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Volume 148, Issue 6

Organization and characterization of the capsule biosynthesis locus of serotype 9V

The GenBank accession number for the sequence reported in this paper is AF402095.

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Abstract

The capsular polysaccharide (CPS) synthesis locus of serotype 9V was amplified by long-range PCR and sequenced. The locus was 17368 bp in size and contained 15 ORFs. The genetic organization of the cluster shared many features with other capsule loci, including the presence of four putative regulatory genes at the 5′ end. Comparative sequence analyses allowed putative functions to be assigned to each of the gene products. The ORFs appeared to encode, besides the four regulatory genes, five glycosyltransferases, two -acetyltransferases, an -acetylglucosamine 2-epimerase, a glucose 6-dehydrogenase, an oligosaccharide transporter protein and a polysaccharide repeating unit polymerase. These functions covered the steps proposed in the CPS biosynthesis of serotype 9V. TLC of carbohydrate intermediates formed after incubation of bacterial membrane preparations with C-labelled precursors demonstrated that the fifth ORF () encoded a UDP-glucosyl-1-phosphate transferase. This function was confirmed with the help of a mutant that carried a deletion of a guanine residue located adjacent to a stretch of adenines. The identification and characterization of the serotype 9V locus is a major step in unravelling the 9V capsule biosynthesis pathway and broadens the insight into the genetic diversity of the capsule loci.

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Keyword(s): capsular polysaccharide biosynthesis , CPS, capsular polysaccharide and glucosyltransferase
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2002-06-01
2024-04-16
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References

  1. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
     [Google Scholar]
  2. Arrecubieta C., Garcia E., Lopez R. 1995; Sequence and transcriptional analysis of a DNA region involved in the production of capsular polysaccharide in Streptococcus pneumoniae type 3. Gene 167:1–7 [CrossRef]
     [Google Scholar]
  3. Arrecubieta C., Garcia E., Lopez R. 1996; Demonstration of UDP-glucose dehydrogenase activity in cell extracts of Escherichia coli expressing the pneumococcal cap3A gene required for the synthesis of type 3 capsular polysaccharide. J Bacteriol 178:2971–2974
     [Google Scholar]
  4. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Smith J. A., Seidman J. G., Struhl K. 1987 Current Protocols in Molecular Biology New York: Wiley;
     [Google Scholar]
  5. Barnes D. M., Whittier S., Gilligan P. H., Soares S., Tomasz A., Henderson F. W. 1995; Transmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence suggesting capsular transformation of the resistant strain in vivo . J Infect Dis 171:890–896 [CrossRef]
     [Google Scholar]
  6. Bender M. H., Yother J. 2001; CpsB is a modulator of capsule-associated tyrosine kinase activity in Streptococcus pneumoniae . J Biol Chem 276:47966–47974
     [Google Scholar]
  7. Butler J. C. 1997; Epidemiology of pneumococcal serotypes and conjugate vaccine formulations. Microb Drug Resist 3:125–129 [CrossRef]
     [Google Scholar]
  8. Butler J. C., Shapiro E. D., Carlone G. M. 1999; Pneumococcal vaccines: history, current status, and future directions. Am J Med 107:69S–76S [CrossRef]
     [Google Scholar]
  9. Campbell R. E., Mosimann S. C., Tanner M. E., Strynadka N. C. J. 2000a; The structure of UDP- N -acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases. Biochemistry 39:14993–15001 [CrossRef]
     [Google Scholar]
  10. Campbell R. E., Mosimann S. C., van De Rijn I., Tanner M. E., Strynadka N. C. J. 2000b; The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation. Biochemistry 39:7012–7023 [CrossRef]
     [Google Scholar]
  11. Charpentier E., Tuomanen E. 2000; Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae . Microbes Infect 2:1855–1864 [CrossRef]
     [Google Scholar]
  12. Cieslewicz M. J., Kasper D. L., Wang Y., Wessels M. R. 2001; Functional analysis in type Ia group B Streptococcus of a cluster of genes involved in extracellular polysaccharide production by diverse species of streptococci. J Biol Chem 276:139–146 [CrossRef]
     [Google Scholar]
  13. Coffey T. J., Dowson C. G., Daniels M., Zhou J., Martin C., Spratt B. G., Musser J. M. 1991; Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae . Mol Microbiol 5:2255–2260 [CrossRef]
     [Google Scholar]
  14. Coffey T. J., Enright M. C., Daniels M., Morona J. K., Morona R., Hryniewicz W., Paton J. C., Spratt B. G. 1998a; Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae . Mol Microbiol 27:73–83 [CrossRef]
     [Google Scholar]
  15. Coffey T. J., Enright M. C., Daniels M., Wilkinson P., Berron S., Fenoll A., Spratt B. G. 1998b; Serotype 19A variants of the Spanish serotype 23F multiresistant clone of Streptococcus pneumoniae . Microb Drug Resist 4:51–55 [CrossRef]
     [Google Scholar]
  16. Cole S. T., Brosch R., Parkhill J. 39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
     [Google Scholar]
  17. Dillard J. P., Vandersea M. W., Yother J. 1995; Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae . J Exp Med 181:973–983 [CrossRef]
     [Google Scholar]
  18. Griffith F. M. B. 1928; The significance of pneumococcal types. J Hyg 27:8–159
     [Google Scholar]
  19. Guidolin A., Morona J. K., Morona R., Hansman D., Paton J. C. 1994; Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F. Infect Immun 62:5384–5396
     [Google Scholar]
  20. Hausdorff W. P., Bryant J., Paradiso P. R., Siber G. R. 2000; Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I. Clin Infect Dis 30:100–121 [CrossRef]
     [Google Scholar]
  21. Iannelli F., Pearce B. J., Pozzi G. 1999; The type 2 capsule locus of Streptococcus pneumoniae . J Bacteriol 181:2652–2654
     [Google Scholar]
  22. Jiang S. M., Wang L., Reeves P. R. 2001; Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters. Infect Immun 69:1244–1255 [CrossRef]
     [Google Scholar]
  23. Kamerling J. P. 1999; Pneumococcal polysaccharides: a chemical view. In Streptococcus pneumoniae: Molecular Biology and Mechanisms of Disease pp 81–114 Edited by Tomasz A. Larchmont, NY: Mary Ann Liebert;
     [Google Scholar]
  24. Kolkman M. A. B., Morrison D. A., Van Der Zeijst B. A. M., Nuijten P. J. M. 1996; The capsule polysaccharide synthesis locus of Streptococcus pneumoniae serotype 14: identification of the glycosyl transferase gene cps14E . J Bacteriol 178:3736–3741
     [Google Scholar]
  25. Kolkman M. A. B., van der Zeijst B. A. M., Nuijten P. J. M. 1997a; Functional analysis of glycosyltransferases encoded by the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae serotype 14. J Biol Chem 272:19502–19508 [CrossRef]
     [Google Scholar]
  26. Kolkman M. A. B., Wakarchuk W., Nuijten P. J. M., van der Zeijst B. A. M. 1997b; Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit. Mol Microbiol 26:197–208 [CrossRef]
     [Google Scholar]
  27. Llull D., Lopez R., Garcia E., Munoz R. 1998; Molecular structure of the gene cluster responsible for the synthesis of the polysaccharide capsule of Streptococcus pneumoniae type 33F. Biochim Biophys Acta 1443217–224 [CrossRef]
     [Google Scholar]
  28. Llull D., Munoz R., Lopez R., Garcia E. 1999; A single gene ( tts ) located outside the cap locus directs the formation of Streptococcus pneumoniae type 37 capsular polysaccharide. Type 37 pneumococci are natural, genetically binary strains. J Exp Med 190:241–251 [CrossRef]
     [Google Scholar]
  29. Mollerach M., Lopez R., Garcia E. 1998; Characterization of the galU gene of Streptococcus pneumoniae encoding a uridine diphosphoglucose pyrophosphorylase: a gene essential for capsular polysaccharide biosynthesis. J Exp Med 188:2047–2056 [CrossRef]
     [Google Scholar]
  30. Morona J. K., Morona R., Paton J. C. 1997a; Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway. Mol Microbiol 23:751–763 [CrossRef]
     [Google Scholar]
  31. Morona J. K., Morona R., Paton J. C. 1997b; Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 19B. J Bacteriol 179:4953–4958
     [Google Scholar]
  32. Morona J. K., Miller D. C., Coffey T. J., Vindurampulle C. J., Spratt B. G., Morona R., Paton J. C. 1999a; Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 23F. Microbiology 145:781–789 [CrossRef]
     [Google Scholar]
  33. Morona J. K., Morona R., Paton J. C. 1999b; Analysis of the 5′ portion of the type 19A capsule locus identifies two classes of cpsC , cpsD , and cpsE genes in Streptococcus pneumoniae . J Bacteriol 181:3599–3605
     [Google Scholar]
  34. Morona J. K., Morona R., Paton J. C. 1999c; Comparative genetics of capsular polysaccharide biosynthesis in Streptococcus pneumoniae types belonging to serogroup 19. J Bacteriol 181:5355–5364
     [Google Scholar]
  35. Morona J. K., Paton J. C., Miller D. C., Morona R. 2000; Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae . Mol Microbiol 35:1431–1442
     [Google Scholar]
  36. Mulholland K. 1999; Strategies for the control of pneumococcal diseases. Vaccine 17:S79–S84 [CrossRef]
     [Google Scholar]
  37. Munoz R., Mollerach M., Lopez R., Garcia E. 1997; Molecular organization of the genes required for the synthesis of type 1 capsular polysaccharide of Streptococcus pneumoniae : formation of binary encapsulated pneumococci and identification of cryptic dTDP-rhamnose biosynthesis genes. Mol Microbiol 25:79–92 [CrossRef]
     [Google Scholar]
  38. Munoz R., Mollerach M., Lopez R., Garcia E. 1999; Characterization of the type 8 capsular gene cluster of Streptococcus pneumoniae . J Bacteriol 181:6214–6219
     [Google Scholar]
  39. Musher D. M. 1992; Infections caused by Streptococcus pneumoniae : clinical spectrum, pathogenesis, immunity, and treatment. Clin Infect Dis 14:801–807 [CrossRef]
     [Google Scholar]
  40. Nesin M., Ramirez M., Tomasz A. 1998; Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo . J Infect Dis 177:707–713 [CrossRef]
     [Google Scholar]
  41. Nielsen S. V., Henrichsen J. 1992; Capsular types of Streptococcus pneumoniae isolated from blood and CSF during 1982–1987. Clin Infect Dis 15:794–798 [CrossRef]
     [Google Scholar]
  42. Obaro S. K. 2000; Confronting the pneumococcus: a target shift or bullet change?. Vaccine 19:1211–1217 [CrossRef]
     [Google Scholar]
  43. Oggioni M. R., Claverys J. P. 1999; Repeated extragenic sequences in prokaryotic genomes: a proposal for the origin and dynamics of the RUP element in Streptococcus pneumoniae . Microbiology 145:2647–2653
     [Google Scholar]
  44. Osborn M. J., Cynkin M. A., Gilbert J. M., Muller L., Singh M. 1972; Synthesis of bacterial O-antigens. Methods Enzymol 28:583–601
     [Google Scholar]
  45. Paton J. C., Morona J. K. 2000; Streptococcus pneumoniae capsular polysaccharide. In Gram-Positive Pathogens pp 201–213 Edited by Fischetti V. A. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  46. Perry M. B., Daoust V., Carlo D. J. 1981; The specific capsular polysaccharide of Streptococcus pneumoniae type 9V. Can J Biochem 59:524–533
     [Google Scholar]
  47. Ramirez M., Tomasz A. 1998; Molecular characterization of the complete 23F capsular polysaccharide locus of Streptococcus pneumoniae . J Bacteriol 180:5273–5278
     [Google Scholar]
  48. Ramirez M., Tomasz A. 1999; Acquisition of new capsular genes among clinical isolates of antibiotic-resistant Streptococcus pneumoniae . Microb Drug Resist 5:241–246 [CrossRef]
     [Google Scholar]
  49. Reeves P. R., Hobbs M., Valvano M. A. 8 other authors 1996; Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol 4:495–503 [CrossRef]
     [Google Scholar]
  50. Robbins J. B., Austrian R., Lee C.-J. 8 other authors 1983; Considerations for formulating the second-generation pneumococcal capsular polysaccharide vaccine with emphasis on the cross-reactive types within groups. J Infect Dis 148:1136–1159 [CrossRef]
     [Google Scholar]
  51. Rodriguez-Herva J. J., Reniero D., Galli E., Ramos J. L. 1999; Cell envelope mutants of Pseudomonas putida : physiological characterization and analysis of their ability to survive in soil. Environ Microbiol 1:479–488 [CrossRef]
     [Google Scholar]
  52. Rutherford T. J., Jones C., Davies D. B., Elliott A. C. 1991; Location and quantitation of the sites of O -acetylation on the capsular polysaccharide from Streptococcus pneumoniae type 9V by 1H-n.m.r. spectroscopy: comparison with type 9A. Carbohydr Res 218:175–184 [CrossRef]
     [Google Scholar]
  53. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  54. Shinefield H. R., Black S. 2000; Efficacy of pneumococcal conjugate vaccines in large scale field trials. Pediatr Infect Dis J 19:394–397 [CrossRef]
     [Google Scholar]
  55. Sulzenbacher G., Gal L., Peneff C., Fassy F., Bourne Y. 2001; Crystal structure of Streptococcus pneumoniae N -acetylglucosamine-1-phosphate uridyltransferase bound to acetyl-coenzyme A reveals a novel active site architecture. J Biol Chem 276:11844–11851 [CrossRef]
     [Google Scholar]
  56. Tettelin H., Nelson K. E., Paulsen I. T. 36 other authors 2001; Complete genome sequence of a virulent isolate of Streptococcus pneumoniae . Science 293:498–506 [CrossRef]
     [Google Scholar]
  57. Tomasz A. 1999; New faces of an old pathogen: emergence and spread of multidrug-resistant Streptococcus pneumoniae . Am J Med 107:55S–62S
     [Google Scholar]
  58. Whitfield C., Roberts I. S. 1999; Structure, assembly and regulation of expression of capsules in Escherichia coli . Mol Microbiol 31:1307–1319 [CrossRef]
     [Google Scholar]
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Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9VThe GenBank accession number for the sequence reported in this paper is AF402095.
Microbiology 148, 1747 (2002); https://doi.org/10.1099/00221287-148-6-1747
/content/journal/micro/10.1099/00221287-148-6-1747
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