Identification of a hypothetical protein of plant pathogenic Xanthomonas campestris as a novel beta-galactosidase.
Xc17L, a lactose-utilizing mutant of Xanthomonas campestris pv. campestris previously isolated by mutagenesis with nitrous acid, displays a level of beta-galactosidase 3.5-fold higher than that in the parental Xc17. In this study, the gene encoding the enzyme displaying a higher specific activity in Xc17L was inactivated by mini-Tn5 transposition. Sequencing revealed that the product (579 aa, 63.5 kDa) of this gene, designated galD, was previously annotated to encode a hypothetical protein on the genome. Mutation of the gene by marker exchange, complementation test and Western blot analysis together confirmed that galD is indeed the gene involved in beta-galactosidase elevation in Xc17L. With only the N-terminal region possessing similarity to the known beta-galactosidases and partially conserved consensus motif, GalD is recognized as a member of the glycosyl hydrolase family 35. Insertion with GmOmega, which causes polar effects, into the upstream genes followed by Western blotting showed that galD is cotranscribed with the upstream genes and expressed constitutively. Mutation in galD causes no significant changes including pathogenicity in the bacterium.
Yang
Tsuey-Ching
TC
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, ROC.
Hu
Rouh-Mei
RM
Weng
Shu-Fen
SF
Tseng
Yi-Hsiung
YH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009154:Mutation; D010944:Plants / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016959:Xanthomonas campestris / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D001616:beta-Galactosidase