Applied Microbiology and Biotechnology

, Volume 97, Issue 5, pp 1941–1952 | Cite as

Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen

  • Zhilan Sun
  • Jian KongEmail author
  • Shumin Hu
  • Wentao Kong
  • Wenwei Lu
  • Wei Liu
Biotechnologically relevant enzymes and proteins


It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB380–501 was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB1–379 peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus.


Lactobacillus crispatus S-layer·cell wall fragments Extracellular matrix protein Enzyme-linked immunosorbent assay 



We would like to thank M. van de Guchte and S. Hazebrouck for their generous gifts of L. delbrueckii subsp. bulgaricus ATCC 11842 and L. casei BL23, respectively. This research was supported by the National Natural Science Foundation of China (31070091), and the 863 Hi-Tech Research and Development Program of China (2011AA100902).


  1. Abbot EL, Smith WD, Siou GP, Chiriboga C, Smith RJ, Wilson JA, Hirst BH, Kehoe MA (2007) Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin. Cell Microbiol 9:1822–1833CrossRefGoogle Scholar
  2. Ames BN, Dubin DT (1960) The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem 235:769–775Google Scholar
  3. Antikainen J, Anton L, Sillanpaa J, Korhonen TK (2002) Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly. Mol Microbiol 46:381–394CrossRefGoogle Scholar
  4. Avall-Jaaskelainen S, Hynonen U, Ilk N, Pum D, Sleytr UB, Palva A (2008) Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287. BMC Microbiol 8:165CrossRefGoogle Scholar
  5. Avall-Jaaskelainen S, Palva A (2005) Lactobacillus surface layers and their applications. FEMS Microbiol Rev 29:511–529CrossRefGoogle Scholar
  6. Barbosa AS, Abreu PA, Neves FO, Atzingen MV, Watanabe MM, Vieira ML, Morais ZM, Vasconcellos SA, Nascimento AL (2006) A newly identified leptospiral adhesin mediates attachment to laminin. Infect Immun 74:6356–6364CrossRefGoogle Scholar
  7. Boot HJ, Kolen CP, Andreadaki FJ, Leer RJ, Pouwels PH (1996a) The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA. J Bacteriol 178:5388–5394Google Scholar
  8. Boot HJ, Kolen CP, Pot B, Kersters K, Pouwels PH (1996b) The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus. Microbiology 142(Pt 9):2375–2384CrossRefGoogle Scholar
  9. Boot HJ, Kolen CP, van Noort JM, Pouwels PH (1993) S-layer protein of Lactobacillus acidophilus ATCC 4356: purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J Bacteriol 175:6089–6096Google Scholar
  10. Buck BL, Altermann E, Svingerud T, Klaenhammer TR (2005) Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 71:8344–8351CrossRefGoogle Scholar
  11. Callegari ML, Riboli B, Sanders JW, Cocconcelli PS, Kok J, Venema G, Morelli L (1998) The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression. Microbiology 144(Pt 3):719–726CrossRefGoogle Scholar
  12. Chen X, Xu J, Shuai J, Chen J, Zhang Z, Fang W (2007) The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium. Int J Food Microbiol 115:307–312CrossRefGoogle Scholar
  13. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386–401CrossRefGoogle Scholar
  14. Goh YJ, Azcarate-Peril MA, O'Flaherty S, Durmaz E, Valence F, Jardin J, Lortal S, Klaenhammer TR (2009) Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol 75:3093–3105CrossRefGoogle Scholar
  15. Golowczyc MA, Mobili P, Garrote GL, Abraham AG, De Antoni GL (2007) Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis. Int J Food Microbiol 118:264–273CrossRefGoogle Scholar
  16. Hagen KE, Guan LL, Tannock GW, Korver DR, Allison GE (2005) Detection, characterization, and in vitro and in vivo expression of genes encoding S-layer proteins in Lactobacillus gallinarum strains isolated from chicken crops. Appl Environ Microbiol 71:6633–6643CrossRefGoogle Scholar
  17. Hazebrouck S, Pothelune L, Azevedo V, Corthier G, Wal JM, Langella P (2007) Efficient production and secretion of bovine beta-lactoglobulin by Lactobacillus casei. Microb Cell Fact 6:6–1CrossRefGoogle Scholar
  18. Horie M, Kajikawa HS, Toba T (2002) Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene. Lett Appl Microbiol 35:57–61CrossRefGoogle Scholar
  19. Huber C, Ilk N, Runzler D, Egelseer EM, Weigert S, Sleytr UB, Sara M (2005) The three S-layer-like homology motifs of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 are not sufficient for binding to the pyruvylated secondary cell wall polymer. Mol Microbiol 55:197–205CrossRefGoogle Scholar
  20. Hynonen U, Avall-Jaaskelainen S, Palva A (2010) Characterization and separate activities of the two promoters of the Lactobacillus brevis S-layer protein gene. Appl Microbiol Biotechnol 87:657–668CrossRefGoogle Scholar
  21. Hynonen U, Westerlund-Wikstrom B, Palva A, Korhonen TK (2002) Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis. J Bacteriol 184:3360–3367CrossRefGoogle Scholar
  22. Khang YH, Park HY, Jeong YS, Kim JA, Kim YH (2009) Recombinant S-layer proteins of Lactobacillus brevis mediating antibody adhesion to calf intestine alleviated neonatal diarrhea syndrome. J Microbiol Biotechnol 19:511–519CrossRefGoogle Scholar
  23. Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681CrossRefGoogle Scholar
  24. Mercenier A, Muller-Alouf H, Grangette C (2000) Lactic acid bacteria as live vaccines. Curr Issues Mol Biol 2:17–25Google Scholar
  25. Narita J, Ishida S, Okano K, Kimura S, Fukuda H, Kondo A (2006) Improvement of protein production in lactic acid bacteria using 5′-untranslated leader sequence of slpA from Lactobacillus acidophilus. Appl Microbiol Biotechnol 73:366–373CrossRefGoogle Scholar
  26. Oozeer R, Furet JP, Goupil-Feuillerat N, Anba J, Mengaud J, Corthier G (2005) Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice. Appl Environ Microbiol 71:1356–1363CrossRefGoogle Scholar
  27. Ribeiro LA, Azevedo V, Le Loir Y, Oliveira SC, Dieye Y, Piard JC, Gruss A, Langella P (2002) Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis. Appl Environ Microbiol 68:910–916CrossRefGoogle Scholar
  28. Sawa N, Zendo T, Kiyofuji J, Fujita K, Himeno K, Nakayama J, Sonomoto K (2009) Identification and characterization of lactocyclicin Q, a novel cyclic bacteriocin produced by Lactococcus sp. strain QU 12. Appl Environ Microbiol 75:1552–1558CrossRefGoogle Scholar
  29. Sillanpaa J, Martinez B, Antikainen J, Toba T, Kalkkinen N, Tankka S, Lounatmaa K, Keranen J, Hook M, Westerlund-wikstrom B, Pouwels PH, Korhonen TK (2000) Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus. J Bacteriol 182:6440–6450CrossRefGoogle Scholar
  30. Sleytr UB, Beveridge TJ (1999) Bacterial S-layers. Trends Microbiol 7:253–260CrossRefGoogle Scholar
  31. Smit E, Oling F, Demel R, Martinez B, Pouwels PH (2001) The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J Mol Biol 305:245–257CrossRefGoogle Scholar
  32. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E (2000) Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289:1352–1355CrossRefGoogle Scholar
  33. Sun Z, Kong J, Kong W (2010) Characterization of a cryptic plasmid pD403 from Lactobacillus plantarum and construction of shuttle vectors based on its replicon. Mol Biotechnol 45:24–33CrossRefGoogle Scholar
  34. Toba T, Virkola R, Westerlund B, Bjorkman Y, Sillanpaa J, Vartio T, Kalkkinen N, Korhonen TK (1995) A collagen-binding S-Layer protein in Lactobacillus crispatus. Appl Environ Microbiol 61:2467–2471Google Scholar
  35. Van de Guchte M, Penaud S, Grimaldi C, Barbe V, Bryson K, Nicolas P, Robert C, Oztas S, Mangenot S, Couloux A, Loux V, Dervyn R, Bossy R, Bolotin A, Batto JM, Walunas T, Gibrat JF, Bessières P, Weissenbach J, Ehrlich SD, Maguin E (2006) The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci U S A 103:9274–9279CrossRefGoogle Scholar
  36. Walter J, Heng NC, Hammes WP, Loach DM, Tannock GW, Hertel C (2003) Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract. Appl Environ Microbiol 69:2044–2051CrossRefGoogle Scholar
  37. Westerlund B, Korhonen TK (1993) Bacterial proteins binding to the mammalian extracellular matrix. Mol Microbiol 9:687–694CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Zhilan Sun
    • 1
  • Jian Kong
    • 1
    Email author
  • Shumin Hu
    • 1
    • 2
  • Wentao Kong
    • 1
  • Wenwei Lu
    • 1
  • Wei Liu
    • 1
  1. 1.State Key Laboratory of Microbial TechnologyShandong UniversityJinanPeople’s Republic of China
  2. 2.Scientific Research CenterTsingtao Brewery Co.LTDQingdaoPeople’s Republic of China

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