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J Bacteriol. Dec 1991; 173(23): 7650–7664.
PMCID: PMC212534

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.

Abstract

Exposure of Enterococcus faecalis cells carrying the tetracycline resistance plasmid pCF10 to the heptapeptide pheromone cCF10 results in an increase in conjugal transfer frequency by as much as 10(6)-fold. Pheromone-induced donor cells also express at least two plasmid-encoded surface proteins, the 130-kDa Sec 10 protein, which is involved in surface exclusion, and the 150-kDa Asc10 protein, which has been associated with the formation of mating aggregates. Previous subcloning and transposon mutagenesis studies indicated that the adjacent EcoRI c (7.5 kb) and e (4.5 kb) fragments of pCF10 encode the structural genes for these proteins and that the EcoRI c fragment also encodes at least two regulatory genes involved in activation of the expression of the genes encoding Asc10 and Sec10. In this paper, the results of physical and genetic analysis of this region of pCF10, along with the complete DNA sequences of the EcoRI c and e fragments, are reported. The results of the genetic studies indicate the location of the structural genes for the surface proteins and reveal important features of their transcription. In addition, we provide evidence here and in the accompanying paper (S. B. Olmsted, S.-M. Kao, L. J. van Putte, J. C. Gallo, and G. M. Dunny, J. Bacteriol. 173:7665-7672, 1991) for a role of Asc10 in mating aggregate formation. The data also reveal a complex positive control system that acts at distances of at least 3 to 6 kb to activate expression of Asc10. DNA sequence analysis presented here reveals the positions of a number of specific genes, termed prg (pheromone-responsive genes) in this region of pCF10. The genes mapped include prgA (encoding Sec10) and prgB (encoding Asc10), as well as four putative regulatory genes, prgX, -R, -S, and -T. Although the predicted amino acid sequences of Sec10 and Asc10 have some structural features in common with a number of surface proteins of gram-positive cocci, and the Asc10 sequence is highly similar to that of a similar protein encoded by the pheromone-inducible plasmid pAD1 (D. Galli, F. Lottspeich, and R. Wirth, Mol. Microbiol. 4:895-904, 1990), the regulatory genes show relatively little resemblance to any previously sequenced genes from either procaryotes or eucaryotes.

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  • Adler B, Sasakawa C, Tobe T, Makino S, Komatsu K, Yoshikawa M. A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol Microbiol. 1989 May;3(5):627–635. [PubMed]
  • Anderson DG, McKay LL. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 1983 Sep;46(3):549–552. [PMC free article] [PubMed]
  • Achtman M, Kusećek B, Timmis KN. Tra cistrons and proteins encoded by the Escherichia coli antibiotic resistance plasmid R6-5. Mol Gen Genet. 1978 Jul 11;163(2):169–179. [PubMed]
  • Biggin MD, Gibson TJ, Hong GF. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. [PMC free article] [PubMed]
  • Brutlag DL, Dautricourt JP, Maulik S, Relph J. Improved sensitivity of biological sequence database searches. Comput Appl Biosci. 1990 Jul;6(3):237–245. [PubMed]
  • Chen CC, Cleary PP. Complete nucleotide sequence of the streptococcal C5a peptidase gene of Streptococcus pyogenes. J Biol Chem. 1990 Feb 25;265(6):3161–3167. [PubMed]
  • Chen EY, Seeburg PH. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. [PubMed]
  • Chen JD, Morrison DA. Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene. 1987;55(2-3):179–187. [PubMed]
  • Christie PJ, Dunny GM. Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions. Plasmid. 1986 May;15(3):230–241. [PubMed]
  • Christie PJ, Kao SM, Adsit JC, Dunny GM. Cloning and expression of genes encoding pheromone-inducible antigens of Enterococcus (Streptococcus) faecalis. J Bacteriol. 1988 Nov;170(11):5161–5168. [PMC free article] [PubMed]
  • Clewell DB, Pontius LT, An FY, Ike Y, Suzuki A, Nakayama J. Nucleotide sequence of the sex pheromone inhibitor (iAD1) determinant of Enterococcus faecalis conjugative plasmid pAD1. Plasmid. 1990 Sep;24(2):156–161. [PubMed]
  • Clewell DB, Weaver KE. Sex pheromones and plasmid transfer in Enterococcus faecalis. Plasmid. 1989 May;21(3):175–184. [PubMed]
  • Clewell DB, Yagi Y, Dunny GM, Schultz SK. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J Bacteriol. 1974 Jan;117(1):283–289. [PMC free article] [PubMed]
  • Doi RH, Wang LF. Multiple procaryotic ribonucleic acid polymerase sigma factors. Microbiol Rev. 1986 Sep;50(3):227–243. [PMC free article] [PubMed]
  • Dunny G, Funk C, Adsit J. Direct stimulation of the transfer of antibiotic resistance by sex pheromones in Streptococcus faecalis. Plasmid. 1981 Nov;6(3):270–278. [PubMed]
  • Dunny GM. Genetic functions and cell-cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis. Mol Microbiol. 1990 May;4(5):689–696. [PubMed]
  • Dunny GM, Brown BL, Clewell DB. Induced cell aggregation and mating in Streptococcus faecalis: evidence for a bacterial sex pheromone. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3479–3483. [PMC free article] [PubMed]
  • Dunny GM, Craig RA, Carron RL, Clewell DB. Plasmid transfer in Streptococcus faecalis: production of multiple sex pheromones by recipients. Plasmid. 1979 Jul;2(3):454–465. [PubMed]
  • Dunny GM, Lee LN, LeBlanc DJ. Improved electroporation and cloning vector system for gram-positive bacteria. Appl Environ Microbiol. 1991 Apr;57(4):1194–1201. [PMC free article] [PubMed]
  • Dunny GM, Zimmerman DL, Tortorello ML. 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. [PMC free article] [PubMed]
  • Ehrenfeld EE, Kessler RE, Clewell DB. Identification of pheromone-induced surface proteins in Streptococcus faecalis and evidence of a role for lipoteichoic acid in formation of mating aggregates. J Bacteriol. 1986 Oct;168(1):6–12. [PMC free article] [PubMed]
  • Freytag JW, Noelken ME, Hudson BG. Physical properties of collagen--sodium dodecyl sulfate complexes. Biochemistry. 1979 Oct 16;18(21):4761–4768. [PubMed]
  • Galli D, Lottspeich F, Wirth R. Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1. Mol Microbiol. 1990 Jun;4(6):895–904. [PubMed]
  • Galli D, Wirth R, Wanner G. Identification of aggregation substances of Enterococcus faecalis cells after induction by sex pheromones. An immunological and ultrastructural investigation. Arch Microbiol. 1989;151(6):486–490. [PubMed]
  • Guo LH, Yang RC, Wu R. An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid. Nucleic Acids Res. 1983 Aug 25;11(16):5521–5540. [PMC free article] [PubMed]
  • Guss B, Uhlén M, Nilsson B, Lindberg M, Sjöquist J, Sjödahl J. Region X, the cell-wall-attachment part of staphylococcal protein A. Eur J Biochem. 1984 Jan 16;138(2):413–420. [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Hollingshead SK, Fischetti VA, Scott JR. Complete nucleotide sequence of type 6 M protein of the group A Streptococcus. Repetitive structure and membrane anchor. J Biol Chem. 1986 Feb 5;261(4):1677–1686. [PubMed]
  • Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. [PubMed]
  • Hynes RO. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. [PubMed]
  • Kessler RE, Yagi Y. Identification and partial characterization of a pheromone-induced adhesive surface antigen of Streptococcus faecalis. J Bacteriol. 1983 Aug;155(2):714–721. [PMC free article] [PubMed]
  • Kornitzer D, Teff D, Altuvia S, Oppenheim AB. Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation. J Bacteriol. 1989 May;171(5):2563–2572. [PMC free article] [PubMed]
  • Krejci E, Garzino V, Mary C, Bennani N, Pradel J. Modulo, a new maternally expressed Drosophila gene encodes a DNA-binding protein with distinct acidic and basic regions. Nucleic Acids Res. 1989 Oct 25;17(20):8101–8115. [PMC free article] [PubMed]
  • Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Mori M, Sakagami Y, Ishii Y, Isogai A, Kitada C, Fujino M, Adsit JC, Dunny GM, Suzuki A. Structure of cCF10, a peptide sex pheromone which induces conjugative transfer of the Streptococcus faecalis tetracycline resistance plasmid, pCF10. J Biol Chem. 1988 Oct 5;263(28):14574–14578. [PubMed]
  • Nakayama J, Nagasawa H, Isogai A, Clewell DB, Suzuki A. Amino acid sequence of pheromone-inducible surface protein in Enterococcus faecalis, that is encoded on the conjugative plasmid pPD1. FEBS Lett. 1990 Jul 2;267(1):81–84. [PubMed]
  • Oda M, Sugishita A, Furukawa K. Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon. J Bacteriol. 1988 Jul;170(7):3199–3205. [PMC free article] [PubMed]
  • Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol. 1985;39:615–648. [PubMed]
  • Olmsted SB, Kao SM, van Putte LJ, Gallo JC, Dunny GM. 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. [PMC free article] [PubMed]
  • Pancholi V, Fischetti VA. Isolation and characterization of the cell-associated region of group A streptococcal M6 protein. J Bacteriol. 1988 Jun;170(6):2618–2624. [PMC free article] [PubMed]
  • Riede I, Eschbach ML. Evidence that TraT interacts with OmpA of Escherichia coli. FEBS Lett. 1986 Sep 15;205(2):241–245. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Shine J, Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. [PMC free article] [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Swinfield TJ, Oultram JD, Thompson DE, Brehm JK, Minton NP. Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1. Gene. 1990 Mar 1;87(1):79–90. [PubMed]
  • Tabor S, Richardson CC. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. [PMC free article] [PubMed]
  • Tinoco I, Jr, Borer PN, Dengler B, Levin MD, Uhlenbeck OC, Crothers DM, Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. [PubMed]
  • Tortorello ML, Dunny GM. Identification of multiple cell surface antigens associated with the sex pheromone response of Streptococcus faecalis. J Bacteriol. 1985 Apr;162(1):131–137. [PMC free article] [PubMed]
  • Uchimura T, Lau PC. Nucleotide sequences from the colicin E8 operon: homology with plasmid ColE2-P9. Mol Gen Genet. 1987 Oct;209(3):489–493. [PubMed]
  • Wanner G, Formanek H, Galli D, Wirth R. Localization of aggregation substances of Enterococcus faecalis after induction by sex pheromones. An ultrastructural comparison using immuno labelling, transmission and high resolution scanning electron microscopic techniques. Arch Microbiol. 1989;151(6):491–497. [PubMed]
  • Watson ME. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. [PMC free article] [PubMed]
  • Wirth R, An FY, Clewell DB. Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector. J Bacteriol. 1986 Mar;165(3):831–836. [PMC free article] [PubMed]
  • Yagi Y, Kessler RE, Shaw JH, Lopatin DE, An F, Clewell DB. Plasmid content of Streptococcus faecalis strain 39-5 and identification of a pheromone (cPD1)-induced surface antigen. J Gen Microbiol. 1983 Apr;129(4):1207–1215. [PubMed]
  • 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. [PubMed]

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