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Genetics and Molecular Biology

Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseriADH

W. M. Russell, T. R. Klaenhammer
W. M. Russell
Department of Microbiology and
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T. R. Klaenhammer
Department of Microbiology and
Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695
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DOI: 10.1128/AEM.67.3.1253-1261.2001
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ABSTRACT

The gusA gene, encoding a new β-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a β-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 β-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 β-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 β-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 β-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in otherLactobacillus species tested.

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Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseriADH
W. M. Russell, T. R. Klaenhammer
Applied and Environmental Microbiology Mar 2001, 67 (3) 1253-1261; DOI: 10.1128/AEM.67.3.1253-1261.2001

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Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseriADH
W. M. Russell, T. R. Klaenhammer
Applied and Environmental Microbiology Mar 2001, 67 (3) 1253-1261; DOI: 10.1128/AEM.67.3.1253-1261.2001
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KEYWORDS

Cloning, Molecular
Glucuronidase
lactobacillus

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