Download effectiveness of transduction and conjugation in genetic transformation

Journal of Pharmaceutical Research and Opinion 1: 7 (2011) 170 – 173.
Contents lists available at www.innovativejournal.in
JOURNAL OF PHARMACEUTICAL RESEARCH AND OPINION
Journal homepage: http://www.innovativejournal.in/index.php/jpro
REVIEW
EFFECTIVENESS OF TRANSDUCTION AND CONJUGATION IN GENETIC
TRANSFORMATION
Shuaib M. Awwal*, Abalaka M.
Department of Microbiology, Federal University of Technology, Minna, P.M.B 65, Niger State. Nigeria
ARTICLE INFO
ABSTRACT
Received 23 Nov 2011
Accepted 11 Dec 2011
Bacteria have mechanisms by which they can 'obtain' extra DNA, which
creates opportunities for recombination to occur. Certain species of bacteria can
become competent to take up high molecular weight DNA from the surrounding.
An event in which one bacterium donates DNA to another bacterium is a type of
genetic transfer termed recombination leading to Genetic transformation.
Transduction is DNA transfer mediated through the action of a bacteriophage
while Conjugation requires the attachment of two related species through a
bridge that can transport DNA. Both phenomenons provide additional genes for
resistance to drugs and metabolic poisons, increased virulence and adaptation to
environment effectively, thus genetic transformation.
Corresponding Author:
Shuaib M. Awwal Department of
Microbiology, Federal University
of Technology, Minna, P.M.B 65,
Niger State. Nigeria
[email protected]
KeyWords: Transformation,
Transduction, Conjugation, DNA
©2011, JPRO, All Right Reserved.
INTRODUCTION
Genetic transformation is a process by which free
DNA is incorporated into a recipient cell and brings about
change. The discovery of genetic transformation in bacteria
was one of the outstanding events in biology, as it led to
experiments proving without a doubt that DNA was the
genetic material. This discovery became the keystone of
molecular biology and modern genetics.
Genes derived from unrelated species and even
other kingdoms, such as bacteria, fungi, plants, animals,
that would otherwise be inaccessible to an organism, can
be combined in the lab usinggenetic transformation
techniques.(National4-Hcouncil,1997).
Bacteria can exchange or transfer DNA between
other bacteria in three different ways. In every case the
source cells of the DNA are called the DONORS and the cells
that receive the DNA are called the RECIPIENTS. In each
case the donor DNA is incorporated into the recipient’s
cell's DNA by recombination exchange. If the exchange
involves an allele of the recipient's gene, the recipient's
genome and phenotype will have changed. The three forms
of bacterial DNA exchange are (1) TRANSFORMATION, (2)
CONJUGATION and (3) TRANSDUCTION. (Hurlbert, 1999).
Natural genetic transformation was originally discovered in
Streptococcus pneumoniae (Griffith, 1928), and ever since
the pneumococcus has served as a paradigm for this
important phenomenon. After (Dawson and Sia, 1931)
achieved transformation in vitro in 1931, the ‘nuts and
bolts’ of the transformation apparatus and its regulation
has gradually been unraveled. However, much remains to
be learned, especially with respect to environmental cues
that promote competence development in situ, and the
complex nature of the DNA uptake machinery.
TRANSDUCTION
Bacteriophages (bacterial viruses) have been
previously described as destructive bacterial parasites.
Infection by a virus does not always kill the host cell,
however, and viruses can in fact serve as genetic vectors
(an entity that can bring foreign DNA into a cell). The
process by which a bacteriophage serves as the carrier of
DNA from a donor cell to a recipient cell is transduction.
Although it occurs naturally in a broad spectrum of
bacteria, the participating bacteria in a single transduction
event must be the same species because of the specificity of
viruses for host cells. (Kathleene and Arthur, 2002).
Transduction is the transfer of bacterial genes by viruses.
Bacterial genes are incorporated into a phage capsid
because of errors made during the virus life cycle. The virus
containing these genes then injects them into another
bacterium, completing the transfer. There are two different
kinds of transduction: generalized and specialized.
(Prescott et al., 2008) .
Generalized Transduction
Sometimes, during bacteriophage replication, a
mistake is made, and a fragment of the host DNA gets
packaged into a viral capsid. The resulting phage would be
able to infect another cell, but it would not have any viral
genes, so it would not be able to replicate. The cell infected
by this phage would survive, and would have an extra piece
of bacterial DNA present, which could undergo
recombination with the host chromosome, and perhaps
cause a gene conversion event. Because it is a random
fragment that gets packaged into the viral capsid, any
segment of the bacterial DNA can be transferred this way
(hence
the
name
'generalized').(emunix
,2011).
170
Awwal et. al / Effectiveness Of Transduction And Conjugation In Genetic Transformation
Generalized transduction in the Enterobacteriaceae is
typified by the action of the lytic bacteriophages PI, P22,
etc. The injected DNA is integrated into the chromosome by
the host recombination mechanism or maintained by
autonomous replication in cases where a replicative
plasmid is transduced. (Philippe and Julian,1991) . As in
transformation, once the DNA has been injected, it must be
incorporated into the recipient cell’s chromosome to
preserve the transferred genes. (Prescott et al., 2008).
Specialized Transduction
Specialized transduction occurs only with certain
types of bacteriophage, such as phage lambda. Lambda has
the ability to establish what is called a lysogenic infection in
a bacterial cell. In a lysogenic infection, the viral DNA
becomes incorporated into the host chromosome, much as
the F factor did in Hfr cells. In a lysogenic infection by
lambda, the DNA integrates into a very specific spot in the
host chromosome. The integrated viral DNA can remain
integrated for long periods of time, without disturbing the
cell. Under the appropriate conditions, the viral DNA will
excise itself from the chromosome, and enter the lytic
phase, in which the virus replicates. The cell gets lysed, and
new bacteriophage particles are released to infect other
cells. As with excision of the F factor (when Hfr cells
become F'), sometimes the excision of lambda is sloppy,
and some bacteria DNA is excised along with it. When the
resulting virus infects another cell, it will pass that
bacterial DNA into the cell, along with its own DNA. If the
infected cell survives, it will contain a new piece of
bacterial DNA, which can undergo recombination and
possibly cause gene conversion. Because the viral DNA
integrates into a specific location, when it excises, the
bacterial DNA removed with it will be the same in all cases.
Therefore, the DNA transferred to the second cell will be
the same segment of the bacterial chromosome. This is why
this process is called 'specialized' transduction. (emunix,
2011).
CONJUGATION
In 1946 Joshua Lederberg and Tatum discovered
that some bacteria can transfer genetic information to
otherbacteria through a process known as conjugation.
(Sridhar, 2006). Conjugation is a mode of sexual mating in
which a plasmid or other genetic material is transferred by
a donor to a recipient cell via a direct
connection.(Kathleene and Arthur, 2002). The contact
between the cells is via a protein tube called an F or sex
pilus, which is also the conduit for the transfer of the
genetic material. (emunix, 2011). Although mating systems
have been studied in the most detail in gram-negative
bacteria, conjugation systems have been identified and
analyzed in streptococci, staphylococci, bacillaceae,
streptomycetes, and halobacteria (Rosenshine et al., 1989).
Pheromone-activated conjugation in streptococci, which
has been extensively studied by Clewell and associates, is
the only identified bacterial conjugation system in which a
diffusible signal is required to activate cell interaction.
(Clewell and Weaver, 1989) . Processes involving diffusible
components might be more susceptible to environmental
inhibition, such as the presence of proteases.
The prototypical conjugative plasmid is the Fplasmid, or F-factor. (Holmes and Jobling, 1996) . The Fplasmid is an episome (a plasmid that can integrate itself
into the bacterial chromosome by homologous
recombination) with a length of about 100 kb. It carries its
own origin of replication, the oriV, and an origin of transfer,
or oriT. Ryan and (Ray, 2004) .There can only be one copy
of the F-plasmid in a given bacterium, either free or
integrated, and bacteria that possess a copy are called Fpositive or F-plus (denoted F+). Cells that lack F plasmids
are called F-negative or F-minus (F-) and as such can
function as recipient cells. Contact is made when a pillus
grows out from the F+ cell, attaches to the surface of the Fcell, contracts, and draws the two cells together. In both
gram-positive and gram-negative cells, an opening is
created between the connected cells, and the replicated
DNA passes across from one cell to the other. Conjugation
is a very conservative process, in that the donor bacterium
generally retains a copy of genetic material being
transferred. (Kathleene and Arthur, 2002).
When conjugation is initiated by a signal the
relaxase enzyme creates a nick in one of the strands of the
conjugative plasmid at the oriT. Relaxase may work alone
or in a complex of over a dozen proteins known collectively
as a relaxosome. In the F-plasmid system the relaxase
enzyme is called TraI and the relaxosome consists of TraI,
TraY, TraM and the integrated host factor IHF. The nicked
strand, or T-strand, is then unwound from the unbroken
strand and transferred to the recipient cell in a 5'-terminus
to 3'-terminus direction. The remaining strand is replicated
either independent of conjugative action (vegetative
replication beginning at the oriV) or in concert with
conjugation (conjugative replication similar to the rolling
circle replication of lambda phage). Conjugative replication
may require a second nick before successful transfer can
occur. A recent report claims to have inhibited conjugation
with chemicals that mimic an intermediate step of this
second nicking event. (Lujan et al., 2007)
If the F-plasmid that is transferred has previously
been integrated into the donor’s genome some of the
donor’s chromosomal DNA may also be transferred with
the plasmid DNA. (Griffiths et al.,1999). The amount of
chromosomal DNA that is transferred depends on how long
the two conjugating bacteria remain in contact. In common
laboratory strains of E. coli the transfer of the entire
bacterial chromosome takes about 100 minutes. The
transferred DNA can then be integrated into the recipient
genome via homologous recombination.
In high-frequency recombination (Hfr) donors, the
fertility has been integrated into the F+ donor chromosome.
The term high-frequency recombination was adopted to
denote that a cell with an integrated F factor transmits its
chromosomal genes at a higher frequency than other cells.
(Kathleene and Arthur, 2002).
Effectiveness of transduction in genetic transformation
Transduction may be important for the exchange
of genetic material between closely related species
(identical or cross-reacting receptors) in nature. (Kokjohn,
1989). DNA carried by the transducing particle is protected
from the environment and may survive for relatively long
periods of time (Zeph et al., 1988). Several cases of
specialized transduction have biomedical importance. The
virulent strains of bacteria such as C. diphtheria,
Clostridium spp., and S. pyogenes all produce toxins with
profound physiological effects, whereas nonvirulent strains
do not produce toxins. It turns out that toxicity arises from
the expression of bacteriophage genes that have been
introduced by transduction. Only those bacteria infected
with a temperate phage are toxin formers. Other instances
of transduction are seen in staphylococcal transfer of drug
171
Awwal et. al / Effectiveness Of Transduction And Conjugation In Genetic Transformation
resistance and in the transmission of gene regulators in
gram-negative rods (Escherichia, Salmonella). (Kathleene
and Arthur, 2002).
Effectiveness of conjugation in genetic transformation
The initial studies of (Wolk et al.,1984) and
(Guiney et al.,1984) demonstrated conjugative transfer
between two distant groups of gram-negative bacteria,
including anerobes; Subsequently, Trieu euot and
collaborators (Trieu-Cuot et al.,1987) designed and
constructed broad-host range conjugative plasmids having
dual gram-negative and gram-positive replication origins
on a mobilizable plasmid possessing a "universal"
antibiotic resistance selection marker. The motivation for
the study was to obtain evidence for intergeneric transfer
of certain antibiotic resistance determinants in nature.
Gene transfer between, distantly related organisms
has also expanded outside the microbial world! The vir
genes of the Agrobacterium tumefaciens Ti plasmid provide
some of the tra functions for conjugal transfer from
bacteria to plants. In an important experiment, BuchananWollaston and collaborators (Buchanan-Wollaston et al.,
1987) showed that the mob and oriT functions of the
broad-host range IncQnplasmid RSFIOIO mediate the
transfer of plasmids from Agrobacterium sp. into plant
cells. The broad-host range plasmid pRK2 (IncP) was
unsuccessful in similar tests of transfer into plant cells.
Conjugation is so successful in promoting diverse gene
exchanges, an important factor is likely to be the
replicative transfer of single-stranded DNA during
conjugation. Single-stranded DNA is known to be refractory
to most restriction systems, thus the transfer of singlestranded DNA during bacterial conjugation may escape
digestion by recipient restriction systems. (Baltz and
McHenney, 1989). The facile transfer of IncQ plasmids to a
variety of gram-positive species is of significance in
considerations of gene transfer in the environment (range
and efficacy) and in aiding the manipulation of such
industrially important microorganisms as Mycobacteria
and Streptomycetes.
CONCLUSION
Transduction and specialized transduction is
especially important because they explain how anti-biotic
drugs become ineffective due to the transfer of resistant
genes between bacteria. In addition, hopes to create
medical methods of genetic modification of diseases such
as Duschenne/Becker Muscular Dystrophy are based upon
these methodologies.
Conjugation has great biomedical importance.
Special resistance (R) plasmids, or factors, that bear genes
for resisting antibiotics and other drugs are commonly
shared among bacteria through conjugation. Transfer of R
factors can confer multiple resistances to antibiotics such
as
tetracycline,
chloramphenicol,
streptomycin,
sulfonamides and penicillin. Other Benefits may include
xenobiotic tolerance or the ability to use new metabolites
that increases the pathogenicity of the bacterial strain.
Conjugation studies have also provided an excellent way to
map the bacterial chromosome. Both transduction and
conjugation have been found to occur in a variety of
bacteria, the two phenomenons are sufficiently widespread
for us to assume that it plays a significant role in genetic
transformation.
REFERENCES
1. Baltz, R. H . , McHenney, M. A. (1989). Transduction of
plasmid DNA in Streptomyces spp. In Genetics and
Molecular Biology of Industrial Microorganisms, ed. C. L.
Hershberger, S. W. Queener, G. Hegeman , pp. 163-67 .
Washington, DC: Am. Soc. Microbiol . 377 pp.
2. Buchanan-Wollaston,V.,
Passiatore,J.E.,
Cannon,F.
(1987) . The mob and oriT mobilization functions of a
bacterial plasmid promote its transfer to plants. Nature
328: 1 72-75
3. Clewell, D. B., Weaver, K. E (1989) . Sex pheromones and
plasmid transfer in Enterococcus faecalis. Plasmid 21: 1
75-84
4. Dawson M.H., Sia R.H (1931) In vitro transformation of
pneumococcal types. I. A technique for inducing
transformation of pneumococcal types in vitro. J Exp
Med 54: 681–699.
5. Griffiths AJF, et al. (1999). An Introduction to genetic
analysis (7th ed.). San Francisco: W.H. Freeman. ISBN 07167-3520-2.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=
Bacterial+conjugation&rid=iga.section.1304.
6. Griffith F (1928) . The significance of pneumococcal
types . J Hyg 27: 108–159
7. Guiney, D. G . , Hasegawa, P. , Davis, C. E. (1984). Plasmid
transfer from Escherichia
8. coli to Bacteroides fragilis: Differential expression of
antibiotic resistancc phenotypes. Proc. Natl. Acad. Sci.
USA 8 1 :7203-6
9. Holmes RK, Jobling MG (1996). Genetics: Conjugation. in:
Baron's Medical Microbiology (Baron S et al., eds.) (4th
ed.). Univ of Texas Medical Branch. ISBN 0-9631172-11.
10. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=
conjugation&rid=mmed.section.468#473
11. Hurlbert R. E. (1999).microbiology 101/102 internet
text chapter ix: microbial exchange of genetic material
http://www.slic2.wsu.edu:82/hurlbert/micro101/page
s/Chap9.html#microbial_sex
12. Kathleene, T. and Arthur T. (2002). Foundations in
Microbiology, 4th Edition, McGraw-Hill United states pp
277-280.
13. Kokjohn, T . A . (1989). Transduction: mechanism and
potential for gene transfer in the environment, pp. 7397
14. Lujan SA, Guogas LM, Ragonese H, Matson SW, Redinbo
MR
(2007).
"Disrupting
antibiotic
resistance
propagation by inhibiting the conjugative DNA
relaxase".
PNAS
104
(30):
12282–7.
doi:10.1073/pnas.0702760104.
JSTOR 25436291.
PMC 1916486.
PMID 17630285.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?t
ool=pmcentrez&artid=1916486
15. National 4-H Council (1997) . Fields of Genes: Making
Sense of Biotechnology inAgriculture.
16. http://www.fourhcouncil.edu/ycc/ffg/FSCI.html
17. Philippe M. and Julian D. (1991) . Gene transfer between
distantly related bacteria. Annu. Rev. Genet. 25:147-71
18. Prescott,L.M.,
Harley,
J.P.,
Klein,
D.A(2008).
Microbiology,7th Edition. McGraw-Hill,United states pp
345.
19. Rosenshine, I., Tchelet, R., Mevarech,M. (1989). The
mechanism of DNA transfers in the mating system of an
archaebacterium. Science 245: 1 387-89
172
Awwal et. al / Effectiveness Of Transduction And Conjugation In Genetic Transformation
20. Ryan KJ, Ray CG (editors) (2004). Sherris Medical
Microbiology (4th ed.). McGraw Hill. pp. 60–4.
ISBN 0838585299
21. Sridhar Rao P.N (2006). bacterial genetics. Department
of Microbiology, JJMC Devangere, pp 6
22. http://www.microrao.com
23. Trieu-Cuot, P . , Carlier, C . , Martin, P . , Courvalin, P.
(1987). Plasmid transfer by conjugation from
Escherichia coli to gram-positive bacteria. FEMS
MicrobioI. Lett. 48:289-94
24. Wolk, C . P., Vonshak, A. , Kehoe, P . , Elhai, J . (1984).
Construction o f shuttle vectors capable of conjugative
transfer from Escherichia coli to nitrogen-fixing
filamentous cyanobacteria. Proc. Natl. Acad. Sci. USA 8
1: 1561-65
25. .Zeph, L. R., Onaga, M. A., Stotzky, G. (1988).
Transduction o f Escherichia coli by bacteriophage PI in
soil. Appl. Environ. Microbiol. 54: 173 1-37
26. http://www.emunix.emich.edu/~rwinning/genetics/ba
ctrec.htm [accessed September 2011]
173