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. 1995 Aug;61(8):2958–2964. doi: 10.1128/aem.61.8.2958-2964.1995

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.

A Gasparic 1, J Martin 1, A S Daniel 1, H J Flint 1
PMCID: PMC167572  PMID: 7487028

Abstract

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.

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Selected References

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  1. Anderson K. L., Salyers A. A. Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes. J Bacteriol. 1989 Jun;171(6):3192–3198. doi: 10.1128/jb.171.6.3192-3198.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Avgustin G., Flint H. J., Whitehead T. R. Distribution of xylanase genes and enzymes among strains of Prevotella (Bacteroides) ruminicola from the rumen. FEMS Microbiol Lett. 1992 Dec 1;78(2-3):137–143. doi: 10.1016/0378-1097(92)90015-g. [DOI] [PubMed] [Google Scholar]
  3. BRYANT M. P., SMALL N., BOUMA C., CHU H. Bacteroides ruminicola n. sp. and Succinimonas amylolytica; the new genus and species; species of succinic acid-producing anaerobic bacteria of the bovine rumen. J Bacteriol. 1958 Jul;76(1):15–23. doi: 10.1128/jb.76.1.15-23.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dehority B. A. Effects of microbial synergism on fibre digestion in the rumen. Proc Nutr Soc. 1991 Aug;50(2):149–159. doi: 10.1079/pns19910026. [DOI] [PubMed] [Google Scholar]
  5. Flint H. J., Martin J., McPherson C. A., Daniel A. S., Zhang J. X. A bifunctional enzyme, with separate xylanase and beta(1,3-1,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens. J Bacteriol. 1993 May;175(10):2943–2951. doi: 10.1128/jb.175.10.2943-2951.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Flint H. J., McPherson C. A., Bisset J. Molecular cloning of genes from Ruminococcus flavefaciens encoding xylanase and beta(1-3,1-4)glucanase activities. Appl Environ Microbiol. 1989 May;55(5):1230–1233. doi: 10.1128/aem.55.5.1230-1233.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flint H. J., McPherson C. A., Martin J. Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13. J Gen Microbiol. 1991 Jan;137(1):123–129. doi: 10.1099/00221287-137-1-123. [DOI] [PubMed] [Google Scholar]
  8. Flipphi M. J., van Heuvel M., van der Veen P., Visser J., de Graaff L. H. Cloning and characterization of the abfB gene coding for the major alpha-L-arabinofuranosidase (ABF B) of Aspergillus niger. Curr Genet. 1993 Dec;24(6):525–532. doi: 10.1007/BF00351717. [DOI] [PubMed] [Google Scholar]
  9. Garcia-Campayo V., McCrae S. I., Zhang J. X., Flint H. J., Wood T. M. Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1-->4)-beta-D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli. Biochem J. 1993 Nov 15;296(Pt 1):235–243. doi: 10.1042/bj2960235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gardner R. G., Wells J. E., Russell J. B., Wilson D. B. The effect of carbohydrates on the expression of the Prevotella ruminicola 1,4-beta-D-endoglucanase. FEMS Microbiol Lett. 1995 Jan 15;125(2-3):305–310. doi: 10.1111/j.1574-6968.1995.tb07373.x. [DOI] [PubMed] [Google Scholar]
  11. Gasparic A., Marinsek-Logar R., Martin J., Wallace R. J., Nekrep F. V., Flint H. J. Isolation of genes encoding beta-D-xylanase, beta-D-xylosidase and alpha-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B1(4). FEMS Microbiol Lett. 1995 Jan 15;125(2-3):135–141. doi: 10.1111/j.1574-6968.1995.tb07349.x. [DOI] [PubMed] [Google Scholar]
  12. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lever M. Carbohydrate determination with 4-hydroxybenzoic acid hydrazide (PAHBAH): effect of bismuth on the reaction. Anal Biochem. 1977 Jul;81(1):21–27. doi: 10.1016/0003-2697(77)90594-2. [DOI] [PubMed] [Google Scholar]
  15. Mannarelli B. M., Evans S., Lee D. Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens. J Bacteriol. 1990 Aug;172(8):4247–4254. doi: 10.1128/jb.172.8.4247-4254.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matsushita O., Russell J. B., Wilson D. B. Cloning and sequencing of a Bacteroides ruminicola B(1)4 endoglucanase gene. J Bacteriol. 1990 Jul;172(7):3620–3630. doi: 10.1128/jb.172.7.3620-3630.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Reilly P. J. Xylanases: structure and function. Basic Life Sci. 1981;18:111–129. doi: 10.1007/978-1-4684-3980-9_8. [DOI] [PubMed] [Google Scholar]
  18. Sakka K., Yoshikawa K., Kojima Y., Karita S., Ohmiya K., Shimada K. Nucleotide sequence of the Clostridium stercorarium xylA gene encoding a bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities, and properties of the translated product. Biosci Biotechnol Biochem. 1993 Feb;57(2):268–272. [PubMed] [Google Scholar]
  19. Strobel H. J. Pentose utilization and transport by the ruminal bacterium Prevotella ruminicola. Arch Microbiol. 1993;159(5):465–471. doi: 10.1007/BF00288595. [DOI] [PubMed] [Google Scholar]
  20. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  21. Utt E. A., Eddy C. K., Keshav K. F., Ingram L. O. Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities. Appl Environ Microbiol. 1991 Apr;57(4):1227–1234. doi: 10.1128/aem.57.4.1227-1234.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Vercoe P. E., Gregg K. DNA sequence and transcription of an endoglucanase gene from Prevotella (Bacteroides) ruminicola AR20. Mol Gen Genet. 1992 May;233(1-2):284–292. doi: 10.1007/BF00587590. [DOI] [PubMed] [Google Scholar]
  23. Weaver J., Whitehead T. R., Cotta M. A., Valentine P. C., Salyers A. A. Genetic analysis of a locus on the Bacteroides ovatus chromosome which contains xylan utilization genes. Appl Environ Microbiol. 1992 Sep;58(9):2764–2770. doi: 10.1128/aem.58.9.2764-2770.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Whitehead T. R. Analyses of the gene and amino acid sequence of the Prevotella (Bacteroides) ruminicola 23 xylanase reveals unexpected homology with endoglucanases from other genera of bacteria. Curr Microbiol. 1993 Jul;27(1):27–33. doi: 10.1007/BF01576830. [DOI] [PubMed] [Google Scholar]
  25. Wong K. K., Tan L. U., Saddler J. N. Multiplicity of beta-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev. 1988 Sep;52(3):305–317. doi: 10.1128/mr.52.3.305-317.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zhang J. X., Flint H. J. A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine-rich sequence. Mol Microbiol. 1992 Apr;6(8):1013–1023. doi: 10.1111/j.1365-2958.1992.tb02167.x. [DOI] [PubMed] [Google Scholar]
  27. von Heijne G. Transcending the impenetrable: how proteins come to terms with membranes. Biochim Biophys Acta. 1988 Jun 9;947(2):307–333. doi: 10.1016/0304-4157(88)90013-5. [DOI] [PubMed] [Google Scholar]

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