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J Bacteriol. Mar 1996; 178(6): 1680–1690.
PMCID: PMC177854

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.


We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bastin DA, Stevenson G, Brown PK, Haase A, Reeves PR. Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length. Mol Microbiol. 1993 Mar;7(5):725–734. [PubMed]
  • Batchelor RA, Alifano P, Biffali E, Hull SI, Hull RA. Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation. J Bacteriol. 1992 Aug;174(16):5228–5236. [PMC free article] [PubMed]
  • Becker A, Kleickmann A, Keller M, Arnold W, Pühler A. Identification and analysis of the Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONP fragment. Mol Gen Genet. 1993 Nov;241(3-4):367–379. [PubMed]
  • Becker A, Niehaus K, Pühler A. Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain. Mol Microbiol. 1995 Apr;16(2):191–203. [PubMed]
  • Brown PK, Romana LK, Reeves PR. Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B. Mol Microbiol. 1992 May;6(10):1385–1394. [PubMed]
  • Cerning J. Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol Rev. 1990 Sep;7(1-2):113–130. [PubMed]
  • Caparon MG, Scott JR. Excision and insertion of the conjugative transposon Tn916 involves a novel recombination mechanism. Cell. 1989 Dec 22;59(6):1027–1034. [PubMed]
  • Collins LV, Hackett J. Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium. J Bacteriol. 1991 Apr;173(8):2521–2529. [PMC free article] [PubMed]
  • Dao ML, Ferretti JJ. Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptococcal genes. Appl Environ Microbiol. 1985 Jan;49(1):115–119. [PMC free article] [PubMed]
  • de Vos WM, Vos P, de Haard H, Boerrigter I. Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase. Gene. 1989 Dec 21;85(1):169–176. [PubMed]
  • Doco T, Wieruszeski JM, Fournet B, Carcano D, Ramos P, Loones A. Structure of an exocellular polysaccharide produced by Streptococcus thermophilus. Carbohydr Res. 1990 May 1;198(2):313–321. [PubMed]
  • Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. [PMC free article] [PubMed]
  • Downie JA. The nodL gene from Rhizobium leguminosarum is homologous to the acetyl transferases encoded by lacA and cysE. Mol Microbiol. 1989 Nov;3(11):1649–1651. [PubMed]
  • Fath MJ, Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. [PMC free article] [PubMed]
  • Frosch M, Müller A. Phospholipid substitution of capsular polysaccharides and mechanisms of capsule formation in Neisseria meningitidis. Mol Microbiol. 1993 May;8(3):483–493. [PubMed]
  • Gagnon Y, Breton R, Putzer H, Pelchat M, Grunberg-Manago M, Lapointe J. Clustering and co-transcription of the Bacillus subtilis genes encoding the aminoacyl-tRNA synthetases specific for glutamate and for cysteine and the first enzyme for cysteine biosynthesis. J Biol Chem. 1994 Mar 11;269(10):7473–7482. [PubMed]
  • Gasson MJ. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. [PMC free article] [PubMed]
  • Gawron-Burke C, Clewell DB. A transposon in Streptococcus faecalis with fertility properties. Nature. 1982 Nov 18;300(5889):281–284. [PubMed]
  • Germond JE, Lapierre L, Delley M, Mollet B. A new mobile genetic element in Lactobacillus delbrueckii subsp. bulgaricus. Mol Gen Genet. 1995 Aug 30;248(4):407–416. [PubMed]
  • Glucksmann MA, Reuber TL, Walker GC. Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis. J Bacteriol. 1993 Nov;175(21):7045–7055. [PMC free article] [PubMed]
  • Gruter M, Leeflang BR, Kuiper J, Kamerling JP, Vliegenthart JF. Structure of the exopolysaccharide produced by Lactococcus lactis subspecies cremoris H414 grown in a defined medium or skimmed milk. Carbohydr Res. 1992 Jul 2;231:273–291. [PubMed]
  • Gruter M, Leeflang BR, Kuiper J, Kamerling JP, Vliegenthart JF. Structural characterisation of the exopolysaccharide produced by Lactobacillus delbrückii subspecies bulgaricus rr grown in skimmed milk. Carbohydr Res. 1993 Feb 1;239:209–226. [PubMed]
  • Guédon G, Bourgoin F, Pébay M, Roussel Y, Colmin C, Simonet JM, Decaris B. Characterization and distribution of two insertion sequences, IS1191 and iso-IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co-cultures? Mol Microbiol. 1995 Apr;16(1):69–78. [PubMed]
  • Guidolin A, Morona JK, Morona R, Hansman D, Paton JC. Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F. Infect Immun. 1994 Dec;62(12):5384–5396. [PMC free article] [PubMed]
  • Hediger MA, Johnson DF, Nierlich DP, Zabin I. DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6414–6418. [PMC free article] [PubMed]
  • Hershfield MS, Chaffee S, Koro-Johnson L, Mary A, Smith AA, Short SA. Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7185–7189. [PMC free article] [PubMed]
  • Hill C, Venema G, Daly C, Fitzgerald GF. Cloning and characterization of the tetracycline resistance determinant of and several promoters from within the conjugative transposon Tn919. Appl Environ Microbiol. 1988 May;54(5):1230–1236. [PMC free article] [PubMed]
  • Hutkins R, Morris HA, McKay LL. Galactokinase activity in Streptococcus thermophilus. Appl Environ Microbiol. 1985 Oct;50(4):777–780. [PMC free article] [PubMed]
  • Israelsen H, Hansen EB. Insertion of Transposon Tn917 Derivatives into the Lactococcus lactis subsp. lactis Chromosome. Appl Environ Microbiol. 1993 Jan;59(1):21–26. [PMC free article] [PubMed]
  • Jiang XM, Neal B, Santiago F, Lee SJ, Romana LK, Reeves PR. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. [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]
  • Kröncke KD, Boulnois G, Roberts I, Bitter-Suermann D, Golecki JR, Jann B, Jann K. Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli. J Bacteriol. 1990 Feb;172(2):1085–1091. [PMC free article] [PubMed]
  • Lazarevic V, Margot P, Soldo B, Karamata D. Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier. J Gen Microbiol. 1992 Sep;138(9):1949–1961. [PubMed]
  • Lee SJ, Romana LK, Reeves PR. Sequence and structural analysis of the rfb (O antigen) gene cluster from a group C1 Salmonella enterica strain. J Gen Microbiol. 1992 Sep;138(9):1843–1855. [PubMed]
  • Lin WS, Cunneen T, Lee CY. Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus. J Bacteriol. 1994 Nov;176(22):7005–7016. [PMC free article] [PubMed]
  • Liu D, Lindqvist L, Reeves PR. Transferases of O-antigen biosynthesis in Salmonella enterica: dideoxyhexosyltransferases of groups B and C2 and acetyltransferase of group C2. J Bacteriol. 1995 Jul;177(14):4084–4088. [PMC free article] [PubMed]
  • MacLachlan PR, Kadam SK, Sanderson KE. Cloning, characterization, and DNA sequence of the rfaLK region for lipopolysaccharide synthesis in Salmonella typhimurium LT2. J Bacteriol. 1991 Nov;173(22):7151–7163. [PMC free article] [PubMed]
  • Mollet B, Delley M. A beta-galactosidase deletion mutant of Lactobacillus bulgaricus reverts to generate an active enzyme by internal DNA sequence duplication. Mol Gen Genet. 1991 May;227(1):17–21. [PubMed]
  • Mollet B, Knol J, Poolman B, Marciset O, Delley M. Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus. J Bacteriol. 1993 Jul;175(14):4315–4324. [PMC free article] [PubMed]
  • Morona R, Mavris M, Fallarino A, Manning PA. Characterization of the rfc region of Shigella flexneri. J Bacteriol. 1994 Feb;176(3):733–747. [PMC free article] [PubMed]
  • Morona R, van den Bosch L, Manning PA. Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri. J Bacteriol. 1995 Feb;177(4):1059–1068. [PMC free article] [PubMed]
  • Müller P, Keller M, Weng WM, Quandt J, Arnold W, Pühler A. Genetic analysis of the Rhizobium meliloti exoYFQ operon: ExoY is homologous to sugar transferases and ExoQ represents a transmembrane protein. Mol Plant Microbe Interact. 1993 Jan-Feb;6(1):55–65. [PubMed]
  • Nakajima H, Hirota T, Toba T, Itoh T, Adachi S. Structure of the extracellular polysaccharide from slime-forming Lactococcus lactis subsp. cremoris SBT 0495. Carbohydr Res. 1992 Feb 7;224:245–253. [PubMed]
  • Neeser JR, Schweizer TF. A quantitative determination by capillary gas-liquid chromatography of neutral and amino sugars (as O-methyloxime acetates), and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries. Anal Biochem. 1984 Oct;142(1):58–67. [PubMed]
  • Neve H, Geis A, Teuber M. Plasmid-encoded functions of ropy lactic acid streptococcal strains from Scandinavian fermented milk. Biochimie. 1988 Mar;70(3):437–442. [PubMed]
  • Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. [PMC free article] [PubMed]
  • Poolman B, Royer TJ, Mainzer SE, Schmidt BF. Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of phosphoenolpyruvate-dependent phosphotransferase systems. J Bacteriol. 1989 Jan;171(1):244–253. [PMC free article] [PubMed]
  • Poolman B, Royer TJ, Mainzer SE, Schmidt BF. Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase. J Bacteriol. 1990 Jul;172(7):4037–4047. [PMC free article] [PubMed]
  • Reeves P. Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet. 1993 Jan;9(1):17–22. [PubMed]
  • Reuber TL, Walker GC. Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell. 1993 Jul 30;74(2):269–280. [PubMed]
  • Robijn GW, Thomas JR, Haas H, van den Berg DJ, Kamerling JP, Vliegenthart JF. The structure of the exopolysaccharide produced by Lactobacillus helveticus 766. Carbohydr Res. 1995 Oct 16;276(1):137–154. [PubMed]
  • Robijn GW, van den Berg DJ, Haas H, Kamerling JP, Vliegenthart JF. Determination of the structure of the exopolysaccharide produced by Lactobacillus sake 0-1. Carbohydr Res. 1995 Oct 16;276(1):117–136. [PubMed]
  • Romero DA, Klaenhammer TR. IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1992 Feb;58(2):699–702. [PMC free article] [PubMed]
  • Roussel Y, Pebay M, Guedon G, Simonet JM, Decaris B. Physical and genetic map of Streptococcus thermophilus A054. J Bacteriol. 1994 Dec;176(24):7413–7422. [PMC free article] [PubMed]
  • Rubens CE, Heggen LM, Haft RF, Wessels MR. Identification of cpsD, a gene essential for type III capsule expression in group B streptococci. Mol Microbiol. 1993 May;8(5):843–855. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Slos P, Bourquin JC, Lemoine Y, Mercenier A. Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus. Appl Environ Microbiol. 1991 May;57(5):1333–1339. [PMC free article] [PubMed]
  • Stevenson G, Neal B, Liu D, Hobbs M, Packer NH, Batley M, Redmond JW, Lindquist L, Reeves P. Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. J Bacteriol. 1994 Jul;176(13):4144–4156. [PMC free article] [PubMed]
  • Stingele F, Mollet B. Homologous integration and transposition to identify genes involved in the production of exopolysaccharides in Streptococcus thermophilus. Dev Biol Stand. 1995;85:487–493. [PubMed]
  • Thomas TD, Crow VL. Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures. Appl Environ Microbiol. 1984 Jul;48(1):186–191. [PMC free article] [PubMed]
  • Triglia T, Peterson MG, Kemp DJ. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988 Aug 25;16(16):8186–8186. [PMC free article] [PubMed]
  • Vedamuthu ER, Neville JM. Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS. Appl Environ Microbiol. 1986 Apr;51(4):677–682. [PMC free article] [PubMed]
  • von Wright A, Tynkkynen S. Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype. Appl Environ Microbiol. 1987 Jun;53(6):1385–1386. [PMC free article] [PubMed]
  • Wang L, Reeves PR. Involvement of the galactosyl-1-phosphate transferase encoded by the Salmonella enterica rfbP gene in O-antigen subunit processing. J Bacteriol. 1994 Jul;176(14):4348–4356. [PMC free article] [PubMed]
  • Yamamoto Y, Murosaki S, Yamauchi R, Kato K, Sone Y. Structural study on an exocellular polysaccharide produced by Lactobacillus helveticus TY1-2. Carbohydr Res. 1994 Aug 3;261(1):67–78. [PubMed]
  • Yao Z, Valvano MA. Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a. J Bacteriol. 1994 Jul;176(13):4133–4143. [PMC free article] [PubMed]

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