MethodsMolBiol conjugacion

8/4/2019 MethodsMolBiol conjugacion

1/5

Bacterial Conjugation 61

61

From: Methods in Molecular Biology, Vol. 235:E. coli Plasmid VectorsEdited by: N. Casali and A. Preston Humana Press Inc., Totowa, NJ

6

DNA Transfer by Bacterial Conjugation

Claire A. Woodall

1. Introduction

Bacterial conjugation is defined as contact-dependent transmission of genetic

information from a donor bacterium to a recipient cell (1). Transfer of DNA by conju-

gation is often termed lateral orhorizontal gene transfer, as opposed to vertical trans-

fer by which genetic information is transferred from mother to daughter cells. When

genetic information is transferred by conjugation from the donor strain to the recipient

strain, this population of recipient bacteria are called transconjugants. The genetic

information is usually transferred to the recipient bacterium on a plasmid; however,

conjugative transposons are also known. The mechanism and proteins involved in con-jugative DNA transfer vary depending on the type of plasmid or transposon present in

the donor strain. For example, RP4 and F (fertility) plasmids isolated from Escheri-

chia coli are self-transmissible plasmids and thus contain genes that encode for all of

the necessary proteins involved in the mobilization and transfer of the plasmid from

one bacterium to another(2). Other plasmids that are conjugative, but non-self-trans-

missible, such as RSF1010 isolated from E. coli, can only be mobilized when the

necessary functions are supplied in trans (3). The cointegration of a conjugative and

nonconjugative circular plasmid can result in the transfer of both plasmids into arecipient strain. For example, in a high-frequency recombinant (Hfr) E. coli K-12

strain, where the F plasmid has integrated into the bacterial chromosome, the whole chro-

mosome can be conjugated to a recipient strain (4,5). Finally, conjugative transposons can

be transferred from one bacterium to another. Examples include Tn916 isolated from

Enterococcus faecalis DS16 and Tn1545 isolated from Streptococcus (611).

A detailed understanding of the regulation and biochemistry of bacterial conjuga-

tion is still emerging (reviewed in ref. 12). However, the basic mechanism and genet-

ics of bacterial conjugation have been established. Early experiments by Lederbergand Tatum in the 1940s showed that DNA from one bacterium could be transferred to

another bacterium (1). The recipient bacterial strain subsequently develops pheno-


2/5

62 Woodall

typic characteristics of the donor strain. A liquid mating system for bacterial conjuga-

tion using the E. coli F plasmid is highly efficient and has been investigated in the

most detail. The F transfer region (33.3 kb) contains 36 open-reading frames that

encode for several proteins involved in bacterial conjugation (414) (GenBank acces-sion number U01159). When a donor bacterium, containing the F plasmid, and a fertil-

ity minus (F) recipient bacterium come into close contact, a conjugative pilus on the

donor bacterium makes contact with the recipient. F factor DNA is transferred though

this pilus into the recipient bacterium. The conjugative pilus consists of several pilin

subunits comprising three proteins encoded by the genes traA, traQ, and traX

(reviewed in ref. 15). Once the conjugative pilus has connected the two bacteria, an

uncharacterized mating signal initiates the formation of a nucleoprotein complex, at

the oriT (origin of transfer) on the F factor. The nucleoprotein complex consists of

F-plasmid-encoded proteins TraI and TraY (16,17). TraY binds to the oriT site, which

initiates the binding of TraI and activates the nucleoprotein complex (18). The oriT on

the F plasmid DNA is nicked by the transesterase function of TraI and duplex DNA

is unwound by the helicase mechanism of TraI (19,20). Single-stranded DNA (ssDNA)

is then transferred in a 5' to 3' direction into the recipient cell. In the donor strain,

replacement strand synthesis occurs, and complementary strand replacement occurs in

the recipient strain. Finally, the transfer of DNA is terminated when the DNA strand is

recircularized at oriT (20).

In the laboratory, conjugation can be used to transfer disrupted genes on a self-

transmissible plasmid, to develop a mutant strain. For example marker-exchange

mutagenesis is a technique that can be used to help elucidate the function of a gene,

based on the characteristics of the mutant strain compared to the wild-type strain. A

gene of interest from a recipientE. coli strain is cloned into a self-transmissible vector

and maintained in a donor strain for genetic manipulation. The deleted gene construct

is then transferred by conjugation from the donor strain back into the recipient strain.

In addition, classic laboratory experiments have exploited the conjugation of Hfr

E. coli K-12 strains to map genes and for complementation studies (21). Genetic map-ping of theE. coli chromosome using interrupted pairing studies with F prime factors

can identify the time of entry and order of genes on the bacterial genome (22).

The transfer of genetic information by conjugation in the environment is important

for bacterial diversity (23). Conjugative plasmids can mediate the lateral transfer of

antibiotic resistance or virulence determinants between bacteria, allowing bacteria to

adapt to otherwise hostile environments. For example, a total of 234 lateral transfer

events were reported to have occurred since Salmonella enterica andE. coli MG1655

diverged 100 million years ago, increasing the potential ofE. coli to inhabit previ-ously unobtainable ecological niches (for reviews, see refs. 2426). Conjugative plas-

mids can also induce biofilm development (27). A biofilm niche is often found in an

aquatic environment or at sites of bacterial infection. At these sites, conjugative plas-

mids may also play an important role in extensive lateral gene transfer, where viru-

lence genes are mobilized between bacterial species. Conjugation can also occur from

bacteria to plant and eukaryotic cells; examples are the conjugative transfer of the Ti

plasmid fromAgrobacterium tumifaciens to plants (28) and F and RP4 plasmids from


3/5


E. coli to the yeast, Sacromyces cerevisiae (29). Recently, the RP4 plasmid (also

known as RK2 and RP1) was conjugated from E. coli into Chinese hamster ovary

(CHO K1) cells (30). Thus, conjugation is an invaluable mechanism for the mobiliza-

tion of DNA among the three kingdoms: bacteria, plants, and animals.

2. Materials

1. LuriaBertani (LB) broth medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium

chloride. Adjust to pH 7.0 by addition of 5 NNaOH and autoclave.

2. Antibiotics for selection of transconjugants.

3. LB agar: Add 15 g/L Bacto agar to LB broth prior to autoclaving. LB agar plates should

contain an antibiotic for transformant selection. Add appropriate antibiotics, once the

agar has cooled to around 50C, prior to pouring plates.

4. Filter mating system and holder, aseptic closed system for biological samples (Millipore,Nalgene, Whatmann, Sartorius, PALL Gelman Laboratories).

5. 0.45-m Pore-size Filter-mating disks (smaller pore sizes can also be used). Polycarbonate or

nitrocelluose membrane disks are recommended (Millipore, Nalgene, Whatmann, Sartorius,

PALL Gelman Laboratories, Nucleopore), also polyvinylidene fluoride (PVDF) Durapore

membranes (Millipore).

3. Method

1. Dilute overnight cultures of the donor and recipient strains 1 in 50 in fresh LB broth.

Incubate at 37C with vigorous shaking until an OD600 of 0.6 0.8 is reached.

2. Mix different ratios of the donor and recipient strains in a sterile universal. For example,

donor/recipient ratios of 1 : 1, 1 : 2 and 1 : 10 might be set up in the first instance (see

Note 1). Dilute strains in LB broth if required.

3. Pour the cell mix into the upper chamber of a filter-mating unit. Use a hydro-powered

suction pump to collect cells on the membrane filter; the liquid medium will flow directly

into the lower chamber. Any remaining cells in the upper chamber can be washed onto the

membrane filter with a small volume of LB broth (see Note 2).

4. Immediately place the membrane filter, cell side upward, onto a nonselective LB agar

plate and incubate at 37C from 30 min to 16 h (see Note 3).

5. Carefully remove the membrane filter from the agar plate and place in a sterile universal

bottle. Aseptically add 5 mL of LB broth. Shake the universal vigorously to wash the

cells off of the membrane filter into the liquid medium.

6. Spread plate serial dilutions (see Note 4) of cells onto selective agar plates and incubate

overnight at 37C to obtain transconjugates (see Note 5).

7. Determine the conjugation frequency (see Note 6).

4. Notes

1. Different ratios of donor to recipient strains are used to optimize the conjugation proce-

dure. It may also be necessary to increase the cell biomass of the bacterial cultures. Opti-

mization is particularly important if the recipient strain is notE. coli.

2. A variation of the filter-mating assay can be carried out using liquid cultures. In this

method, the mixed cell suspension of donor and recipient cells is added to a sterile uni-

versal and incubated at 37C from 30 min to 16 h (see Note 3). After incubation, the

conjugated cell mix should be vortexed vigorously to disrupt the mating pairs. The cell

mix is spread plate as described in step 6.


4/5

64 Woodall

3. For optimal recovery of conjugated cells, the incubation period may vary. It may be worth

setting up several plates and harvesting the bacteria at time periods from 30 min to 16 h.

4. Serial dilutions of the conjugated cells are done best as a 10-fold dilution series (i.e., 1

mL cell mix in 9 mL LB broth). Spread plate a portion of the diluted mix (e.g., 200 L).

5. Antibiotic resistance selection must not only select for bacteria carrying the conjugating

DNA but also select against the donor bacteria. All donor bacteria will carry the antibiotic

resistance conferred by the DNA to be conjugated. Thus, the donor and recipient bacteria

must have different antibiotic sensitivities or be distinguishable in another way (e.g.,

different abilities to grow on the medium on which the transconjugants are selected).

6. The conjugation frequency can be calculated as the number of transconjugants divided by

the total number of bacteria isolated on nonselective medium.

Acknowledgments

I gratefully acknowledge Dr. Andrew Grant for proofreading this chapter.

References

1. Lederberg, J. and Tatum, E. L. (1946) Gene recombination in Escherichia coli. Nature

158, 558.

2. Firth, N., Ippen-Ihler, K., and Skurray, R. A. (1996) Structure and function of the F factor

and mechanism of conjugation, inEscherichia coli and Salmonella: Celluar and Molecu-

lar Biology (Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., et al., eds.), ASM, Washing-

ton, DC, pp. 23772401.3. Haase, J. Lurz, R. Grahn, A. M., et al. (1995) Bacterial conjugation mediated by plasmid

RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production

require the same Tra2 core components of a proposed DNA transport complex. J

Bacteriol. 177, 47794791.

4. Gross, J. D. and Caro, L. G. (1966) DNA transfer in bacterial conjugation.J. Mol. Biol. 16,

269284.

5. Lloyd, R. G. and Buckman, C. (1995) Conjugational recombination inEscherichia coli:

genetic analysis of recombinant formation in Hfr F crosses. Genetics 139, 11231148.

6. Franke, A. E. and Clewell, D. B. (1981) Evidence for a chromosome-borne resistancetransposon (Tn916) in Streptococcus faecalis that is capable of conjugal transfer in the

absence of a conjugative plasmid.J. Bacteriol. 145, 494502.

7. Courvalin, P. and Carlier, C. (1986) Transposable multiple antibiotic resistance in Strep-

tococcus pneumoniae.Mol. Gen. Genet. 205, 291297.

8. Courvalin, P. and Carlier, C. (1987) Tn1545: a conjugative shuttle transposon.Mol. Gen.

Genet. 206, 259264.

9. Caillaud, F., Carlier, C., and Courvalin, P. (1987) Physical analysis of the conjugative

shuttle transposon Tn1545.Plasmid17, 5860.

10. Clewell, D. B., Flannagan, S.E., and Jaworski, D.D. (1995) Unconstrained bacterial pro-miscuity: The Tn916Tn1545 family of conjugative transposons. Trends Microbiol. 3,

229236.

11. Hinerfeld, D. and Churchward, G. (2001) Xis protein of the conjugative transposon Tn916

plays dual opposing roles in transposon excision.Mol. Microbiol. 41, 14591467.

12. Frost, L. S., Ippen-Ihler, K., and Skurray, R. A. (1994) Analysis of the sequence and gene

products of the transfer region of the F sex factor. Microbiol. Rev. 58, 162210.

13. Penfold, S. S., Simon, J., and Frost, J. S. (1996) Regulation of the expression of the traM

gene of the F sex factor ofEscherichia coli.Mol. Microbiol. 20, 549558.


5/5


14. Anthony, K. G., Klimke, W. A., Manchak, J., et al. (1999) Comparison of proteins involved

in pilus synthesis and mating pair stabilization from the related plasmids F and R100-1:

insights into the mechanism of conjugation.J. Bacteriol. 181, 51495159.

15. Silverman, P. M. (1997) Towards a structural biology of bacterial conjugation. Mol.

Microbiol. 23, 423429.

16. Nelson, W., Howard, M., Sherman, J., et al. (1995) The traYgene product and integration

host factor stimulateEscherichia coli DNA helicase I-catalyzed nicking at the F plasmid

oriT.J. Biol. Chem. 270, 28,37428,380.

17. Howard, M., Nelson, W., and Matson, S. (1995) Stepwise assembly of a relaxosome at the

F plasmid origin of transfer.J. Biol. Chem. 270, 28,38128,386.

18. Lahue, E. E. and Matson S. W. (1990) Purified Escherichia coli F-factor TraY protein

binds oriT.J. Bacteriol. 172, 13851391.

19. Tsai, M. M, Fu, Y. H., and Deonier, R. C. (1990) Intrinsic bends and integration host

factor binding at F plasmid oriT.J. Bacteriol. 172, 46034609.

20. Matson, S. W., Sampson, J. K., and Byrd, D. R. N. (2001) F plasmid conjugative DNA

transfer.J. Biol. Chem. 276, 23722379.

21. Dosch, D. C, Helmer, G. L., Sutton, S.H., et al. (1991) Genetic analysis of potassium

transport loci inE. coli: evidence for three constitutive systems mediating uptake of potas-

sium.J. Bacteriol. 173, 687696.

22. Holloway, B. and Low, B. (1996) F-prime and R-prime factors, inEscherichia coli and

Salmonella: Celluar and Molecular Biology (Neidhardt, F. C., Curtiss, R., III, Ingraham,

J. L., et al., eds.), ASM, Washington DC, pp. 24132419.

23. Davison, J. (1999) Genetic exchange between bacteria in the environment. Plasmid42,

7391.

24. Lawerence, J. G. and Ochman, H. (1998) Molecular archaeology of theEscherichia coli

genome.Proc. Natl. Acad. Sci. USA 95, 94139417.

25. Ochman, H. and Moran, N. A. (2001) Genes lost and genes found: evolution of bacterial

pathogenesis and symbiosis. Science 292, 10961099.

26. Ochman, H., Lawrence, J. G., and Groisman, E. A. (2000) Lateral gene transfer and the

nature of bacterial innovation.Nature 405, 299304.

27. Ghigo, J. M. (2001) Natural conjugative plasmids induce bacterial biofilm development.

Nature 412, 442445.

28. Stachel, S. E. and Zambryski, P. C. (1986)Agrobacterium tumefaciens and the susceptible

plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell 47,

155157.

29. Heinemann, J. A. and Sprague, G. F. (1989) Bacterial conjugative plasmids mobilize DNA

transfer between bacteria and yeast.Nature 340, 205209.

30. Waters, V. L. (2001) Conjugation between bacterial and mammalian cells.Nat. Genet. 29,

375376.

MethodsMolBiol conjugacion

Documents

Transcript of MethodsMolBiol conjugacion