Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH.
The gusA gene, encoding a new beta-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a beta-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored beta-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to beta-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a beta-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified beta-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.
Department of Microbiology, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695, USA.
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WM
Russell
T R
TR
Klaenhammer
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D005966:Glucuronidase