Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Apr;171(4):2116–2123. doi: 10.1128/jb.171.4.2116-2123.1989

Cell-associated pullulanase from Bacteroides thetaiotaomicron: cloning, characterization, and insertional mutagenesis to determine role in pullulan utilization.

K A Smith 1, A A Salyers 1
PMCID: PMC209865  PMID: 2703467

Abstract

We have cloned a pullulanase gene from Bacteroides thetaiotaomicron. The pullulanase expressed from this clone in Escherichia coli was cell associated and soluble and had a molecular mass of 72 kilodaltons by gel filtration. Maxicell analysis of proteins coded by the cloned insert showed that a 71.6- to 73.2-kilodalton doublet was associated with pullulanase activity. Thus, the pullulanase is probably a monomer. The cloned pullulanase produced maltotriose as an end product of pullulan digestion. In B. thetaiotaomicron the pullulanase activity was cell associated. Approximately 80% of the activity was soluble, and 16 to 18% was membrane associated. The molecular mass of the soluble pullulanase was 77 kilodaltons by gel filtration. To determine whether the cloned pullulanase gene was essential for pullulan utilization, we used directed insertional mutagenesis to inactivate the B. thetaiotaomicron pullulanase gene. The pullulanase specific activity of the mutant was approximately 45% of that of wild-type B. thetaiotaomicron. However, the pullulanase-negative insertional mutant 95-1 was still able to grow on pullulan at a rate similar to that of wild-type B. thetaiotaomicron. Thus, there must be a second pullulanase in B. thetaiotaomicron.

Full text

PDF
2116

Images in this article

2119Image
on p.2119
2120Image
on p.2120
2121Image
on p.2121

Selected References

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

  1. Dygert S., Li L. H., Florida D., Thoma J. A. Determination of reducing sugar with improved precision. Anal Biochem. 1965 Dec;13(3):367–374. doi: 10.1016/0003-2697(65)90327-1. [DOI] [PubMed] [Google Scholar]
  2. Eisele B., Rasched I. R., Wallenfels K. Molecular characterization of pullulanase from Aerobacter aerogenes. Eur J Biochem. 1972 Mar 15;26(1):62–67. doi: 10.1111/j.1432-1033.1972.tb01739.x. [DOI] [PubMed] [Google Scholar]
  3. Englyst H. N., Cummings J. H. Digestion of polysaccharides of potato in the small intestine of man. Am J Clin Nutr. 1987 Feb;45(2):423–431. doi: 10.1093/ajcn/45.2.423. [DOI] [PubMed] [Google Scholar]
  4. Gherardini F. C., Salyers A. A. Characterization of an outer membrane mannanase from Bacteroides ovatus. J Bacteriol. 1987 May;169(5):2031–2037. doi: 10.1128/jb.169.5.2031-2037.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gherardini F. C., Salyers A. A. Purification and characterization of a cell-associated, soluble mannanase from Bacteroides ovatus. J Bacteriol. 1987 May;169(5):2038–2043. doi: 10.1128/jb.169.5.2038-2043.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Guthrie E. P., Shoemaker N. B., Salyers A. A. Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron. J Bacteriol. 1985 Nov;164(2):510–515. doi: 10.1128/jb.164.2.510-515.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hashimoto F., Horigome T., Kanbayashi M., Yoshida K., Sugano H. An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem. 1983 Feb 15;129(1):192–199. doi: 10.1016/0003-2697(83)90068-4. [DOI] [PubMed] [Google Scholar]
  9. Holdeman L. V., Good I. J., Moore W. E. Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl Environ Microbiol. 1976 Mar;31(3):359–375. doi: 10.1128/aem.31.3.359-375.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hope G. C., Dean A. C. Pullulanase synthesis in klebsiella (aerobacter) aerogenes strains growing in continuous culture. Biochem J. 1974 Nov;144(2):403–411. doi: 10.1042/bj1440403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kasahara M., Anraku Y. Succinate dehydrogenase of Escherichia coli membrane vesicles. Activation and properties of the enzyme. J Biochem. 1974 Nov;76(5):959–966. [PubMed] [Google Scholar]
  12. Katsuragi N., Takizawa N., Murooka Y. Entire nucleotide sequence of the pullulanase gene of Klebsiella aerogenes W70. J Bacteriol. 1987 May;169(5):2301–2306. doi: 10.1128/jb.169.5.2301-2306.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. McCarthy R. E., Kotarski S. F., Salyers A. A. Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron. J Bacteriol. 1985 Feb;161(2):493–499. doi: 10.1128/jb.161.2.493-499.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Michaelis S., Chapon C., D'Enfert C., Pugsley A. P., Schwartz M. Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae. J Bacteriol. 1985 Nov;164(2):633–638. doi: 10.1128/jb.164.2.633-638.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pugsley A. P., Chapon C., Schwartz M. Extracellular pullulanase of Klebsiella pneumoniae is a lipoprotein. J Bacteriol. 1986 Jun;166(3):1083–1088. doi: 10.1128/jb.166.3.1083-1088.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shoemaker N. B., Guthrie E. P., Salyers A. A., Gardner J. F. Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element. J Bacteriol. 1985 May;162(2):626–632. doi: 10.1128/jb.162.2.626-632.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  27. Speer B. S., Salyers A. A. Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli. J Bacteriol. 1988 Apr;170(4):1423–1429. doi: 10.1128/jb.170.4.1423-1429.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takizawa N., Murooka Y. Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes. Appl Environ Microbiol. 1985 Feb;49(2):294–298. doi: 10.1128/aem.49.2.294-298.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Valentine P. J., Shoemaker N. B., Salyers A. A. Mobilization of Bacteroides plasmids by Bacteroides conjugal elements. J Bacteriol. 1988 Mar;170(3):1319–1324. doi: 10.1128/jb.170.3.1319-1324.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Varel V. H., Bryant M. P. Nutritional features of Bacteroides fragilis subsp. fragilis. Appl Microbiol. 1974 Aug;28(2):251–257. doi: 10.1128/am.28.2.251-257.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wood W. B. Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol. 1966 Mar;16(1):118–133. doi: 10.1016/s0022-2836(66)80267-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES