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. 1990 Dec;56(12):3657–3663. doi: 10.1128/aem.56.12.3657-3663.1990

Extracellular beta-galactosidase activity of a Fibrobacter succinogenes S85 mutant able to catabolize lactose.

P Javorsky 1, S F Lee 1, A M Gibbins 1, C W Forsberg 1
PMCID: PMC185048  PMID: 2128006

Abstract

Fibrobacter succinogenes S85 is unable to grow with lactose as the source of carbohydrate, although it does exhibit low beta-galactosidase (EC 3.2.1.23) activity. Spontaneous mutants of strain S85 able to grow on lactose were isolated after spreading cells on a chemically defined agar medium with lactose as the carbohydrate source. A lactose-catabolizing isolate, designated L2, exhibited a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile and an immunoblot profile with polyclonal antibodies to whole cells of S85 which were identical to those observed for S85. Strain L2 exhibited both cell-associated and extracellular beta-galactosidase activity with either p-nitrophenyl-beta-D-galactopyranoside or lactose as the substrate. The cell-associated enzyme exhibited the greatest activity in the periplasmic space. Enzyme production was partially inhibited by glucose. The beta-galactosidase was activated by divalent cations and exhibited a pH optimum of 6.5. Analysis of the extracellular culture fluid revealed that glucose derived from the hydrolysis of lactose was used for growth, but galactose was not metabolized further. Cells were unable to take up the lactose analog, methyl-beta-D-thiogalactopyranoside. These data suggest that beta-galactosidase of F. succinogenes L2 cleaves lactose outside the cells and that the glucose released is catabolized while the galactose accumulates in the extracellular culture fluid.

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Selected References

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  1. ANEMA P. J. PURIFICATION AND SOME PROPERTIES OF BETA-GLACTOSIDASE OF BACILLUS SUBTILIS. Biochim Biophys Acta. 1964 Sep 18;89:495–502. [PubMed] [Google Scholar]
  2. Begbie R., Stewart C. S. Polyacrylamide gel electrophoresis of Bacteroides succinogens. Can J Microbiol. 1984 Jun;30(6):863–866. doi: 10.1139/m84-134. [DOI] [PubMed] [Google Scholar]
  3. Biermann L., Glantz M. D. Isolation and characterization of beta-galactosidase from Saccharomyces lactis. Biochim Biophys Acta. 1968 Oct 8;167(2):373–377. doi: 10.1016/0005-2744(68)90216-7. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. CRAVEN G. R., STEERS E., Jr, ANFINSEN C. B. PURIFICATION, COMPOSITION, AND MOLECULAR WEIGHT OF THE BETA-GALACTOSIDASE OF ESCHERICHIA COLI K12. J Biol Chem. 1965 Jun;240:2468–2477. [PubMed] [Google Scholar]
  6. Forsberg C. W., Crosby B., Thomas D. Y. Potential for manipulation of the rumen fermentation through the use of recombinant DNA techniques. J Anim Sci. 1986 Jul;63(1):310–325. doi: 10.2527/jas1986.631310x. [DOI] [PubMed] [Google Scholar]
  7. Franklund C. V., Glass T. L. Glucose uptake by the cellulolytic ruminal anaerobe Bacteroides succinogenes. J Bacteriol. 1987 Feb;169(2):500–506. doi: 10.1128/jb.169.2.500-506.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goodman R. E., Pederson D. M. beta-Galactosidase from Bacillus stearothermophilus. Can J Microbiol. 1976 Jun;22(6):817–825. doi: 10.1139/m76-118. [DOI] [PubMed] [Google Scholar]
  9. Groleau D., Forsberg C. W. Cellulolytic activity of the rumen bacterium Bacteroides succinogenes. Can J Microbiol. 1981 May;27(5):517–530. doi: 10.1139/m81-077. [DOI] [PubMed] [Google Scholar]
  10. Harrap G. J., Watkins W. M. Enzymes of Trichomonas foetus. Separation and properties of two beta-galactosidases. Biochem J. 1970 May;117(4):667–675. doi: 10.1042/bj1170667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huang L., Forsberg C. W. Isolation of a Cellodextrinase from Bacteroides succinogenes. Appl Environ Microbiol. 1987 May;53(5):1034–1041. doi: 10.1128/aem.53.5.1034-1041.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huang L., McGavin M., Forsberg C. W., Lam J. S., Cheng K. J. Antigenic nature of the chloride-stimulated cellobiosidase and other cellulases of Fibrobacter succinogenes subsp. succinogenes S85 and related fresh isolates. Appl Environ Microbiol. 1990 May;56(5):1229–1234. doi: 10.1128/aem.56.5.1229-1234.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Irvin J. E., Teather R. M. Cloning and expression of a Bacteroides succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene in Escherichia coli. Appl Environ Microbiol. 1988 Nov;54(11):2672–2676. doi: 10.1128/aem.54.11.2672-2676.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LANDMAN O. E. Properties and induction of beta-galactosidase in Bacillus megaterium. Biochim Biophys Acta. 1957 Mar;23(3):558–569. doi: 10.1016/0006-3002(57)90377-3. [DOI] [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. Lee C., Li P., Inouye H., Brickman E. R., Beckwith J. Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane. J Bacteriol. 1989 Sep;171(9):4609–4616. doi: 10.1128/jb.171.9.4609-4616.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee S. F., Progulske-Fox A., Bleiweis A. S. Molecular cloning and expression of a Streptococcus mutans major surface protein antigen, P1 (I/II), in Escherichia coli. Infect Immun. 1988 Aug;56(8):2114–2119. doi: 10.1128/iai.56.8.2114-2119.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. MALAMY M., HORECKER B. L. The localization of alkaline phosphatase in E. coli K12. Biochem Biophys Res Commun. 1961 Jun 2;5:104–108. doi: 10.1016/0006-291x(61)90020-1. [DOI] [PubMed] [Google Scholar]
  19. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  20. Pollard H. B., Steers E., Jr Bacillus megaterium, KM beta-galactosidase: purification by affinity chromatography and characterization of the active species. Arch Biochem Biophys. 1973 Oct;158(2):650–661. doi: 10.1016/0003-9861(73)90557-2. [DOI] [PubMed] [Google Scholar]
  21. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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