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. 1992 Nov;174(22):7185–7193. doi: 10.1128/jb.174.22.7185-7193.1992

A locus that contributes to colonization of the intestinal tract by Bacteroides thetaiotaomicron contains a single regulatory gene (chuR) that links two polysaccharide utilization pathways.

Q Cheng 1, V Hwa 1, A A Salyers 1
PMCID: PMC207410  PMID: 1429442

Abstract

Previously, we isolated two Tn4351-generated mutants of Bacteroides thetaiotaomicron (46-1 and CS3) that were unable to grow either on heparin or on chondroitin sulfate. This phenotype was unexpected, since the heparin and chondroitin sulfate utilization pathways had appeared from earlier studies to be independent of each other. Mutants 46-1 and CS3 were also of interest because both were unable to compete successfully with wild-type B. thetaiotaomicron in the intestinal tracts of germfree mice. Thus, both appeared to have a colonization defect. We have now cloned the chromosomal locus in which the transposon insertions in 46-1 and CS3 occurred. Southern blot analysis showed that the Tn4351 insertions in 46-1 and CS3 were about 100 bp apart. Using complementation and insertional mutagenesis, we localized the region affected by the 46-1 and CS3 insertions to within 2.5 kbp. This DNA segment was sequenced and found to contain a 401-codon open reading frame (ORF1) and the N-terminal segment of a second open reading frame (ORF2), which was downstream of ORF1 and transcribed in the same direction. The deduced amino acid sequence of ORF1 showed significant homology to that of a putative positive regulator of an arylsulfatase gene in Klebsiella aerogenes. ORF2 was at least 381 amino acids long and did not exhibit homology to any proteins in the data bases searched. Transposon insertions in both mutants 46-1 and CS3 disrupted ORF1. The results of insertional mutagenesis and complementation experiments indicated that ORF2 was not essential for growth on chondroitin sulfate or heparin. Thus, the chondroitin sulfate-negative and heparin-negative phenotypes of 46-1 and CS3 appear to be due to the interruption of a regulatory gene encoded by ORF1 and not to a polar effect of the insertions on a downstream gene(s). The gene encoding ORF1 has been designated chuR, for regulation of chondroitin sulfate and heparin utilization. Transcriptional fusion studies showed that the expression of chuR occurred at the same level under inducing and noninducing conditions, in contrast to the regulated expression of structural genes of the chondroitin sulfate utilization system. chuR was not autoregulated, nor was its expression affected by a mutation (46-4) that eliminated the expression of all chondroitin sulfate utilization genes but did not affect the utilization of heparin.

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Selected References

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  1. Adachi T., Murooka Y., Harada T. Derepression of arylsulfatase synthesis in Aerobacter aerogenes by tyramine. J Bacteriol. 1973 Oct;116(1):19–24. doi: 10.1128/jb.116.1.19-24.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Davison J., Heusterspreute M., Chevalier N., Ha-Thi V., Brunel F. Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene. 1987;51(2-3):275–280. doi: 10.1016/0378-1119(87)90316-7. [DOI] [PubMed] [Google Scholar]
  3. Feldhaus M. J., Hwa V., Cheng Q., Salyers A. A. Use of an Escherichia coli beta-glucuronidase gene as a reporter gene for investigation of Bacteroides promoters. J Bacteriol. 1991 Jul;173(14):4540–4543. doi: 10.1128/jb.173.14.4540-4543.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Guiney D. G., Jr, Hasegawa P., Davis C. E. Expression in Escherichia coli of cryptic tetracycline resistance genes from bacteroides R plasmids. Plasmid. 1984 May;11(3):248–252. doi: 10.1016/0147-619x(84)90031-3. [DOI] [PubMed] [Google Scholar]
  5. Guiney D. G., Yakobson E. Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3595–3598. doi: 10.1073/pnas.80.12.3595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  8. Hwa V., Salyers A. A. 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. Appl Environ Microbiol. 1992 Mar;58(3):869–876. doi: 10.1128/aem.58.3.869-876.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Jefferson R. A., Burgess S. M., Hirsh D. beta-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8447–8451. doi: 10.1073/pnas.83.22.8447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Moore W. E., Holdeman L. V. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbiol. 1974 May;27(5):961–979. doi: 10.1128/am.27.5.961-979.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Murooka Y., Ishibashi K., Yasumoto M., Sasaki M., Sugino H., Azakami H., Yamashita M. A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene. J Bacteriol. 1990 Apr;172(4):2131–2140. doi: 10.1128/jb.172.4.2131-2140.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shoemaker N. B., Getty C., Gardner J. F., Salyers A. A. Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome. J Bacteriol. 1986 Mar;165(3):929–936. doi: 10.1128/jb.165.3.929-936.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shoemaker N. B., Getty C., Guthrie E. P., Salyers A. A. Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element. J Bacteriol. 1986 Jun;166(3):959–965. doi: 10.1128/jb.166.3.959-965.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stevens A. M., Sanders J. M., Shoemaker N. B., Salyers A. A. Genes involved in production of plasmidlike forms by a Bacteroides conjugal chromosomal element share amino acid homology with two-component regulatory systems. J Bacteriol. 1992 May;174(9):2935–2942. doi: 10.1128/jb.174.9.2935-2942.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tancula E., Feldhaus M. J., Bedzyk L. A., Salyers A. A. Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron. J Bacteriol. 1992 Sep;174(17):5609–5616. doi: 10.1128/jb.174.17.5609-5616.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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