Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase.
In the accompanying paper [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607-614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides. Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P. cellulosa (Deltaabf51A). The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium. Arabinofuranosidase activity in Deltaabf51A against aryl-alpha-arabinofuranosides, arabinan and alpha1,5-linked arabino-oligosaccharides was approx. 1% of the wild-type bacterium. The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P. cellulosa. These data indicate that P. cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.
Beylot
M H
MH
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK.
Emami
K
K
McKie
V A
VA
Gilbert
H J
HJ
Pell
G
G
D015139:Blotting, Southern; D050260:Carbohydrate Metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D003470:Culture Media; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase