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ATTIVITA’ DI RICERCA SVOLTA ED IN CORSO DI SVOLGIMENTO
NELL’AMBITO DEL PROGETTO
“Organismi geneticamente modificati ed alimentazione: valutazione degli effetti
diretti sull’ospite e sulla microflora intestinale”
Università degli Studi di
Milano
Dipartimento di Scienze e Tecnologie Alimentari e
Microbiologiche
Via Celoria, 2 - 20133 Milano
Internet:
www.distam.unimi.it
Claudia Sorlini; professore ordinario
DISTAM Sez. MAAE
Tel.: 02 50316500
Fax: 02 50310238
e-mail: [email protected]
Milano, 05/07/2007
Milano, 15 giugno 2007
Spett.le Consiglio Diritti Genetici Onlus
All’attenzione del
Dott. Carlo Modonesi
Via Tadino 24
20124 Milano
Con la presente si allega la relazione conclusiva al primo anno di ricerca
nell’ambito del progetto “Organismi geneticamente modificati ed alimentazione:
valutazione degli effetti diretti sull’ospite e sulla microflora intestinale”,
finanziato da Consiglio Diritti Genetici / Fondazione Cariplo.
Cordiali saluti,
Prof.ssa Claudia Sorlini
1
Università degli Studi di
Milano
Dipartimento di Scienze e Tecnologie Alimentari e
Microbiologiche
Via Celoria, 2 - 20133 Milano
Internet:
www.distam.unimi.it
Claudia Sorlini; professore ordinario
DISTAM Sez. MAAE
Tel.: 02 50316500
Fax: 02 50310238
e-mail: [email protected]
Milano, 16/07/2007
ATTIVITA’ DI RICERCA SVOLTA ED IN CORSO DI SVOLGIMENTO
NELL’AMBITO DEL PROGETTO
“Organismi geneticamente modificati ed alimentazione: valutazione degli effetti
diretti sull’ospite e sulla microflora intestinale”
Al progetto finanziato da Consiglio Diritti Genetici - Fondazione Cariplo e coordinato da
Consiglio Diritti Genetici, hanno partecipato tre equipe di ricerca: l’equipe della prof. C.
Sorlini, afferente al Distam (Università degli Studi di Milano) e composta dai prof. C. Sorlini,
D. Daffonchio e da Dott. L. Brusetti e Dott.ssa A. Rizzi; l’equipe del prof. K.M. Nielsen,
afferente come sopra e composta dal prof. K.M. Nielsen e dai Dott. N. Raddadi e F. Mapelli;
l’equipe del prof. T. Traavik, afferente all’Institute of Gene Ecology (Genok) di cui fanno
parte il prof. Traavik e il prof. K.M. Nielsen (dell’Università di Tromso, Norvegia, ma
attualmente in forza all’università di Milano) e i Dott. L. Brusetti e N. Raddadi, i quali hanno
lavorato per complessivi sei mesi (3+3) presso l’università di Tromso e l’Institute of Gene
Ecology..
Il principale obiettivo del primo anno di ricerca è stato quello di porre le basi ad un efficace
disegno sperimentale per la valutazione approfondita degli eventuali effetti che Piante
Geneticamente Modificate (diversi tipi di mais transgenico con particolare attenzione al Btmais MON810) possono avere sulla struttura e sulla diversità della popolazione microbica
residente nel tratto gastrointestinale di mammiferi (nello specifico, del ratto), e sul
trasferimento genico orizzontale di frammenti di DNA esogeno di origine alimentare.
L’attività di ricerca effettuata dalle tre equipe può essere riassunta nei seguenti punti:
-
Definizione del disegno sperimentale da adottarsi durante la ricerca di laboratorio; in
esso sono definiti il numero di ratti da utilizzare, la loro biosicurezza durante le fasi
sperimentali, il tipo di alimentazione e le modalità di amministrazione del cibo, le
indagini sperimentali propedeutiche all’avvio della sperimentazione sui ratti. Questa
fase del lavoro ha prodotto la redazione dei documenti necessari per ottenere
l’autorizzazione universitaria e governativa per la sperimentazione (application
2
-
-
document). La definizione del progetto sperimentale ha coinvolto attivamente i
membri dell’equipe del Prof. Traavik presso l’università di Tromso, in Norvegia.
Per meglio determinare, quantificare e interpretare gli eventuali effetti che il mais
MON810 potrà avere sulla struttura e diversità batterica della microflora intestinale
dei ratti, si è definito un opportuno sistema di controlli positivi basato su un plasmide
che porta una cassetta genica di nuova concezione. La cassetta genica è costituita dalla
proteina di fusione aadA::gfp inserita all’interno del gene codificante il 16S rRNA di
Proteus mirabilis, tassonomicamente affine a molte specie batteriche naturalmente
residenti nel tratto gastrointestinale dei mammiferi. Questo costrutto genico permetterà
di stabilire e quantificare l’eventuale sopravvivenza di DNA esogeno lungo l’intero
tratto gastrointestinale dei ratti, quantificare i fenomeni di trasferimento genico
orizzontale in vivo, osservare in vivo la topologia del trasferimento genico, identificare
le specie batteriche responsabili del fenomeno a prescindere dalla loro coltivabilità in
laboratorio, determinare l’effetto dell’assunzione di DNA esogeno sulla microflora
batterica intestinale a prescindere dalla sua origine vegetale. Questo costrutto
plasmidico è stato ideato e parzialmente realizzato (il lavoro è attualmente ancora in
corso) dalle equipe dei prof. Sorlini, Daffonchio e Nielsen in forza al Distam di
Milano.
In accordo col responsabile scientifico della Fondazione Diritti Genetici Dott.
Modonesi, l’equipe dei prof. Sorlini e Daffonchio ha proceduto ad un’attività di
ricerca diretta alla definizione di eventuali effetti delle Piante Geneticamente
Modificate in ambienti diversi dal tratto gastrointestinale dei mammiferi, ma
comunque di interesse relativo ad esso. Tali ricerche sono state definite in accordo con
il Dott. Modonesi, in sostituzione della parte di progetto riguardante il sistema
immunitario dei ratti alimentati con il Bt-mais, di cui era responsabile l’equipe del
Prof. Monastra dell’INRAN, che non partecipa più al progetto. Tali ricerche hanno
riguardato:
A)
gli effetti delle Piante Geneticamente Modificate sulla microflora
fermentativa degli insilati destinati alla alimentazione animale;
B)
uno studio sulla dispersione in suoli agricoli e compost del gene blaTEM-1
che consente la resistenza verso gli antibiotici •-lattamici e che è
comunemente usato come marcatore di trasformazione nella produzione di
cultivar transgeniche;
C)
la caratterizzazione della struttura e diversità della microflora epifitica
naturalmente presente sul mais transgenico.
In allegato vengono riportate le singole attività di ricerca svolte dalle equipe coinvolte durante
il primo anno di attività (giugno 2006 – maggio 2007) e finanziati da Fondazione Diritti
Genetici - Fondazione Cariplo. Ogni attività di ricerca conclusa ha permesso la pubblicazione
di articoli scientifici su riviste peer-reviewed, i cui autori, titoli e riviste sono elencati al
termine dell’allegato. In ogni articolo è stato adeguatamente riconosciuto il supporto
finanziario del Consiglio dei Diritti Genetici e Fondazione Cariplo.
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ALLEGATI
Vengono allegati: A) Attività di ricerca svolta dalle tre equipe di Milano e di Tromso; B)
Application Document sottoposto alle autorità norvegesi per lo svolgimento della
sperimentazione sugli animali; C) elenco delle pubblicazioni realizzate durante il primo anno
di lavoro.
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A) ALLEGATO 1
Attività svolta presso il DISTAM dell’Università di Milano
Attività svolta dall’equipe dei prof. C. Sorlini e D. Daffonchio
L’equipe è composta dai prof. C. Sorlini, D. Daffonchio e da Dott. L. Brusetti e Dott.ssa A.
Rizzi
- Costruzione di una cassetta costituita da una fusione genica aadA::gfp per il
monitoraggio del trasferimento genico orizzontale in ceppi naturalmente competenti
[articolo 2].
- Costruzione di un plasmide vettore della fusione genica, in cui la cassetta aadA::gfp è
inserita all’interno di un gene codificante il 16S rRNA di Proteus mirabilis.
- Completamento delle sequenze di batteri lattici epifitici isolati da mais transgenico per
la caratterizzazione della popolazione batterica naturalmente presente. [articolo 1].
- Completamento della caratterizzazione delle sequenze del gene codificante per la
resistenza all’ampicillina (blaTEM-1) di isolati batterici resistenti all’antibiotico
ampicillina, ed isolati dalla rizosfera di mais transgenici e parentali non transgenici. Il
gene blaTEM-1 è adottato come marcatore nella costruzione di cassette geniche
comunemente utilizzate nelle piante geneticamente trasformate. [articolo 3].
- Stesura di articoli di rassegna sui rischi del rilascio e persistenza di DNA ricombinante
extracellulare nell’ambiente [articoli 5 e 6].
Attività svolta dall’equipe del prof. K.M. Nielsen
L’equipe è composta dal prof. K.M. Nielsen e dai Dott. N. Raddadi e F. Mapelli
- Verifica del funzionamento della fusione genica aadA::gfp in ceppi batterici
naturalmente competenti [articolo 2 dell’allegato].
- Identificazione mediante sequenziamento del gene codificante il 16S rRNA di un ceppo
enterico vitale in ratto, il cui gene 16S rRNA è stato poi utilizzato per la costruzione del
plasmide bioreporter precedentemente riportato. La ricerca ha portato all’identificazione
del ceppo di Proteus mirabilis CIP103181 type strain.
- Identificazione a livello di specie batterica dei ceppi ampicillino-resistenti isolati da
mais e precedentemente caratterizzati per la presenza del gene blaTEM-1.
Attività svolta presso la Facoltà di Farmacia dell’Università di Tromso, Norvegia
Attività svolta dall’equipe del prof. T. Traavik di cui è responsabile scientifico il Prof. Nielsen
L’equipe del prof. T. Traavik è composta dal prof. K.M. Nielsen (attualmente presso
l’università di Milano) e dai Dott. L. Brusetti e N. Raddadi che hanno lavorato per
complessivi sei mesi (3+3) presso l’università di Tromso.
- Definizione del progetto di alimentazione di ratti di laboratorio e stesura dei documenti
necessari per ottenere l’autorizzazione universitaria e governativa per la
sperimentazione (application document).
- Caratterizzazione fenotipica dei ceppi resistenti all’ampicillina isolati dalla rizosfera di
mais.
- Test preliminari per verificare il potenziale trasferimento in batteri enterici di plasmidi
contenenti geni gfp per la produzione di proteine fluorescenti. Tali costrutti saranno
utilizzati per definire e quantificare la stabilità del DNA esogeno nell’intestino dei ratti,
nonché per evidenziare eventuali fenomeni di trasferimento genico orizzontale, secondo
le modalità descritte nell’application document.
- Test preliminari all’esperimento di alimentazione dei ratti che considerano la capacità di
fitness di ceppi di Acinetobacter sprovvisti della loro naturale capacità di competenza,
per comprendere l’importanza della competenza sulla fitness assoluta nei batteri.
- Identificazione dei punti di inserzione del DNA eterologo nel DNA di Acinetobacter per
meglio comprendere le barriere dell’uptake di DNA esogeno.
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B) ALLEGATO 2
AN INVESTIGATION OF THE STABILITY AND HORIZONTAL TRANSFER OF
FEED-DERIVED DNA IN THE GASTROINTESTINAL TRACT OF RATS
Prof. Terje Traavik, Norwegian Institute of Gene Ecology and Department of Medical
Biology, and Prof. Kaare M. Nielsen, Department of Pharmacy, University of Tromsø,
Tromsø.
Several recent studies have reported on the stability of DNA in the gastrointestinal tract (GIT)
of mammals (Duggan et al., 2000; Reuter and Aulrich, 2003; Einspanier et al., 2004; Mazza et
al., 2005, Nordgård et al., accepted). However, few have examined quantitative aspects of
DNA stability and degradation and the possibility of functional horizontal transfers of feedderived DNA into the genome of the gut microbiota.
Objective
The main objective of this investigation is to further characterize the fate of feed-derived
DNA in the GIT of mammals and examine if horizontal transfer of feed-derived occur at
detectable levels into the genome of bacteria naturally present in the GIT.
Specific objectives
The specific objectives to be addressed in this investigation are:
Specific objective 1
To describe the quantitative and qualitative degradation kinetics of DNA molecules in the
GIT by specifically addressing:
•
How conformation affect DNA stability (circular or linear DNA)
•
How various types of food and the different gut compartments contribute to DNA
fragmentation
•
How the concentration of DNA change during passage through the GIT and
establishment of a lower detection limit of DNA molecules of different sizes in the
various compartments examined
Specific objective 2.
To determine to what extent bacterial members of the GIT microbiota develop competence for
uptake of feed-derived DNA by specifically addressing:
•
If fragmented DNA molecules are physically available to competent bacteria
•
If regions of high DNA similarity between indigenous bacteria and feed-derived DNA
can lead to recombination (and additive integration) of a selectable marker gene
•
If such HGT process occurs at a frequency that can be detected by direct selective
plating or PCR analysis of bacterial community DNA, and if selective in situ
enrichment of antibiotic resistance phenotypes can improve the recovery of bacterial
transformants
Specific objective 3.
To determine if and how the microbial structure of the GIT change during in vivo
administration of specific antimicrobial agents, and describe the nature, reversibility and
dynamics of such process by specifically addressing:
6
•
The qualitative changes occurring in the microbiota during antibiotic administration,
and possibly relate those to health effects
•
The bacterial community responses in rats (undergoing antibiotic treatment) with
feed containing the corresponding antibiotic resistance gene, and to determine if the
response is identical to the bacterial community response seen in those rats not
exposed to feed-derived DNA with the antibiotic resistance gene.
Specific objective 4.
To determine if and how the microbial community structure and diversity of the GIT change
during in vivo administration of genetic modified corn (GMC) in comparison with traditional
non-modified varieties, by specifically addressing:
•
Potential qualitative changes occurring in the GIT microbiota during GMC
administration
Figure 1. The digestive system of rats
General experimental design
Rat model system
Adult Wistar rats will be kept in conventional cages, with two rats per cage. Rats will
introduced to the cages one week before the start of the trials, to normalize them to the
changed environmental condition. The adult rats will be feed standard rat feed (Scanbur Inc.,
UK) with 30 – 40 grams of dried powder (AIN-93M) per day.
Source and concentration of monitored DNA
The rat feed (AIN-93M) has earlier been tested and found free of DNA (Nordgård et al.,
accepted). Moreover, DNA added to the dry feed pellets has been found to be stable. Two
types of DNA sources will be used, depending on the objective of the study.
1). DNA source is isolated plasmid and PCR products. A combination of plasmid DNA (PLA)
and PCR fragments of DNA, both carrying an antibiotic resistance marker gene (flanked by
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16S rRNA gene sequence and a short unique identifier). This DNA has the potential of
undergoing homologous recombination with a range of enterobacterial species, as well as
other bacterial genera normally present in the rat gut. The purpose of the plasmid design is
explained in objective 2. Plasmids will be extracted from a growing culture of transformed E.
coli. Plasmids will be designed as explained in Appendix 1.
2). DNA source is corn. The second type of DNA will be that naturally present in the
proportion of conventional (parental and isogenic or from other corn cultivar) (UMC) or
genetically-modified corn (GMC) (MON810 Bt-corn) meal that will be added to the feed
pellets. Five types of feed pellets will be prepared:
1. Feed pellet only
2. Feed pellet added both DNA sources (1,2)
3. Feed pellet with added GMC (2)
4. Feed pellet with added unmodified parental corn UMC (2)
5. Feed pellet with added a third non-transgenic corn cultivar
The concentration of plasmid/PCR fragments to be added to the feed pellet will be determined
on the basis of known DNA concentrations in food. The aim is to add an amount of
plasmid/PCR fragments comparable with that of the genomes added in a normal feed pellet
containing GM-Corn. Quantitative Real-Time PCR on the maize genome will be performed to
assess the concentration of DNA and recombinant elements (in MON810 corn only).
Sample collection
Rats will be collected before the first feeding (0 days), and at regular intervals according to
the research objectives. Two replicates per sampling time point is the minimum required to
ensure that enough gut content material can be recovered for further analysis. Samples will be
collected from four parts of GIT system; stomach, middle small intestine (jejunum), lower
small intestine (ileum) and colon (Figure 1). The samples will be stored at -80°C until further
analysis. In samples where viable bacteria are examined, 20% glycerol will be added prior to
freezing.
Experimental design for specific objective 1.
To determine on a quantitative and qualitative basis how stable DNA molecules are in
the GIT
A 24 h study is designed to determine the stability of various types of DNA in the GIT of rats
during a normal digestion period. Rats will be fed:
A) DNA-free feed pellet will be used as a control
B) Plasmid DNA and PCR products will be added to the feed pellet
Samples will be collected after 3, 6, 12 and 24 hours from four different parts of GIT, and
stored at -70 degrees before DNA extraction. Samples will be subjected to DNA extraction
with the QIAamp DNA Stool Mini Kit (Qiagen).
8
Quantitative measurements. A quantitative Sybr-Green Real-Time PCR approach will be
applied to assess the concentration of the aadA gene and recombinant junction (A, B) with
aadA-specific primers (Hollingshead and Vapnek, 1985), with expected band size of 284 bp:
Primer F:
aadA-F 5’ – TGA TTT GCT GGT TAC GGT GAC – 3’
Primer R:
aadA-R 5’ – CGC TAT GTT CTC TTG CTT TTG – 3’
Qualitative measurements. In addition to concentration based measurements, qualitative
descriptors of DNA size distribution will be performed by PCR amplification of fragments of
1 kb or larger of each of the added DNA targets. Such amplifications may be further verified
by more sensitive hybridization based assays of both generated PCR products and total DNA
extractions (e.g. examining the banding patterns produced by the added plasmid). Finally,
natural transformation or electroporation assays will be performed to determine if the isolated
DNA is in a state that can be used by competent bacteria.
12 rats will be studied (2 treatment, and 1 control) per time point
Experimental design for specific objective 2.
To determine to what extent members of the indigenous bacteria develop competence
for uptake of feed-derived DNA
A homologous recombination based HGT system is developed to aid in the identification of
putative naturally competent bacteria in the GIT of rats.
The recombination system is based on the insertion of an antibiotic resistance marker gene
with two flanking recombination sites with high DNA similarity to 16S rRNA genes
(Appendix 1), in a plasmid. Different plasmids will be further designed: the first type have the
antibiotic resistance gene flanked by 16S rRNA gene regions that are most conserved and
reflect universal 16S rRNA gene sequence of bacteria. Additional plasmids will carry more
family of genera specific flanking sequences, for instance, 16S rRNA genes closely related to
the Enterobacteria, Clostridia, Enterococci (i.e. representing the dominant members of the
GIT of mammals). A positive transformation control will be to expose the developed plasmids
to naturally competent Acinetobacter baylyi BD413 cells.
The inserted aadA antibiotic resistance marker gene (Clarck et al., 1999) will be used
to select transformants on LB agar plates with antibiotics (streptomycin and spectinomycin).
A short DNA fragment with unique base pair composition will be inserted immediately next
to the aadA gene to provide an extra tag that can ensure 100% confidence in the origin of the
resistance gene in expected transformants (Appendix 3). Alternatively, the reporter cassette
will be an aadA gene fused with a gene encoding the Green Fluorescent Protein, GFP (Rizzi
et al., submitted; Appendix 4). In this latter approach, a functional Prrn promoter allow
expression of the aadA::gfp gene fusion and the streptomycin/spectinomycin-resistant and
fluorescent phenotype, enhancing the possibility to detect rare HGT events. Observed gfp
gene expression also ensure confidence in the origin of the resistance gene in putative
bacterial transformants recovered from the GIT.
8 rats will be studied:
C) two rats given DNA-free feed pellet
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D) six rats given the same feed pellet, but also containing the above described plasmid
(circular and linearized) and PCR products of the same gene regions.
The rats will be collected after 7 days. Samples will be collected from the four parts of the
GIT. The high number of treatment rats is necessary to detect low frequency events.
Analysis of the Culturable fraction of bacteria for HGT: The content of the various gut
compartments (C, D) will be plated on agar media with streptomycin and spectinomycin to
enable selective growth of transformants. Colonies emerging on selective media will be
isolated, and the presence of the aadA gene and the artificial tag will be confirmed by PCR,
and southern blotting. If the the aadA::gfp gene fusion is used, single transformant cells will
be observed by fluorescence microscopy. Putative transformed isolates will be identified at
the species level by 16S rRNA gene sequencing and further characterization of competence
development in the isolate will be performed in vitro. If competent isolates of a genera or
species are identified, further efforts will be made to selectively enrich such bacteria in vitro
(from GIT samples), and a broad screen for competence development in such strains will be
initiated.
Analysis of the unculturable fraction of bacteria for HGT: To examine the possible
occurrence of natural transformation of bacteria in the GIT (unable to growth on the media
chosen for the previous experiment) non-culture based methods will be applied to total
genomic DNA isolated from the GIT. A PCR with primers specifically annealing to the extra
tag (adjacent to the aadA gene) or the aadA cassette and the 16S rRNA gene will reveal the
presence of recombinant bacterial DNA in the sample. Subsequent DGGE-based separation
followed DNA sequencing can resolve the number of transformant species present. We will
also apply Rolling Circle Amplification as described by Maruyama et al. (2006) to possibly
identify non culturable bacteria in total DNA extracted from the GIT samples.
Experimental design for specific objective 3.
To determine if and how the microbial community structure and diversity of the GIT
change during in vivo administration of antibiotics, and describe the nature, reversibility
and dynamics of such process.
To record how the overall microbial diversity in the rat GIT responds to antibiotic treatment
(of limited duration), an 18-days experiment has been designed.
E) 12 rats fed with DNA-free pellets
F) 12 rats fed the same pellet but with added plasmid DNA and PCR products.
Rats will be kept for up to 18 days and samples will be collected from four parts of GIT at
different time intervals. Sampling times (2 rats, per time point) will be after 0 (controls), 3, 4,
8, 13 and 18 days.
After the 3rd day of feeding, all the diets will be supplemented with an antibiotic
(streptomycin and spectinomycin, or similar aminoglycoside, that are not absorbed over the
GIT) for 5 days (5 day treatment period). The dose will be calculated as for recommended
dose/kg bodyweight and great care will be taken to ensure that the treatment dose is as low as
possible to ensure that the animals do not suffer and develop symptoms of diarrhoea during
the limited 5 day treatment. The antibiotics to be used are not taken up from the
gastrointestinal tract.
10
Samples from the various parts of the GIT will be plated on selective media to determine total
CFU, and the proportion of resistant phenotypes. The changes occurring in the bacterial
diversity and species prevalence will be measured with ARISA and DGGE based PCRs of
total DNA isolated form the 4 different sections of the GIT. The derived ARISA peak
matrices will be analyzed with cluster analysis and multivariate analyses, and observed
changes in community composition will be described by further analysis of relevant bacterial
species clusters. DGGE bands that are casually linked to the antibiotic treatment regime will
be sequenced to identify the relevant antibiotic-responsive bacterial species that either
decrease or increase in numbers.
If qualitative changes are seen between gut systems that are under similar antibiotic exposure,
but with different feed-derived resistance gene exposure, we will focus particularly the causal
explanation for such phenomenon. In the latter context, the experimental approach serves as
an in situ model for potential enrichment of rare transformants arising in a bacterial
community under strong directional selection. If the hypothesized HGT events occurs, further
model systems will be developed to more accurately determine both HGT frequencies, and
the population dynamics aspects of the process (in situ selection coefficients, absolute and
relative bacterial population sizes, growth responses etc.).
Experimental design for Objective 4.
To determine on a quantitative and qualitative basis how various corn cultivars affect
microbial communities of the GIT
For this purpose, a seven-days experiment is designed. Rats will be grown and fed with:
G) DNA-free feed pellet will be used as a general control (2 rats per timepoint)
H) MON810 corn powder will be added to feed pellet (2 rats per timepoint)
I) Parental unmodified corn powder will be added to feed pellet (2 rats per timepoint)
L) Non parental and unmodified corn powder will be added to the feed pellet (2 rats per
timepoint)
In total, 16 rats will be studied.
MON810, parental non-transgenic and a non-parental and unmodified corns will be provided
to feeding studies H, I and L with concentration of 50% transgenic corn weight/total weight.
Corn meal will be mixed with rat feeding powder. The mixture will be pressed into pellets of
homogeneous size and airdried. Samples will be collected after 0 and 7 days from four
different parts of GIT. Samples will be subjected to a total DNA extraction with QIAamp
DNA Stool Mini Kit (Qiagen).
Quantitative measurements. A quantitative Sybr-Green Real-Time PCR approach will be
followed to assess the concentration of the housekeeping gene Zein in sample (G, H, I, L)
with
- zein-specific primers (Mazza et al., 2005), with an expected band size of 439 bp.
Primer F:
ZeinF 5’ – GCT TGC ATT GTT CGC TCT C – 3’
Primer R:
ZeinR 5’ – CTA GAA TGC AGC ACC AAC AAA G – 3’
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- MON810 transgene specific sequence (G, H, I, L) accordingly to Holck et al., 2002; the
Mon810 event specific PCR system overlapped the junction region between endogenous
maize DNA and sequences originating from the cauliflower mosaic virus DNA, with an
expected band size of 154 bp.
Primer F: Mon810F1311 5’ – CCT TCA TAA CCT TCG CCC G – 3’
Primer R: Mon810R1311 5’ – AAT AAA GTG ACA GAT AGC TGG GCA – 3’
Qualitative measures. In addition to concentration based measures, qualitative descriptors of
DNA size distribution (G, H, I, L) will be performed by PCR amplification of fragments of 1
kb or larger of each of the added DNA targets. Such amplifications may be further verified by
more sensitive hybridization based assays of gel separated PCR products.
12
Sampling scheme
Objective 1
Conditions
A No DNA addition
B 16S rRNA sources
3 hrs
1
2
Objective 2
Conditions
C No DNA addition
D 16S rRNA sources
7 days
2
6
Objective 3
Conditions
E No DNA addition
F 16S rRNA sources
Objective 4
Conditions
G No DNA addition
H MON810 GM corn
I Conventional corn
L Third non GM cultivar
6 hrs
1
2
12 hrs
1
2
24 hrs
1
2
0 days
2
2
3 days* 4 days
2
2
2
2
8 days
2
2
0 days
4
2
2
2
7 days
4
2
2
2
13 days 18 days
2
2
2
2
*After 3th day, streptomycin and spectinomycin or other relevant antibiotics will be added to
the diets, for 5 days.
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Appendix 1
Design of 16S rRNA-aadA-tag cassette.
16S
Tag or ::gfp
aadA
16S
16S. 16S rRNA in bacteria is characterized by nine diverse variable regions (VR) interspersed
in the entire gene. The first three VRs are the best for taxonomy characterization. In previous
studies, it has been shown that the microbiota of rat is mainly composed of bacteria belonging
to genera: Clostridium, Proteus, Roseburia, Ruminococcus, Fusobacter, Eubacterium,
Lactobacillus, Bacteroides, Prevotella. Moreover many 16S rRNA gene sequences in the rat
gut are similar to uncultivated bacteria. Universal 16S rRNA regions conserved among
bacterial isolates are the preferred choice as a substrate for homologous recombination.
Consequently, the first 16S flanking region could be the part of the inter-VR V3-V4, while
the second flanking region could be the V8-V9 (Neefs et al., 1993). Alternatively
Enterobacterial-like inter-VR V3-V4 and V8-V9 could be considered. In Proteus mirabilis
universal regions are eight: U4 (764-815), U5 (878-991), U6 (1217-1240), U7 (1310-1420)
and U8 (1485-1536) will be considered (Gray et al., 1984).
aadA. Marker gene to be used in order to select positive events of recombination. A multiresistant gene will give a better approach since more antibiotics could be spread on agar
plates. For these reasons, the aadA gene (Clarck et al., 1999) was chosen. In the cassette
aadA::gfp (Rizzi et al., submitted), the aadA gene is fused with a gfp gene, allowing the
detection of transformants by fluorescent microscopy. In both cases, the aadA gene will be
driven by a strong promoter such as Prrn of the ribosomal operon, and it will be followed by
the ribosomal operon terminator Trrn.
TAG. An artificial DNA Tag to assure the transgene origin of aadA. A relatively long tag can
avoid misinterpretation due to random mutation events, and could be useful in specific primer
or probe design in successive experiments. Artificial DNA tag will be used only in the first
type of cassette, while in the aadA::gfp cassette, the gene gfp ensure the transgene origin of
aadA.
Plasmid cassette construction will be done by splicing-PCR as explained by Metzgar et al.
(2004).
PCR fragments will be obtained by amplifying about 1500 bp (primer forward on 16S rRNA,
primer reverse on extra tag; see Appendix 2) and a larger fragment (1800 bp; primer forward
on the plasmid vector, primer reverse on extra tag).
14
Appendix 2
See figure below for a description of the construction of the 16S-aadA construct.
Plasmid 16S
aadA*
Tag
Plasmid
16S rRNA of recipient
Plasmid
16S rRNA of recipient
16S
aadA
Tag
Plasmid
16S
16S rRNA
Species identification targets
aadA-specific primer
V1 V2 V3
10F 27F
519R
786R
aadA-specific primer
Tag-specific primer
Q-PCR fragment
Point E of Experimental flowchart
GC-clamp
519R
27F
First DGGE fragment
Point H of Experimental flowchart
Nested-DGGE fragment
Point H of Experimental flowchart
Sequencing
16S rRNA regions flanked by aadA-Tag sequences will facilitate recombination events as
explained above. Once the cassette is inserted into exposed novel recipient genomes, the
presence of the cassette will be detected by a PCR with a forward primer designed on aadA
and a reverse primer on the artificial Tag. The DGGE will be done in a nested-PCR approach:
first PCR will be done with the 10F universal primer and the reverse artificial Tag primer.
Later the universal primer 27F linked to a GC-clamp will be used coupled with 519R or 343R
universal primers.
* In case of aadA::gfp cassette, tag sequence will be omitted and primers will be designed on
the gfp gene sequence, or on the aminoacidic linker between the two genes.
15
Appendix 3
TAG SEQUENCE ANALYSIS
The uniqueness of the Tag sequence is confirmed by sequence similarity searches to known
sequences deposited in Genebank-EMBL databases.
The sequence could be designed based on this sequence that has no hairpin or self-dimer
characteristics:
5’ – ctactacccagcaccttagctatctcggttcattacctactacccagcaccttagctatctcggttcattac – 3’
The BLASTN result of this sequence is: No significant similarity found.
Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS,
environmental samples or phase 0, 1 or 2 HTGS sequences), Number of letters in database: 1,653,660,871
Number of sequences in database: 5,001,276 Lambda K H 1.37 0.711 1.31 Gapped Lambda K
H 1.37 0.711 1.31 Matrix: blastn matrix:1 -3 Gap Penalties: Existence: 5, Extension: 2
Number of Sequences: 5001276, Number of Hits to DB: 927755, Number of extensions:
37632
Number of successful extensions: 7331, Number of sequences better than 10: 0 , Number of
HSP's better than 10 without gapping: 0., Number of HSP's gapped: 7331, Number of HSP's
successfully gapped: 0, Length of query: 72, Length of database: 19821175605 , Length
adjustment: 20
Effective length of query: 52, Effective length of database: 19721150085
Effective search space: 1025499804420, Effective search space used: 1025499804420 A: 0
X1: 11 (21.8 bits) X2: 15 (29.7 bits) X3: 25 (49.6 bits) S1: 12 (24.3 bits) S2: 19 (38.2 bits)
16
Appendix 4
16S rRNA-aadA::gfp-16S rRNA CASSETTE SEQUENCE
The 16S rRNA gene sequence considered for this cassette is that of Proteus mirabilis
ATCC35659 (99% of homology with P. mirabilis CIP103181T – Ac. N. AJ301682). P.
mirabilis is a common inhabitant of mammalian guts. The genus Proteus (Proteobacteria;
Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae) is constituted by six species of
motile Gram-negative rods with peritrichous flagella. Proteus is widely distributed in the
environment and have been isolated from the intestinal tract of mammals, birds and reptiles
(Manos and Belas, 2006).
>16S-aadA::gfp (530F=907R/16S-Prrn-aadA-Linker-gfp-Trrn-926F=1494/16S)
GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCAATTAAGTCAGATGTGAA
AGCCCCGAGCTTAACTTGGGAATTGCATCTGAAACTGGTTGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCATGTGTAGCGGTGAAATGCGTA
GAGATGTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACC
CTGGTAGTCCACGCTGTAAACGATGTCGATTTAGAGGTTGTGGTCTTGAACCGTGGCTTCTGGAGCTAACGCGTTAAATCGACCGCCTGGGGAG
TACGGCCGCAAGGTTAAAACTCAAATGAATTGACGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTGAGGGGG
CAGGGATGGCTATATTTCTGGGAGCGAACTCCGGGCGAATACGAAGCGCTTGGATACAGTTGTAGGGAGGGATTTATGGATCCCGAAGCGGTGA
TCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGC
AGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAAC
GACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGC
GTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCT
GGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTA
TTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTT
GGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACT
TGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAG
ATCACCAAGGTAGTGGGCAAAGAACTTGTTGAAGGAAAATTGGAGCTAGTAGAAGGTCTTAAAGTCGGCGCCATGAGTAAAGGAGAAGAACTTT
TCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATA
CGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTGACTTATGGTGTTCAATGC
TTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAG
ATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGA
TGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAAC
TTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTAC
CAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGC
TGGGATTACACATGGCATGGATGAACTATACAAATAGGAATTAATTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTC
GTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTG
GCGGGCAGGACGCCCGCCATAAACTGCCAAAACTTAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACG
CGAAGAACCTTACCTACTCTTGACATCCAGCGAATCCTTTAGAGATAGAGGAGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGT
CAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAAGGAGAC
TGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAAA
GAGAAGCGACCTCGCGAGAGCAAGCGGAACTCATAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTA
GTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGT
AGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAG
PRIMERS to be used in splicing-PCR (underlined into the sequence):
A -> 530F-Pmirab (16S): GTGCCAGCAGCCGCGG
B -> 907R-Pmirab (16S): GTCAATTCATTTGAGTTTTA
C -> 907R+Prrn(R) (16S-Prrn): TTGAACGACGGCGGGGGAGCGTCAATTCATTTGAGTTTTA
D -> Prrn(R) (Prrn): TTGAACGACGGCGGGGGAGC
E -> 907R+Prrn(F) (16S-Prrn): TAAAACTCAAATGAATTGACGCTCCCCCGCCGTCGTTCAA
F -> Trrn+926F(F) (Trrn-16S): ACGCCCGCCATAAACTGCCAAAACTTAAAGGAATTGACGG
G -> Trrn+926F(R) (Trrn-16S): CCGTCAATTCCTTTAAGTTTTGGCAGTTTATGGCGGGCGT
H -> 926F-Pmirab (Trrm-16S): AAACTCAAATGAATTGACGG
I -> 1494R-Universal (16S): CTACGGTTACCTTGTTACGA
PCR PRIMER ANNEALING:
A+B:
- A self annealing: No, - B self annealing: Weak, - Primer Dimer: Very Weak
- Expected PCR fragment: 430 bp
17
F+I:- F self annealing: Very Weak, - I self annealing: No, - Primer Dimer: Very Weak
- Expected PCR fragment: 629 bp
18
Table 1. Overview of the samples and the specific analysis to be performed
Obj. Exp. Food type
N. Time Total
Antibiotic Total
Q-RT-PCR
Rats pts
Suppl.
N.
Gut
Sampl.1
DNA
Extract.
RCA
Zein MON810 aadA
1 Kb PCR
/ Blotting
Electro- Plating Isolation Total Microflora analysis
on
of Ant
porat.
Antib. Resistant
bacteria
Zein MON810 aadA
Enrich.
of
transf.
16S
aadA::tag ARISA
DGGE DGGE
1
A
DNA Free
1
4
16
+
+
+
+
1
B
Plasmid/PCR 2
4
32
+
+
+
+
2
C
DNA Free
2
1
16
+
+
+
+
+
2
D
16S plasmid
6
1
16
+
+
+
+
+
3
E
DNA Free
2
6
48
+
+
+
+
+
3
F
16S plasmid
2
6
48
+
+
+
+
+
4
G
DNA free
2
2
16
+
+
+
+
+
4
H
MON810
2
2
16
+
+
+
+
+
4
I
Parental
2
2
16
+
+
+
+
+
4
L
3rd Cultivar
2
2
16
+
+
+
+
+
1 – Total number of samples calculated as number of rats × number of time points × number of gut samples taken from each rat (stomach, jejunum, ileum and colon).
19
References:
1.
Clark N.C. et al., 1999. Detection of a Streptomycin/Spectinomycin Adenylyltransferase Gene (aadA) in
Enterococcus faecalis. Antimicrob. Agents Chemother. 43: 157 – 160.
2.
Duggan P.S. et al., 2000. Survival of free DNA encoding antibiotic resistance from transgenic maize and the
transformation activity of DNA in ovine saliva, ovine rumen fluid and silage effluent. FEMS Microbiol. Lett. 191:
71 – 77.
3.
Einspanier R. et al., 2004. Tracing residual recombinant feed molecules during digestion and rumen bacterial
diversity in cattle fed transgene maize. Eur. Food Res. Technol. 218: 269 – 273.
4.
Gray, M. W. et al., 1984. On the evolutionary descent of organisms and organelles: a global phylogeny based on a
highly conserved structural core in small subunit ribosomal RNA. Nucl. Acids Res. 12: 5837 – 5852.
5.
Holck A. et al., 1992. 5'-Nuclease PCR for quantitative event-specific detection of the genetically modified
Mon810 MaisGard maize. Eur Food Res Technol 214: 449 – 453.
6.
Hollingshead S. and D. Vapnek. 1985. Nucleotide sequence
streptomycin/spectinomycin adenyltransferase. Plasmid 13: 17 – 30.
7.
Manos J. and R. Belas. 2006. The Genera Proteus, Providencia, and Morganella. In Prokaryotes. An Evolving
Electronic Database for the Microbiological Community, 3rd edition (release 3.6), Dworkin M. et al. (eds.).
Springer-Verlag New York.
8.
Maruyama F. et al., 2006. Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ
Rolling Circle Amplification. Appl. Environ. Microbiol. 71: 7933 – 7940.
9.
Mazza R. et al., 2005. Assessing the transfer of genetically modified DNA from feed to animal tissues. Transgenic
Research 14: 775 – 784.
analysis
of
a
gene
encoding
10. Metzgar D. et al., (2004). Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome
engineering Nucl. Acids Res. 32: 5780 – 5790.
11. Neefs J. et al., 1993. Compilation of small ribosomal subunit RNA structures. Nucl. Acids Res. 21: 3025 – 3049.
12. Nielsen K.M. et al. In preparation.
13. Nordgård L. et al. Lack of detectable uptake of DNA by bacterial gut isolates grown in vitro and by Acinetobacter
baylyi colonizing rodents in situ. Environ. Biosafety Res. Submitted.
14. Reuter T. and K. Aulrich. 2003. Investigations on genetically modified maize (Bt-maize) in pig nutrition: Fate of
feed ingested foreign DNA in pig bodies. Eur. Food Res. Technol. 216:185 – 192.
15. Rizzi A. et al. A strategy for in situ localization of natural transformation-based horizontal gene transfer events.
Appl. Environ. Microbiol. Submitted.
20
C) ALLEGATO 3
Articoli prodotti dai gruppi di ricerca nel corso del primo anno di ricerca.
Di seguito vengono riportati gli articoli prodotti, indicandone gli autori, il titolo, il giornale
scientifico a cui l’articolo è stato sottomesso e lo stato di avanzamento del lavoro (sottomesso,
accettato, in stampa). Successivamente sono riportati in allegato, il frontespizio, il riassunto e i
ringraziamenti di ogni articolo. Si noti che la Fondazione Diritti Genetici viene ringraziata con la
passata denominazione “Consiglio per i Diritti Genetici” in quanto la sottomissione ed accettazione
degli articoli sono avvenute prima del cambiamento sociale dell’organizzazione.
1. Brusetti L., S. Borin, A. Rizzi, D. Mora, C. Sorlini e D. Daffonchio. Exploration of methods
to assess the effects of transgenic modification on bacterial communities in silage of various
maize cultivars (submitted to Environmental Biosafety Research).
2. Rizzi A., Pontiroli A., Brusetti L., Borin S., Sorlini C., Abruzzese A., Sacchi G.A., Vogel
T., Simonet P., Bazzicalupo M., Nielsen K.M., Monier J.-M. e Daffonchio D. A strategy for
in situ localization of natural transformation-based horizontal gene transfer events
(submitted to Applied and Environmental Microbiology).
3. Brusetti, L., T. Glad, S. Borin, P. Myren, A. Rizzi, P. Johnsen, P. Carter, D. Daffonchio, e
K. M. Nielsen. Low prevalence of blaTEM genes in arctic and agricultural soil and
rhizosphere (submitted to Microbial Ecology in Health and Disease).
4. Nordgård, L, Nguyen, T., Midtvedt, T., Benno, Y., Traavik, T., K. M. Nielsen. Lack of
detectable uptake of DNA by bacterial gut isolates grown in vitro and by Acinetobacter
baylyi colonizing rodents in situ (accepted in Environmental Biosafety Research).
5. Nielsen, K. M, P. Johnsen, D. Bensasson, D. Daffonchio. Release and persistence of
extracellular DNA in the open environment (66 pp. review; accepted in Environmental
Biosafety Research).
6. Nielsen, K. M., Daffonchio, D. Unintended horizontal transfer of recombinant DNA.
Capitolo di libro in: Traavik, T. e Lim, L.C. (eds.) Biosafety First: Holistic Approaches to
Risk and Uncertainty in Genetic Engineering and Genetically Modified Organisms. Tapir
Academic Press, Trondheim (In press).
21
Exploration of methods to assess the effects of transgenic modification on bacterial communities in
silage of various maize cultivars
Lorenzo Brusetti1, Sara Borin1, Aurora Rizzi1, Diego Mora1, Claudia Sorlini1, and Daniele
Daffonchio1,*
1
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM),
Università degli Studi di Milano, via Celoria 2, 20133, Milano, Italy
Abstract: An evaluation of different techniques to assess the cultivar effect of silages prepared with
transgenic (cv. Tundra, event Bt-176) and three conventional maize (Zea mays) cultivars on the bacterial
community structure and diversity were investigated by ensiling plants cultivated in greenhouse and
harvested after 30 days of growth. Silage samples collected at successive times during maize fermentation
were analyzed for bacterial counts and DNA-based population fingerprinting profiles. Bacterial counts found
some minor differences between maize cultivars for the total culturable bacteria, sporeforming, and
mesophilic and thermophilic lactic acid bacteria (LAB). Moreover, the analysis of species composition of
388 LAB strains isolated from cv. Tundra (event Bt-176) and its parental cultivar, characterized by intergenic
transcribed spacer (ITS) PCR followed by sequencing of 16S rRNA gene, showed only minor differences
between Bt-176 containing maize and its parental cultivar. On the opposite, innovative methodologies like
automated ribosomal intergenic spacers analysis (ARISA) and 16S rRNA gene length heterogeneity-PCR
(LH-PCR) of the whole bacterial communities, which profile were analyzed with multivariate analysis,
indicated that each maize cultivar selected a different bacterial population. Despite further studies are
required to assess how experimental variability influences the evaluation of the effects of transgenic plants
on silage bacterial community, the overall data indicated that automated techniques such as ARISA and LHPCR offer more detailed views of the bacterial community structure to distinguish slight bacterial
community differences during ensiling fermentation.
ACNOWLEDGEMENTS
The work was supported by the Italian Ministry for University and Scientific Research. Partial support
comes also from the EU TRANSBAC QLK3-CT-2001-02242 and from Consiglio dei Diritti Genetici in the
ambit of the project “Organismi geneticamente modificati ed alimentazione: valutazione degli effetti diretti
sull’ospite e sulla microflora intestinale”, funded by the Cariplo Foundation. We thank K. M. Nielsen for
comments on the manuscript.
22
A strategy for in situ localization of natural transformation-based horizontal gene transfer events
Aurora Rizzi,1 Alessandra Pontiroli,1,2 Lorenzo Brusetti,1 Sara Borin,1 Claudia Sorlini,1 Alessandro
Abruzzese,3 Gian Attilio Sacchi,3 Tim Vogel,2 Pascal Simonet, 2 Jean Michel Monier,2 Marco
Bazzicalupo,4 Kaare Magne Nielsen,5,6, and Daniele Daffonchio1*
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM),1 and Dipartimento di
Produzione Vegetale (DIPROVE),3 Università degli Studi di Milano, 20133 Milan, Italy; Écologie
Microbienne, UMR CNRS 5557, Universitè Claude Bernard, Lyon 1, 69622 Villeurbanne Cedex, France2;
Dipartimento di Biologia Animale e Genetica (DBAG), Università degli Studi di Firenze, 50125 Florence,
Italy; Department of Pharmacy, University of Tromsø,5, and Norwegian Institute of Gene Ecology, Research
Park, 9294 Tromsø, Norway6
Abstract: Natural transformation of bacteria in complex environments has previously been quantified ex situ
with sample-destructive approaches that does not allow the localization of gene transfer sites at the microscale level. We here present a natural transformation model that enables both in situ localization and
quantification of transformants. The model system consists of a green fluorescent protein gene (gfp) linked to
the selectable spectinomycin-resistance gene (aadA) in the genome of the highly transformable bacterium
Acinetobacter baylyi strain BD413. Specifically, a promoter-less aadA gene was fused in frame with a gfp
gene, downstream of the rbcL gene of the tobacco plastid genome, and inserted into strain BD413
chromosome. The resulting BD413(rbcL-∆PaadA::gfp) strain was employed in transformation experiments
using DNA extracted from transplastomic tobacco or bacterial DNA from different species, all sources
containing the Prrn::aadA cassette downstream of the plastid rbcL gene. Following double-crossover and
recombination in the rbcL and aadA flanking loci, insertion of a functional Prrn promoter allowed
expression of the aadA::gfp gene fusion and the spectinomycin-resistant and fluorescent phenotype. A
transformation frequency of 10-5 (transformants/recipients) was obtained in vitro with bacterial plasmids, and
8.4 × 10-10 with transplastomic leaf homogenate. Single transformant cells obtained after the addition of
exogenous bacterial DNA were localized by fluorescence microscopy on defrosted leaf tissues amended with
donor DNA; where single transformant cells and microcolonies were observed scattered on the leaf and close
to stoma cells.
ACKNOWLEDGEMENTS
We are indebted to L. N. Ornston and R. G. Kok for providing the pZR80 plasmidand to L. J. Halverson for
plasmid pPnptII::gfp. This work was supported by the EU research project TRANSBAC QLK3-CT-200102242). D.D. and K.M.N. acknowledge financial support from the Consiglio dei Diritti Genetici, Italy,
project “Organismi geneticamente modificati ed alimentazione: valutazione degli effetti diretti sull’ospite e
sulla microflora intestinale” funded by the Cariplo Foundation, Italy.
23
Low prevalence of blaTEM genes in Arctic environments and agricultural soil and rhizosphere
Lorenzo Brusetti1, Trine Glad2, Sara Borin1, Petter Myren2,3, Aurora Rizzi1, Pål J. Johnsen2, Phil
Carter3, Daniele Daffonchio1, Kaare M. Nielsen2,4
1
Department of Food Science, Technology and Microbiology (DISTAM), University of Milan, via Celoria 2,
20133, Milan, Italy.
2
Department of Pharmacy, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway
3
ESR Kenepuru Science Centre PO Box 50-348 Porirua, New Zealand.
4
Norwegian Institute of Gene Ecology, Science Park, 9294 Tromsø, Norway
Abstract: The prevalence of blaTEM genes conferring ampicillin resistance (Ampr) in different soils was
determined to clarify the environmental distribution of resistance determinants of major clinical importance.
Samples were collected from 16 sites in New Zealand, mainland Norway, Svalbard, and Italy. The Ampr
bacteria represented 1.7% to 100% of the cultivable microflora with an average of 28%. Approximately 1200
Ampr isolates were further analyzed. Although >50% of the resistant isolates were capable of •-lactam-ring
(nitrocefin) degradation, none carried a PCR-detectable blaTEM gene. The proportion of blaTEM genes in the
culturable Ampr isolates was <0.07%. The overall blaTEM gene prevalence was determined by blaTEM-specific
PCR of DNA extracted directly from the environmental sample. DNA hybridization was performed on
selected samples with a detection limit of ~11 blaTEM genes per PCR sample. Our analysis indicates that the
prevalence of blaTEM carrying bacteria is less than 1 per 1000 to 100000 bacteria in the samples analyzed.
The study suggests that blaTEM genes are rare in soil environments in contrast to their increasing prevalence
in some clinical and commensal bacterial populations. The frequent observation of nitrocefin-degrading
capacity among the sampled isolates suggests other enzymatic mechanisms conferring resistance to •lactams antibiotics are widespread in Arctic and agricultural soil environments.
ACKNOWLEDGEMENTS
We thank M. Pajoro, A. Pagliuca and P. Francia for technical assistance in plant cultivation, bacterial counts
and strain isolation performed at the University of Milan. The work was supported by the Italian Ministry for
University and Scientific Research within the project “Risposta della comunità microbica del suolo a
differenti pressioni antropiche: effetti su struttura, dinamica e diversità della microflora” and the Consiglio
dei Diritti Genetici, Italy. Partial support comes also from the EU project TRANSBAC QLK3-CT-200102242. T.G and K.M.N acknowledge financial support from the Research Council of Norway. Support for
the New Zealand work was provided by the Foundation for Research, Science and Technology.
24
Lack of detectable DNA uptake by bacterial gut isolates grown in vitro and by Acinetobacter baylyi
colonizing rodents in situ.
Lise Nordgård1, Thuy Nguyen1,3, Tore Midtvedt2, Yoshimi Benno3, Terje Traavik1,4 and Kaare M.
Nielsen*1,5
1
Norwegian Institute of Gene Ecology, Science Park, N9294 Tromsø, Norway, 2Department of Microbiology,
Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden, 3Microbe division, Japan Collection of
Microorganisms, RIKEN Bioresource Center, Wako, Saitama 351-0198, Japan, 4Department of
Microbiology and Virology, or 5Department of Pharmacy, Faculty of Medicine, University of Tromsø, N9037
Tromsø, Norway.
Abstract: Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps
related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is
presented that are designed to determine if genetic transformation of the naturally competent bacterium
Acinetobacter baylyi BD413 occurs, with feed-introduced bacterial DNA containing a kanamycin resistance
gene (nptII), in the GIT of mice and rats. Strain BD413 was found in various gut locations in germfree mice
at 103-105 CFU per gram GIT content 24-48 h after administration. However, subsequent DNA exposure of
the colonized mice did not result in detectable bacterial transformants with a detection limit of 1
transformant per 103-105 bacteria. Further attempts to increase the likelihood of detection by introducing
weak positive selection with kanamycin of putative transformants arising in situ during a 4 weeks long
feeding experiment (where the mice received DNA and the recipient cells regularly) did not yield
transformants either. Moreover, the in vitro exposure of actively growing A. baylyi cells to gut contents from
the stomach, small intestine, cecum or colon contents of rats (with a normal microbiota) feed either purified
DNA (50 µg) or bacterial cell lysates did not produce bacterial transformants. The presence of gut content of
germfree mice was also highly inhibitory to transformation of A. baylyi indicating that microbially-produced
nucleases are not responsible for the sharp 500 to 1 000 000-fold reduction of transformation frequencies
seen. Finally, a range of isolates from the genera Enterococcus, Streptococcus and Bifidobacterium spp. was
examined for competence expression in vitro without yielding any transformants. In conclusion, model
choice and methodological constraints severely limit the sample size and, hence, transfer frequencies that can
be measured experimentally in the GIT. Our observations suggest the contents of the GIT shields or adsorbs
DNA, preventing detectable exposure of feed-derived DNA fragments to competent bacteria.
ACKNOWLEDGEMENTS
Financial support was received from the Research Council of Norway (140890/720 and 140870/130). We
thank Elisabeth Norin, MTC, Karolinska Institute, Stockholm, Sweden, for excellent advice. We
acknowledge support from the Consiglio dei Diritti Genetici (CdG) project “Organismi geneticamente
modificati ed alimentazione: valutazione degli effetti diretti sull’ospite e sulla microflora intestinale”, funded
by the Cariplo Foundation.
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Release and persistence of extracellular DNA in the environment
Kaare M. Nielsen1,2*, Pål J. Johnsen1, Douda Bensasson3, Daniele Daffonchio4
1
Department of Pharmacy, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway
2
Norwegian Institute of Gene Ecology, 9037 Tromsø, Norway
3
Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK
4
Department of Food Science, Technology and Microbiology, University of Milan, Via Celoria 2, 20133
Milan, Italy
Abstract: The introduction of genetically modified organisms (GMOs) has called for an improved
understanding of the fate of DNA in various environments because extracellular DNA may also be important
for transferring genetic information between individuals and species. Accumulating nucleotide sequence data
suggest that acquisition of foreign DNA by horizontal gene transfer (HGT) is of considerable importance in
bacterial evolution. The uptake of extracellular DNA by natural transformation is one out of several ways
bacteria can acquire new genetic information given sufficient size, concentration and integrity of the DNA.
We review studies on the release, breakdown and persistence of bacterial and plant DNA in soil, sediment
and water with a focus on the accessibility of the extracellular nucleic acids as substrate for competent
bacteria. DNA fragments often persist over time in many environments, thereby facilitating their detection
and characterization. Nevertheless, the long-term physical persistence of DNA fragments of limited size
observed by PCR and Southern hybridization often contrasts with the short-term availability of extracellular
DNA to competent bacteria studied in microcosms. The main factors leading to breakdown of extracellular
DNA are presented. There is a need for improved methods for accurately determining the degradation routes
and the persistence, integrity and potential for horizontal transfer of DNA released from various organisms
throughout their lifecycles.
ACKNOWLEDGMENTS
K. M. N. and P. J. J. were supported by The Research Council of Norway´s Biodiversity Programme. K. M.
N. and D. D. acknowledge support from the CdG, Italy, and D. B. acknowledges support from a NERC
fellowship, UK. We thank M. Choi, G. Pietramellara, and P. Nannipieri for comments on an earlier version
of the manuscript.
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Chapter 13: Unintended horizontal transfer of recombinant DNA
Kaare Magne Nielsen,1 Daniele Daffonchio2
1. Department of Pharmacy, University of Tromsø, Norway, and the Norwegian Institute of Gene Ecology,
Tromsø, Norway
2. Department of Food Science and Microbiology, University of Milan, Italy
Abstract: DNA is usually transferred over generations following the normal reproduction pathway of the
organism involved (e.g. sexual reproduction/inheritance by descent). This process is called vertical gene
transfer and an example is pollen flow between the same or related plant species. Thus, vertical gene transfer
is the normal mode in which DNA is shared among individuals and passed on to the following generations.
DNA can, however, also more infrequently spread to unrelated species through a process called horizontal
gene transfer (HGT). HGT, sometimes also called lateral gene transfer, occurs independently of normal
sexual reproduction and is more common among single-celled organisms such as bacteria. HGT is a one-way
transfer of a limited amount of DNA from a donor cell/organism into single recipient cells. Examples of
HGT are the spread of antibiotic resistance among bacterial species, gene therapy in humans, and
Agrobacterium-infection in plants. HGT of recombinant DNA from GMOs to bacteria is a potential biosafety
concern (Nielsen et al. 2005). In this chapter we introduce the main biosafety aspects of unintended HGT
processes as they relate to the use of recombinant DNA, as follows:
1. Introduction to some biosafety aspects of recombinant DNA
2. Recombinant DNA introduction and potential impact in various environments
Human exposure to foreign DNA
DNA in food
DNA stability in the digestive tract
3. HGT of recombinant DNA to eukaryotic cells (e.g. human cells)
4. HGT of recombinant DNA to prokaryotic cells (e.g. bacterial cells)
5. Conclusions
ACKNOWLEDGEMENTS
The authors acknowledge financial support from the Research Council of Norway and the Consiglio dei
Diritti Genetici, Italy.
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