Bacterial Conjugation

61

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 conjugation is often termed lateral or horizontal gene transfer, as opposed to vertical transfer 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 conjugative 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 Escherichia 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-transmissible, 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 a recipient 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 chromosome 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 conjugation is still emerging (reviewed in ref. 12). However, the basic mechanism and genetics of bacterial conjugation have been established. Early experiments by Lederberg and Tatum in the 1940s showed that DNA from one bacterium could be transferred to another bacterium (1). The recipient bacterial strain subsequently develops phenoFrom: Methods in Molecular Biology, Vol. 235: E. coli Plasmid Vectors Edited by: N. Casali and A. Preston Humana Press Inc., Totowa, NJ

61

62

Woodall

typic characteristics of the donor strain. A liquid mating system for bacterial conjugation 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 accession number U01159). When a donor bacterium, containing the F plasmid, and a fertility 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 selftransmissible 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 recipient E. 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 mapping of the E. 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 and E. coli MG1655 diverged 100 million years ago, increasing the potential of E. coli to inhabit previously unobtainable ecological niches (for reviews, see refs. 2426). Conjugative plasmids 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 plasmids may also play an important role in extensive lateral gene transfer, where virulence 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 from Agrobacterium tumifaciens to plants (28) and F and RP4 plasmids from

Bacterial Conjugation

63

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 mobilization 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 N NaOH 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 procedure. It may also be necessary to increase the cell biomass of the bacterial cultures. Optimization is particularly important if the recipient strain is not E. 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 universal 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.

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. 2. Lederberg, J. and Tatum, E. L. (1946) Gene recombination in Escherichia coli. Nature 158, 558. Firth, N., Ippen-Ihler, K., and Skurray, R. A. (1996) Structure and function of the F factor and mechanism of conjugation, in Escherichia coli and Salmonella: Celluar and Molecular Biology (Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., et al., eds.), ASM, Washington, DC, pp. 23772401. 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. Gross, J. D. and Caro, L. G. (1966) DNA transfer in bacterial conjugation. J. Mol. Biol. 16, 269284. Lloyd, R. G. and Buckman, C. (1995) Conjugational recombination in Escherichia coli: genetic analysis of recombinant formation in Hfr F crosses. Genetics 139, 11231148. Franke, A. E. and Clewell, D. B. (1981) Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of conjugal transfer in the absence of a conjugative plasmid. J. Bacteriol. 145, 494502. Courvalin, P. and Carlier, C. (1986) Transposable multiple antibiotic resistance in Streptococcus pneumoniae. Mol. Gen. Genet. 205, 291297. Courvalin, P. and Carlier, C. (1987) Tn1545: a conjugative shuttle transposon. Mol. Gen. Genet. 206, 259264. Caillaud, F., Carlier, C., and Courvalin, P. (1987) Physical analysis of the conjugative shuttle transposon Tn1545. Plasmid 17, 5860. Clewell, D. B., Flannagan, S.E., and Jaworski, D.D. (1995) Unconstrained bacterial promiscuity: The Tn916Tn1545 family of conjugative transposons. Trends Microbiol. 3, 229236. Hinerfeld, D. and Churchward, G. (2001) Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision. Mol. Microbiol. 41, 14591467. 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. Penfold, S. S., Simon, J., and Frost, J. S. (1996) Regulation of the expression of the traM gene of the F sex factor of Escherichia coli. Mol. Microbiol. 20, 549558.

3.

4. 5. 6.

7. 8. 9. 10.

11. 12. 13.

Bacterial Conjugation

65

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 traY gene product and integration host factor stimulate Escherichia 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 in E. coli: evidence for three constitutive systems mediating uptake of potassium. J. Bacteriol. 173, 687696. 22. Holloway, B. and Low, B. (1996) F-prime and R-prime factors, in Escherichia 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. Plasmid 42, 7391. 24. Lawerence, J. G. and Ochman, H. (1998) Molecular archaeology of the Escherichia 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.