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Applied and Environmental Microbiology, July 2001, p. 3033-3040, Vol. 67, No. 7
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.7.3033-3040.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Analysis of the Lactococcus lactis galU and galE Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis

Ingeborg C. Boels,1,2 Ana Ramos,3 Michiel Kleerebezem,1,2,* and Willem M. de Vos1

Wageningen Centre for Food Sciences, Wageningen,1 and NIZO Food Research, Ede,2 The Netherlands, and Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa and Instituto de Biologia Experimental e Technólogica, Oeiras, Portugal3

Received 8 November 2000/Accepted 20 April 2001

We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.

* Corresponding author. Mailing address: NIZO Food Research, Department of Flavor and Natural Ingredients, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31-318-659511. Fax: 31-318-650400. E-mail: kleerebe{at}nizo.nl.

Applied and Environmental Microbiology, July 2001, p. 3033-3040, Vol. 67, No. 7
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.7.3033-3040.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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