Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 May;179(10):3310–3316. doi: 10.1128/jb.179.10.3310-3316.1997

Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11.

M S Turner 1, P Timms 1, L M Hafner 1, P M Giffard 1
PMCID: PMC179112  PMID: 9150229

Abstract

Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an apparent molecular mass of 32 kDa. A clone encoding an immunoreactive 32-kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.

Full Text

The Full Text of this article is available as a PDF (815.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Atlan D., Gilbert C., Blanc B., Portalier R. Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397. Microbiology. 1994 Mar;140(Pt 3):527–535. doi: 10.1099/00221287-140-3-527. [DOI] [PubMed] [Google Scholar]
  3. Boot H. J., Kolen C. P., van Noort J. M., Pouwels P. H. S-layer protein of Lactobacillus acidophilus ATCC 4356: purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J Bacteriol. 1993 Oct;175(19):6089–6096. doi: 10.1128/jb.175.19.6089-6096.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fachon-Kalweit S., Elder B. L., Fives-Taylor P. Antibodies that bind to fimbriae block adhesion of Streptococcus sanguis to saliva-coated hydroxyapatite. Infect Immun. 1985 Jun;48(3):617–624. doi: 10.1128/iai.48.3.617-624.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fenno J. C., Shaikh A., Spatafora G., Fives-Taylor P. The fimA locus of Streptococcus parasanguis encodes an ATP-binding membrane transport system. Mol Microbiol. 1995 Mar;15(5):849–863. doi: 10.1111/j.1365-2958.1995.tb02355.x. [DOI] [PubMed] [Google Scholar]
  6. Ganeshkumar N., Song M., McBride B. C. Cloning of a Streptococcus sanguis adhesin which mediates binding to saliva-coated hydroxyapatite. Infect Immun. 1988 May;56(5):1150–1157. doi: 10.1128/iai.56.5.1150-1157.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Giffard P. M., Simpson C. L., Milward C. P., Jacques N. A. Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975. J Gen Microbiol. 1991 Nov;137(11):2577–2593. doi: 10.1099/00221287-137-11-2577. [DOI] [PubMed] [Google Scholar]
  8. Gilbert C., Atlan D., Blanc B., Portailer R., Germond J. E., Lapierre L., Mollet B. A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus. J Bacteriol. 1996 Jun;178(11):3059–3065. doi: 10.1128/jb.178.11.3059-3065.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holck A., Naes H. Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151. J Gen Microbiol. 1992 Jul;138(7):1353–1364. doi: 10.1099/00221287-138-7-1353. [DOI] [PubMed] [Google Scholar]
  10. Jenkinson H. F., Baker R. A., Tannock G. W. A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides. J Bacteriol. 1996 Jan;178(1):68–77. doi: 10.1128/jb.178.1.68-77.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jenkinson H. F. Cell surface protein receptors in oral streptococci. FEMS Microbiol Lett. 1994 Aug 15;121(2):133–140. doi: 10.1111/j.1574-6968.1994.tb07089.x. [DOI] [PubMed] [Google Scholar]
  12. Kolenbrander P. E., Andersen R. N., Ganeshkumar N. Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette. Infect Immun. 1994 Oct;62(10):4469–4480. doi: 10.1128/iai.62.10.4469-4480.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  14. Navarre W. W., Schneewind O. Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in gram-positive bacteria. Mol Microbiol. 1994 Oct;14(1):115–121. doi: 10.1111/j.1365-2958.1994.tb01271.x. [DOI] [PubMed] [Google Scholar]
  15. Nohno T., Saito T., Hong J. S. Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ). Mol Gen Genet. 1986 Nov;205(2):260–269. doi: 10.1007/BF00430437. [DOI] [PubMed] [Google Scholar]
  16. Pei Z. H., Ellison R. T., 3rd, Blaser M. J. Identification, purification, and characterization of major antigenic proteins of Campylobacter jejuni. J Biol Chem. 1991 Sep 5;266(25):16363–16369. [PubMed] [Google Scholar]
  17. Pei Z., Blaser M. J. PEB1, the major cell-binding factor of Campylobacter jejuni, is a homolog of the binding component in gram-negative nutrient transport systems. J Biol Chem. 1993 Sep 5;268(25):18717–18725. [PubMed] [Google Scholar]
  18. Rodriguez F., Grandi G. An operon encoding a novel ABC-type transport system in Bacillus subtilis. Microbiology. 1995 Jul;141(Pt 7):1781–1784. doi: 10.1099/13500872-141-7-1781. [DOI] [PubMed] [Google Scholar]
  19. Roos S., Aleljung P., Robert N., Lee B., Wadström T., Lindberg M., Jonsson H. A collagen binding protein from Lactobacillus reuteri is part of an ABC transporter system? FEMS Microbiol Lett. 1996 Oct 15;144(1):33–38. doi: 10.1111/j.1574-6968.1996.tb08505.x. [DOI] [PubMed] [Google Scholar]
  20. Rush C. M., Hafner L. M., Timms P. Genetic modification of a vaginal strain of Lactobacillus fermentum and its maintenance within the reproductive tract after intravaginal administration. J Med Microbiol. 1994 Oct;41(4):272–278. doi: 10.1099/00222615-41-4-272. [DOI] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sutcliffe I. C., Russell R. R. Lipoproteins of gram-positive bacteria. J Bacteriol. 1995 Mar;177(5):1123–1128. doi: 10.1128/jb.177.5.1123-1128.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tam R., Saier M. H., Jr Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol Rev. 1993 Jun;57(2):320–346. doi: 10.1128/mr.57.2.320-346.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Valdivia R. H., Wang L., Winans S. C. Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6. J Bacteriol. 1991 Oct;173(20):6398–6405. doi: 10.1128/jb.173.20.6398-6405.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vidgrén G., Palva I., Pakkanen R., Lounatmaa K., Palva A. S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence. J Bacteriol. 1992 Nov;174(22):7419–7427. doi: 10.1128/jb.174.22.7419-7427.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Zanker H., von Lintig J., Schröder J. Opine transport genes in the octopine (occ) and nopaline (noc) catabolic regions in Ti plasmids of Agrobacterium tumefaciens. J Bacteriol. 1992 Feb;174(3):841–849. doi: 10.1128/jb.174.3.841-849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES