1887

Microbiology Society logo

Volume 151, Issue 6

Immobilization-based isolation of capsule-negative mutants of Free

Abstract

The capsular polysaccharide (CPS) is the most important identified virulence factor of , a human pathogen of the upper respiratory tract. One limitation in studies of surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0·04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CPS, capsular polysaccharide and CSP, competence-stimulating peptide
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27862-0
2005-06-01
2024-04-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/6/mic1511911.html?itemId=/content/journal/micro/10.1099/mic.0.27862-0&mimeType=html&fmt=ahah

References

  1. Adamou J. E., Wizemann T. M., Barren P., Langermann S. 1998; Adherence of Streptococcus pneumoniae to human bronchial epithelial cells (BEAS-2B. Infect Immun 66:820–822
     [Google Scholar]
  2. Adeyeye A., Jansson P.-E., Lindberg B., Henrichsen J. 1988; Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 37. Carbohydr Res 180:295–299 [CrossRef]
     [Google Scholar]
  3. Amann R. I., Krumholz L., Stahl D. A. 1990; Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770
     [Google Scholar]
  4. Avery O. T., MacLeod C. M., McCarty M. 1944; Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med 79:137–158 [CrossRef]
     [Google Scholar]
  5. Barany F., Tomasz A. 1980; Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid. J Bacteriol 144:698–709
     [Google Scholar]
  6. Caimano M. J., Hardy G. G., Yother J. 1998; Capsule genetics in Streptococcus pneumoniae and a possible role for transposition in the generation of the type 3 locus. Microb Drug Resist 4:11–23 [CrossRef]
     [Google Scholar]
  7. Coffey T. J., Enright M. C., Daniels M., Wilkinson P., Berron S., Fenoll A., Spratt B. G. 1998; Serotype 19A variants of the Spanish serotype 23F multiresistant clone of Streptococcus pneumoniae. Microb Drug Resist 4:51–55 [CrossRef]
     [Google Scholar]
  8. Coffey T., Daniels M., Enright C., Spratt B. 1999; Serotype 14 variants of the Spanish penicillin-resistant serotype 9V clone of Streptococcus pneumoniae arose by large recombinational replacements of the cpsA-pbp1a region. Microbiology 145:2023–2031 [CrossRef]
     [Google Scholar]
  9. Finkelstein R. A., Sulkin S. E. 1957; Characteristics of coagulase positive and coagulase negative staphylococci in serum-soft agar. J Bacteriol 75:339–344
     [Google Scholar]
  10. García E., García P., López R. 1993; Cloning and sequencing of a gene involved in the synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 3. Mol Gen Genet 239:188–195
     [Google Scholar]
  11. García E., Llull D., Muñoz R., Mollerach M., López R. 2000; Current trends in capsular polysaccharide biosynthesis of Streptococcus pneumoniae . Res Microbiol 151:429–435 [CrossRef]
     [Google Scholar]
  12. Granlund-Edstedt M., Selin M., Holm A., Hakansson S. 1993; Adherence and surface properties of buoyant density subpopulations of group B streptococci, type III. Acta Pathol Microbiol Immunol Scand 101:141–148 [CrossRef]
     [Google Scholar]
  13. Griffith F. 1928; The significance of pneumococcal types. J Hyg 27:113–159 [CrossRef]
     [Google Scholar]
  14. Henrichsen J. 1995; Six newly recognized types of Streptococcus pneumoniae. J Clin Microbiol 33:2759–2762
     [Google Scholar]
  15. Llull D., Muñoz R., López R., García 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]
  16. Llull D., López R, García E. 2000; Clonal origin of the type 37 Streptococcus pneumoniae. Microb Drug Resist 6:269–275 [CrossRef]
     [Google Scholar]
  17. Llull D., García E., López R. 2001; Tts, a processive beta-glucosyltransferase of Streptococcus pneumoniae, directs the synthesis of the branched type 37 capsular polysaccharide in Pneumococcus and other gram-positive species. J Biol Chem 276:21053–21061 [CrossRef]
     [Google Scholar]
  18. Lund E., Henrichsen J. 1978; Laboratory diagnosis, serology and epidemiology of Streptococcus pneumoniae. Methods Microbiol 12:241–262
     [Google Scholar]
  19. Mercier C., Durrieu C., Briandet R., Domakova E., Tremblay J., Buist G., Kulakauskas S. 2002; Positive role of peptidoglycan breaks in lactococcal biofilm formation. Mol Microbiol 46:235–243 [CrossRef]
     [Google Scholar]
  20. Muñoz R., Mollerach M., López R., García 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]
  21. Muñoz R., García E., López R. 1998; Evidence for horizontal transfer from Streptococcus to Escherichia coli of the kfiD gene encoding the K5-specific UDP-glucose dehydrogenase. J Mol Evol 46:432–436 [CrossRef]
     [Google Scholar]
  22. Narikawa S., Suzuki Y., Takahashi M., Furukawa A., Sakane T., Mizushima Y. 1995; Streptococcus oralis previously identified as uncommon ‘Streptococcus sanguis’. in Behcet's disease. Arch Oral Biol 40:685–690 [CrossRef]
     [Google Scholar]
  23. Pearce B. J., Iannelli F., Pozzi G. 2002; Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae. Res Microbiol 153:243–247 [CrossRef]
     [Google Scholar]
  24. Pozzi G., Masala L., Iannelli F., Manganelli R., Havarstein L. S., Piccoli L., Simon D., Morrison D. A. 1996; Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone. J Bacteriol 178:6087–6090
     [Google Scholar]
  25. Ravin A. W. 1959; Reciprocal capsular transformations of pneumococci. J Bacteriol 77:296–309
     [Google Scholar]
  26. Ring A., Weiser J. N., Tuomanen E. I. 1998; Pneumococcal trafficking across the blood-brain barrier. Molecular analysis of a novel bidirectional pathway. J Clin Invest 102:347–360 [CrossRef]
     [Google Scholar]
  27. Sellin M., Hakansson S., Norgren M. 1995; Phase-shift of polysaccharide capsule expression in group B streptococci, type III. Microb Pathog 18:401–415 [CrossRef]
     [Google Scholar]
  28. Sung C. K., Li H., Claverys J. P., Morrison D. A. 2001; An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67:5190–5196 [CrossRef]
     [Google Scholar]
  29. Trzcinski K., Thompson C. M., Lipsitch M. 2003; Construction of otherwise isogenic serotype 6B, 7F, 14, and 19F capsular variants of Streptococcus pneumoniae strain TIGR4. Appl Environ Microbiol 69:7364–7370 [CrossRef]
     [Google Scholar]
  30. Waite R. D., Struthers J. K., Dowson C. G. 2001; Spontaneous sequence duplication within an open reading frame of the pneumococcal type 3 capsule locus causes high-frequency phase variation. Mol Microbiol 42:1223–1232
     [Google Scholar]
  31. Waite R. D., Penfold D. W., Struthers J. K., Dowson C. G. 2003; Spontaneous sequence duplications within capsule genes cap8I and tts control phase variation in Streptococcus pneumoniae serotypes 8 and 37. Microbiology 149:497–504 [CrossRef]
     [Google Scholar]
  32. Yother J., McDaniel L. S., Briles D. E. 1986; Transformation of encapsulated Streptococcus pneumoniae. J Bacteriol 168:1463–1465
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27862-0
Loading
Immobilization-based isolation of capsule-negative mutants of Streptococcus pneumoniae
Microbiology 151, 1911 (2005); https://doi.org/10.1099/mic.0.27862-0
/content/journal/micro/10.1099/mic.0.27862-0
/content/journal/micro/10.1099/mic.0.27862-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error