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Applied and Environmental Microbiology, March 2001, p. 1253-1261, Vol. 67, No. 3
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.3.1253-1261.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification and Cloning of gusA, Encoding a New beta -Glucuronidase from Lactobacillus gasseri ADH†

W. M. Russell1 and T. R. Klaenhammer1,2,*

Department of Microbiology1 and Department of Food Science,2 Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695

Received 29 September 2000/Accepted 8 January 2001

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.

* Corresponding author. Mailing address: Department of Food Science, North Carolina State University, Box 7624, Raleigh, NC 27695. Phone: (919) 515-2972. Fax: (919) 515-7124. E-mail: klaenhammer{at}ncsu.edu.

dagger Paper no. FSR-00-26 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh, NC 27695-7624.

Applied and Environmental Microbiology, March 2001, p. 1253-1261, Vol. 67, No. 3
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.3.1253-1261.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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