Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 May;179(9):2863–2871. doi: 10.1128/jb.179.9.2863-2871.1997

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

S Ramaswamy 1, M Dworkin 1, J Downard 1
PMCID: PMC179047  PMID: 9139901

Abstract

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Arnold J. W., Shimkets L. J. Cell surface properties correlated with cohesion in Myxococcus xanthus. J Bacteriol. 1988 Dec;170(12):5771–5777. doi: 10.1128/jb.170.12.5771-5777.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnold J. W., Shimkets L. J. Inhibition of cell-cell interactions in Myxococcus xanthus by congo red. J Bacteriol. 1988 Dec;170(12):5765–5770. doi: 10.1128/jb.170.12.5765-5770.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Avery L., Kaiser D. In situ transposon replacement and isolation of a spontaneous tandem genetic duplication. Mol Gen Genet. 1983;191(1):99–109. doi: 10.1007/BF00330896. [DOI] [PubMed] [Google Scholar]
  4. Behmlander R. M., Dworkin M. Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus. J Bacteriol. 1994 Oct;176(20):6295–6303. doi: 10.1128/jb.176.20.6295-6303.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Behmlander R. M., Dworkin M. Extracellular fibrils and contact-mediated cell interactions in Myxococcus xanthus. J Bacteriol. 1991 Dec;173(24):7810–7820. doi: 10.1128/jb.173.24.7810-7820.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Campos J. M., Geisselsoder J., Zusman D. R. Isolation of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus. J Mol Biol. 1978 Feb 25;119(2):167–178. doi: 10.1016/0022-2836(78)90431-x. [DOI] [PubMed] [Google Scholar]
  7. Chang B. Y., Dworkin M. Isolated fibrils rescue cohesion and development in the Dsp mutant of Myxococcus xanthus. J Bacteriol. 1994 Dec;176(23):7190–7196. doi: 10.1128/jb.176.23.7190-7196.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang B. Y., Dworkin M. Mutants of Myxococcus xanthus dsp defective in fibril binding. J Bacteriol. 1996 Feb;178(3):697–700. doi: 10.1128/jb.178.3.697-700.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dana J. R., Shimkets L. J. Regulation of cohesion-dependent cell interactions in Myxococcus xanthus. J Bacteriol. 1993 Jun;175(11):3636–3647. doi: 10.1128/jb.175.11.3636-3647.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dobson W. J., McCurdy H. D., MacRae T. H. The function of fimbriae in Myxococcus xanthus. II. The role of fimbriae in cell-cell interactions. Can J Microbiol. 1979 Dec;25(12):1359–1372. doi: 10.1139/m79-214. [DOI] [PubMed] [Google Scholar]
  11. Downard J., Ramaswamy S. V., Kil K. S. Identification of esg, a genetic locus involved in cell-cell signaling during Myxococcus xanthus development. J Bacteriol. 1993 Dec;175(24):7762–7770. doi: 10.1128/jb.175.24.7762-7770.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Downard J., Toal D. Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development. Mol Microbiol. 1995 Apr;16(2):171–175. doi: 10.1111/j.1365-2958.1995.tb02290.x. [DOI] [PubMed] [Google Scholar]
  13. Dworkin M. Recent advances in the social and developmental biology of the myxobacteria. Microbiol Rev. 1996 Mar;60(1):70–102. doi: 10.1128/mr.60.1.70-102.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Hartzell P. L., Youderian P. Genetics of gliding motility and development in Myxococcus xanthus. Arch Microbiol. 1995 Nov;164(5):309–323. doi: 10.1007/BF02529977. [DOI] [PubMed] [Google Scholar]
  16. Hodgkin J., Kaiser D. Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2938–2942. doi: 10.1073/pnas.74.7.2938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaiser D. Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5952–5956. doi: 10.1073/pnas.76.11.5952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kalman L. V., Cheng Y. L., Kaiser D. The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo. J Bacteriol. 1994 Mar;176(5):1434–1442. doi: 10.1128/jb.176.5.1434-1442.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kroos L., Kaiser D. Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5816–5820. doi: 10.1073/pnas.81.18.5816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuner J. M., Kaiser D. Fruiting body morphogenesis in submerged cultures of Myxococcus xanthus. J Bacteriol. 1982 Jul;151(1):458–461. doi: 10.1128/jb.151.1.458-461.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Long S., Reed J. W., Himawan J., Walker G. C. Genetic analysis of a cluster of genes required for synthesis of the calcofluor-binding exopolysaccharide of Rhizobium meliloti. J Bacteriol. 1988 Sep;170(9):4239–4248. doi: 10.1128/jb.170.9.4239-4248.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. MacNeil S. D., Mouzeyan A., Hartzell P. L. Genes required for both gliding motility and development in Myxococcus xanthus. Mol Microbiol. 1994 Nov;14(4):785–795. doi: 10.1111/j.1365-2958.1994.tb01315.x. [DOI] [PubMed] [Google Scholar]
  23. Shi W., Zusman D. R. The two motility systems of Myxococcus xanthus show different selective advantages on various surfaces. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3378–3382. doi: 10.1073/pnas.90.8.3378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shimkets L. J. Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus. J Bacteriol. 1986 Jun;166(3):837–841. doi: 10.1128/jb.166.3.837-841.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shimkets L. J. Social and developmental biology of the myxobacteria. Microbiol Rev. 1990 Dec;54(4):473–501. doi: 10.1128/mr.54.4.473-501.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Toal D. R., Clifton S. W., Roe B. A., Downard J. The esg locus of Myxococcus xanthus encodes the E1 alpha and E1 beta subunits of a branched-chain keto acid dehydrogenase. Mol Microbiol. 1995 Apr;16(2):177–189. doi: 10.1111/j.1365-2958.1995.tb02291.x. [DOI] [PubMed] [Google Scholar]
  27. Wu S. S., Kaiser D. Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus. Mol Microbiol. 1995 Nov;18(3):547–558. doi: 10.1111/j.1365-2958.1995.mmi_18030547.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES