Abstract
TrbK is the only plasmid-encoded gene product involved in entry exclusion of the broad-host-range plasmid RP4. The corresponding gene, trbK, coding for a protein of 69 amino acid residues maps in the Tra2 region within the mating pair formation genes. TrbK carries a lipid moiety at the N-terminal cysteine of the mature 47-residue polypeptide. The mutant protein TrbKC23G cannot be modified or proteolytically processed but still acts in entry exclusion with reduced efficiency. An 8-amino-acid truncation at the C terminus of TrbK results in a complete loss of the entry exclusion activity but still allows the protein to be processed. TrbK localizes predominately to the cytoplasmic membrane. Its function depends on presence in the recipient cell but not in the donor cell. TrbK excludes plasmids of homologous systems of the P complex; it is inert towards the IncI system. The likely target for TrbK action is the mating pair formation system, because DNA or any of the components of the relaxosome were excluded as possible targets.
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- Achtman M., Kennedy N., Skurray R. Cell--cell interactions in conjugating Escherichia coli: role of traT protein in surface exclusion. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5104–5108. doi: 10.1073/pnas.74.11.5104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Achtman M., Manning P. A., Edelbluth C., Herrlich P. Export without proteolytic processing of inner and outer membrane proteins encoded by F sex factor tra cistrons in Escherichia coli minicells. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4837–4841. doi: 10.1073/pnas.76.10.4837. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Achtman M., Manning P. A., Kusecek B., Schwuchow S., Willetts N. A genetic analysis of F sex factor cistrons needed for surface exclusion in Escherichia coli. J Mol Biol. 1980 Apr 25;138(4):779–795. doi: 10.1016/0022-2836(80)90065-0. [DOI] [PubMed] [Google Scholar]
- Alt-Mörbe J., Stryker J. L., Fuqua C., Li P. L., Farrand S. K., Winans S. C. The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes. J Bacteriol. 1996 Jul;178(14):4248–4257. doi: 10.1128/jb.178.14.4248-4257.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Chatfield L. K., Orr E., Boulnois G. J., Wilkins B. M. DNA primase of plasmid ColIb is involved in conjugal DnA synthesis in donor and recipient bacteria. J Bacteriol. 1982 Dec;152(3):1188–1195. doi: 10.1128/jb.152.3.1188-1195.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication. Gene. 1982 Jan;17(1):79–89. doi: 10.1016/0378-1119(82)90103-2. [DOI] [PubMed] [Google Scholar]
- Cowan P., Krishnapillai V. Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18. Plasmid. 1982 Sep;8(2):164–174. doi: 10.1016/0147-619x(82)90054-3. [DOI] [PubMed] [Google Scholar]
- Datta N., Hedges R. W., Shaw E. J., Sykes R. B., Richmond M. H. Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol. 1971 Dec;108(3):1244–1249. doi: 10.1128/jb.108.3.1244-1249.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Derbyshire K. M., Hatfull G., Willetts N. Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region. Mol Gen Genet. 1987 Jan;206(1):161–168. doi: 10.1007/BF00326552. [DOI] [PubMed] [Google Scholar]
- Dev I. K., Ray P. H. Rapid assay and purification of a unique signal peptidase that processes the prolipoprotein from Escherichia coli B. J Biol Chem. 1984 Sep 10;259(17):11114–11120. [PubMed] [Google Scholar]
- Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunny G. M., Zimmerman D. L., Tortorello M. L. Induction of surface exclusion (entry exclusion) by Streptococcus faecalis sex pheromones: use of monoclonal antibodies to identify an inducible surface antigen involved in the exclusion process. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8582–8586. doi: 10.1073/pnas.82.24.8582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Furuya N., Komano T. Surface exclusion gene of IncI1 plasmid R64: nucleotide sequence and analysis of deletion mutants. Plasmid. 1994 Jul;32(1):80–84. doi: 10.1006/plas.1994.1047. [DOI] [PubMed] [Google Scholar]
- Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
- Gennity J. M., Inouye M. The protein sequence responsible for lipoprotein membrane localization in Escherichia coli exhibits remarkable specificity. J Biol Chem. 1991 Sep 5;266(25):16458–16464. [PubMed] [Google Scholar]
- HJERTEN S., MOSBACH R. "Molecular-sieve" chromatography of proteins on colums of cross-linked polyacrylamide. Anal Biochem. 1962 Feb;3:109–118. doi: 10.1016/0003-2697(62)90100-8. [DOI] [PubMed] [Google Scholar]
- Haase J., Lurz R., Grahn A. M., Bamford D. H., Lanka E. 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. 1995 Aug;177(16):4779–4791. doi: 10.1128/jb.177.16.4779-4791.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
- Hartskeerl R. A., Bergmans J. E., Kamp M. C., Hoekstra W. P. Cloning of an exclusion-determining fragment of the IncI plasmid, R144. Plasmid. 1983 Jul;10(1):11–20. doi: 10.1016/0147-619x(83)90053-7. [DOI] [PubMed] [Google Scholar]
- Hartskeerl R. A., Hoekstra W. P. Exclusion in IncI-type Escherichia coli conjugations: the stage of conjugation at which exclusion operates. Antonie Van Leeuwenhoek. 1984;50(2):113–124. doi: 10.1007/BF00400171. [DOI] [PubMed] [Google Scholar]
- Hartskeerl R., Overduin P., Hoekstra W., Tommassen J. Nucleotide sequence of the exclusion-determining locus of IncI plasmid R144. Gene. 1986;42(1):107–111. doi: 10.1016/0378-1119(86)90156-3. [DOI] [PubMed] [Google Scholar]
- Hartskeerl R., Tommassen J., Hoekstra W. Relationship between the proteins encoded by the exclusion determining locus of the IncI plasmid R144 and the cellular localization of these proteins in Escherichia coli K-12. Mol Gen Genet. 1985;200(1):138–144. doi: 10.1007/BF00383327. [DOI] [PubMed] [Google Scholar]
- Hartskeerl R., Zuidweg E., van Geffen M., Hoekstra W. The IncI plasmids R144, R64 and ColIb belong to one exclusion group. J Gen Microbiol. 1985 Jun;131(6):1305–1311. doi: 10.1099/00221287-131-6-1305. [DOI] [PubMed] [Google Scholar]
- Hedges R. W., Jacob A. E. Transposition of ampicillin resistance from RP4 to other replicons. Mol Gen Genet. 1974;132(1):31–40. doi: 10.1007/BF00268228. [DOI] [PubMed] [Google Scholar]
- Howland C. J., Rees C. E., Barth P. T., Wilkins B. M. The ssb gene of plasmid ColIb-P9. J Bacteriol. 1989 May;171(5):2466–2473. doi: 10.1128/jb.171.5.2466-2473.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hussain M., Ichihara S., Mizushima S. Accumulation of glyceride-containing precursor of the outer membrane lipoprotein in the cytoplasmic membrane of Escherichia coli treated with globomycin. J Biol Chem. 1980 Apr 25;255(8):3707–3712. [PubMed] [Google Scholar]
- Jalajakumari M. B., Guidolin A., Buhk H. J., Manning P. A., Ham L. M., Hodgson A. L., Cheah K. C., Skurray R. A. Surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12. Determination of the nucleotide sequence and promoter and terminator activities. J Mol Biol. 1987 Nov 5;198(1):1–11. doi: 10.1016/0022-2836(87)90452-9. [DOI] [PubMed] [Google Scholar]
- Jobanputra R. S., Datta N. Trimethoprim R factors in enterobacteria from clinical specimens. J Med Microbiol. 1974 May;7(2):169–177. doi: 10.1099/00222615-7-2-169. [DOI] [PubMed] [Google Scholar]
- Kao S. M., Olmsted S. B., Viksnins A. S., Gallo J. C., Dunny G. M. Molecular and genetic analysis of a region of plasmid pCF10 containing positive control genes and structural genes encoding surface proteins involved in pheromone-inducible conjugation in Enterococcus faecalis. J Bacteriol. 1991 Dec;173(23):7650–7664. doi: 10.1128/jb.173.23.7650-7664.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karas M., Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15;60(20):2299–2301. doi: 10.1021/ac00171a028. [DOI] [PubMed] [Google Scholar]
- LaVallie E. R., DiBlasio E. A., Kovacic S., Grant K. L., Schendel P. F., McCoy J. M. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N Y) 1993 Feb;11(2):187–193. doi: 10.1038/nbt0293-187. [DOI] [PubMed] [Google Scholar]
- Lederberg J, Cavalli L L, Lederberg E M. Sex Compatibility in Escherichia Coli. Genetics. 1952 Nov;37(6):720–730. doi: 10.1093/genetics/37.6.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lessl M., Balzer D., Lurz R., Waters V. L., Guiney D. G., Lanka E. Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans. J Bacteriol. 1992 Apr;174(8):2493–2500. doi: 10.1128/jb.174.8.2493-2500.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lessl M., Balzer D., Pansegrau W., Lanka E. Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem. 1992 Oct 5;267(28):20471–20480. [PubMed] [Google Scholar]
- Lessl M., Balzer D., Weyrauch K., Lanka E. The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation. J Bacteriol. 1993 Oct;175(20):6415–6425. doi: 10.1128/jb.175.20.6415-6425.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lessl M., Krishnapillai V., Schilf W. Identification and characterization of two entry exclusion genes of the promiscuous IncP plasmid R18. Mol Gen Genet. 1991 May;227(1):120–126. doi: 10.1007/BF00260716. [DOI] [PubMed] [Google Scholar]
- Lu M. J., Henning U. Superinfection exclusion by T-even-type coliphages. Trends Microbiol. 1994 Apr;2(4):137–139. doi: 10.1016/0966-842x(94)90601-7. [DOI] [PubMed] [Google Scholar]
- Lyras D., Chan A. W., McFarlane J., Stanisich V. A. The surface exclusion system of RP1: investigation of the roles of trbJ and trbK in the surface exclusion, transfer, and slow-growth phenotypes. Plasmid. 1994 Nov;32(3):254–261. doi: 10.1006/plas.1994.1064. [DOI] [PubMed] [Google Scholar]
- Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
- Miele L., Strack B., Kruft V., Lanka E. Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751. DNA Seq. 1991;2(3):145–162. doi: 10.3109/10425179109039685. [DOI] [PubMed] [Google Scholar]
- Novick R. P. Extrachromosomal inheritance in bacteria. Bacteriol Rev. 1969 Jun;33(2):210–263. doi: 10.1128/br.33.2.210-263.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Olmsted S. B., Kao S. M., van Putte L. J., Gallo J. C., Dunny G. M. Role of the pheromone-inducible surface protein Asc10 in mating aggregate formation and conjugal transfer of the Enterococcus faecalis plasmid pCF10. J Bacteriol. 1991 Dec;173(23):7665–7672. doi: 10.1128/jb.173.23.7665-7672.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
- Pansegrau W., Balzer D., Kruft V., Lurz R., Lanka E. In vitro assembly of relaxosomes at the transfer origin of plasmid RP4. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6555–6559. doi: 10.1073/pnas.87.17.6555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pansegrau W., Lanka E., Barth P. T., Figurski D. H., Guiney D. G., Haas D., Helinski D. R., Schwab H., Stanisich V. A., Thomas C. M. Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. J Mol Biol. 1994 Jun 24;239(5):623–663. doi: 10.1006/jmbi.1994.1404. [DOI] [PubMed] [Google Scholar]
- Perumal N. B., Minkley E. G., Jr The product of the F sex factor traT surface exclusion gene is a lipoprotein. J Biol Chem. 1984 May 10;259(9):5357–5360. [PubMed] [Google Scholar]
- Pohlman R. F., Genetti H. D., Winans S. C. Entry exclusion of the IncN plasmid pKM101 is mediated by a single hydrophilic protein containing a lipid attachment motif. Plasmid. 1994 Mar;31(2):158–165. doi: 10.1006/plas.1994.1017. [DOI] [PubMed] [Google Scholar]
- Rees C. E., Bradley D. E., Wilkins B. M. Organization and regulation of the conjugation genes of IncI1 plasmid colIb-P9. Plasmid. 1987 Nov;18(3):223–236. doi: 10.1016/0147-619x(87)90065-5. [DOI] [PubMed] [Google Scholar]
- Riede I., Eschbach M. L. Evidence that TraT interacts with OmpA of Escherichia coli. FEBS Lett. 1986 Sep 15;205(2):241–245. doi: 10.1016/0014-5793(86)80905-x. [DOI] [PubMed] [Google Scholar]
- Sankaran K., Wu H. C. Lipid modification of bacterial prolipoprotein. Transfer of diacylglyceryl moiety from phosphatidylglycerol. J Biol Chem. 1994 Aug 5;269(31):19701–19706. [PubMed] [Google Scholar]
- Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schnaitman C. A. Examination of the protein composition of the cell envelope of Escherichia coli by polyacrylamide gel electrophoresis. J Bacteriol. 1970 Nov;104(2):882–889. doi: 10.1128/jb.104.2.882-889.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
- Skurray R. A., Hancock R. E., Reeves P. Con--mutants: class of mutants in Escherichia coli K-12 lacking a major cell wall protein and defective in conjugation and adsorption of a bacteriophage. J Bacteriol. 1974 Sep;119(3):726–735. doi: 10.1128/jb.119.3.726-735.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strack B., Lessl M., Calendar R., Lanka E. A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4. J Biol Chem. 1992 Jun 25;267(18):13062–13072. [PubMed] [Google Scholar]
- Sukupolvi S., O'Connor C. D. TraT lipoprotein, a plasmid-specified mediator of interactions between gram-negative bacteria and their environment. Microbiol Rev. 1990 Dec;54(4):331–341. doi: 10.1128/mr.54.4.331-341.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tokunaga M., Tokunaga H., Wu H. C. Post-translational modification and processing of Escherichia coli prolipoprotein in vitro. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2255–2259. doi: 10.1073/pnas.79.7.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waters V. L., Strack B., Pansegrau W., Lanka E., Guiney D. G. Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity. J Bacteriol. 1992 Oct;174(20):6666–6673. doi: 10.1128/jb.174.20.6666-6673.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weidlich G., Wirth R., Galli D. Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis. Mol Gen Genet. 1992 May;233(1-2):161–168. doi: 10.1007/BF00587575. [DOI] [PubMed] [Google Scholar]
- Willetts N., Maule J. Interactions between the surface exclusion systems of some F-like plasmids. Genet Res. 1974 Aug;24(1):81–89. doi: 10.1017/s0016672300015093. [DOI] [PubMed] [Google Scholar]
- Winans S. C., Walker G. C. Conjugal transfer system of the IncN plasmid pKM101. J Bacteriol. 1985 Jan;161(1):402–410. doi: 10.1128/jb.161.1.402-410.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winans S. C., Walker G. C. Entry exclusion determinant(s) of IncN plasmid pKM101. J Bacteriol. 1985 Jan;161(1):411–416. doi: 10.1128/jb.161.1.411-416.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamada Y., Yamada M., Nakazawa A. A ColE1-encoded gene directs entry exclusion of the plasmid. J Bacteriol. 1995 Nov;177(21):6064–6068. doi: 10.1128/jb.177.21.6064-6068.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamaguchi K., Yu F., Inouye M. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell. 1988 May 6;53(3):423–432. doi: 10.1016/0092-8674(88)90162-6. [DOI] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
- Ziegelin G., Pansegrau W., Strack B., Balzer D., Kröger M., Kruft V., Lanka E. Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4. DNA Seq. 1991;1(5):303–327. doi: 10.3109/10425179109020786. [DOI] [PubMed] [Google Scholar]
- de Maagd R. A., Lugtenberg B. Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol. 1986 Sep;167(3):1083–1085. doi: 10.1128/jb.167.3.1083-1085.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]