists. The situation is the same
whether one looks at vectors infecting mammals - e.g vectors for gene therapy, for the
generation of transgenic livestock and for the construction of live viral vaccines - or at vectors
having another hostrange (e.g. manipulated insectile viruses). Especially the development of
live viral vaccines for the treatment of fish represents somewhat of a challenge.
In order to make reliable risk assessments and perform sensible risk managements with regard
to the interactions between genetically engineered viruses and indigenous viruses in the
environment the following questions should be asked:
• Is virus-mediated DNA transfer common in the environment, and what is the significance
of the phenomena?
• How well substantiated is the area?
• Have realistic and standardized tests been established for the determination of transfer
frequencies?
The answers to these questions, however, are not within the frames of this report.
10.7 Conclusion and suggestions
The lack of standardized assays for the detection of potentially replication-competent viruses
is the prime concern. Statistically significant validations of the safety of viral gene therapy
might be obtainable by the application of different model systems, but not necessarily in
clinical tests and not at all in connection with actual treatments - unless the systems are so
leaky/permeable that they ought not be used at all. The rather limited number of persons in
this type of clinical tests and the tightness of the highly contained systems causes the rare
complications to be seldomly registered. Practically, this is supported by the fact that useful
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clinical data on possible complications arising from viral gene therapy are not easily
available.
In spite of this apparent lack of statistically significant data there is, nonetheless, a real
theoretical possibility of spreading the viral vector to other individuals – particularly via
immune-deficient individuals. Therefore, it is a problem that the risk cannot be quantified in a
standardized way. This supports the need for the development of standardized in vitro and in
vivo model systems for quantitative description of the level of safety. Furthermore,
standardized model systems would allow comparative studies; thus enabling a more realistic
choice between related vector systems. Concerns about germ-line transmission also require
further study as do the use of lentivirus-based vectors, the use of phage-based vectors, the
sustained long-term use of viral vehicles in human beings, and the administration of viral
vectors to infants. Finally, new approaches for purification of viral particles should be applied
to generate clinical-grade reagents (Summerford & Samulski, 1999).
In connection with more specific analyses of current clinical experiments or treatments
(which is not within the frames of this report) the following questions are of relevance:
•
Are the safest designs applied, - or is efficiency prevailing?
•
Is safety validated in a standardized way or does anarchy reign?
•
Do in vitro and in vivo models show that recordable problems in clinical tests can be
expected?
•
Is the scientific aim clearly defined: safety study and/or clinical benefit study?.
•
Has the viral vector been purified by non-toxic means to generate a clinical grade batch?.
As all the above questions represent serious concerns about safety none should be looked
upon as unimportant and hence, any deviation from proper practice requires solid
argumentation. Many safety concerns may ultimately prove to be of only theoretical
importance but confidence with these viral systems can only be achieved through continued
safety assessments.
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