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. 1992 Mar;58(3):869–876. doi: 10.1128/aem.58.3.869-876.1992

Analysis of two chondroitin sulfate utilization mutants of Bacteroides thetaiotaomicron that differ in their abilities to compete with the wild type in the gastrointestinal tracts of germfree mice.

V Hwa 1, A A Salyers 1
PMCID: PMC195347  PMID: 1575488

Abstract

Previously, we isolated two mutants of Bacteroides thetaiotaomicron that were unable to grow on the mucopolysaccharide chondroitin sulfate (CS). One of these mutants (46-1) was outcompeted by the wild type in the intestinal tracts of germfree mice, whereas the other mutant (46-4) competed equally with the wild type. In the present article, we report a detailed characterization of these two mutants. Assays of enzymes in the CS utilization pathway revealed that 46-1 did not express one of these enzymes, chondro-6-sulfatase. The absence of chondro-6-sulfatase activity in extracts from 46-1 allowed us to detect a previously unknown activity of another enzyme in the CS breakdown pathway, beta-glucuronidase. In addition to hydrolyzing its normal substrate (an unsulfated disaccharide), beta-glucuronidase also hydrolyzed the 6-sulfated disaccharide subunit of CS. Two-dimensional gel analysis of polypeptides produced by 46-1 showed that several proteins other than the 6-sulfatase were either missing or expressed aberrantly. Thus, 46-1 could be a regulatory mutant. Mutant 46-4 was unable to grow on CS, hyaluronic acid, or disaccharides of CS. Thus, expression of the CS pathway enzymes could not be induced. Nonetheless, the growth pattern of 46-4 and some other findings indicate that the structural genes for these enzymes were still intact. The most likely target of mutant 46-4 is a regulatory locus that is required for expression of CS utilization genes. A surprising characteristic of 46-1 was its inability to grow on heparin, a mucopolysaccharide which is structurally similar to CS but is utilized by a different pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Ames G. F., Nikaido K. Two-dimensional gel electrophoresis of membrane proteins. Biochemistry. 1976 Feb 10;15(3):616–623. doi: 10.1021/bi00648a026. [DOI] [PubMed] [Google Scholar]
  2. Gherardini F., Babcock M., Salyers A. A. Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus. J Bacteriol. 1985 Feb;161(2):500–506. doi: 10.1128/jb.161.2.500-506.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Glaser J. H., Conrad H. E. Chondroitin SO4 catabolism in chick embryo chondrocytes. J Biol Chem. 1979 Apr 10;254(7):2316–2325. [PubMed] [Google Scholar]
  4. Guthrie E. P., Salyers A. A. Evidence that the Bacteroides thetaiotaomicron chondroitin lyase II gene is adjacent to the chondro-4-sulfatase gene and may be part of the same operon. J Bacteriol. 1987 Mar;169(3):1192–1199. doi: 10.1128/jb.169.3.1192-1199.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Guthrie E. P., Salyers A. A. Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron. J Bacteriol. 1986 Jun;166(3):966–971. doi: 10.1128/jb.166.3.966-971.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hwa V., Salyers A. A. Evidence for differential regulation of genes in the chondroitin sulfate utilization pathway of Bacteroides thetaiotaomicron. J Bacteriol. 1992 Jan;174(1):342–344. doi: 10.1128/jb.174.1.342-344.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kotarski S. F., Linz J., Braun D. M., Salyers A. A. Analysis of outer membrane proteins which are associated with growth of Bacteroides thetaiotaomicron on chondroitin sulfate. J Bacteriol. 1985 Sep;163(3):1080–1086. doi: 10.1128/jb.163.3.1080-1086.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kotarski S. F., Salyers A. A. Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates. J Bacteriol. 1984 Apr;158(1):102–109. doi: 10.1128/jb.158.1.102-109.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Linn S., Chan T., Lipeski L., Salyers A. A. Isolation and characterization of two chondroitin lyases from Bacteroides thetaiotaomicron. J Bacteriol. 1983 Nov;156(2):859–866. doi: 10.1128/jb.156.2.859-866.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ludwigs U., Elgavish A., Esko J. D., Meezan E., Rodén L. Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose. Biochem J. 1987 Aug 1;245(3):795–804. doi: 10.1042/bj2450795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  13. Salyers A. A., Guthrie E. P. A deletion in the chromosome of Bacteroides thetaiotaomicron that abolishes production of chondroitinase II does not affect survival of the organism in gastrointestinal tracts of exgermfree mice. Appl Environ Microbiol. 1988 Aug;54(8):1964–1969. doi: 10.1128/aem.54.8.1964-1969.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Salyers A. A., Kotarski S. F. Induction of chondroitin sulfate lyase activity in Bacteroides thetaiotaomicron. J Bacteriol. 1980 Aug;143(2):781–788. doi: 10.1128/jb.143.2.781-788.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Salyers A. A., O'Brien M. Cellular location of enzymes involved in chondroitin sulfate breakdown by Bacteroides thetaiotaomicron. J Bacteriol. 1980 Aug;143(2):772–780. doi: 10.1128/jb.143.2.772-780.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Salyers A. A., Pajeau M. Competitiveness of different polysaccharide utilization mutants of Bacteroides thetaiotaomicron in the intestinal tracts of germfree mice. Appl Environ Microbiol. 1989 Oct;55(10):2572–2578. doi: 10.1128/aem.55.10.2572-2578.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Salyers A. A., Pajeau M., McCarthy R. E. Importance of mucopolysaccharides as substrates for Bacteroides thetaiotaomicron growing in intestinal tracts of exgermfree mice. Appl Environ Microbiol. 1988 Aug;54(8):1970–1976. doi: 10.1128/aem.54.8.1970-1976.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Salyers A. A., Vercellotti J. R., West S. E., Wilkins T. D. Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl Environ Microbiol. 1977 Feb;33(2):319–322. doi: 10.1128/aem.33.2.319-322.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]

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