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. 2001 Jul;67(7):3033-40.
doi: 10.1128/AEM.67.7.3033-3040.2001.

Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis

I C Boels  1 , A Ramos, M Kleerebezem, W M de Vos
Affiliations

Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis

I C Boels et al. Appl Environ Microbiol. 2001 Jul.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Schematic representation of pathways involved in glucose fermentation via glycolysis (upper grey box), galactose fermentation via the Leloir pathway (lower grey box), and biosynthesis of EPS in L. lactis. Cell membranes are indicated by vertical lines. The following enzymes are involved (encoding genes are indicated): phosphoglucomutase (pgm); UDP-glucose pyrophosphorylase (galU); UDP-galactose epimerase (galE); galactose-1-phosphate uridyltransferease (galT); galactokinase (galK); galactose mutarotase (galM); and dTDP-rhamnose biosynthetic enzyme system (rfb). Multiple-step reactions are lumped together and are indicated by dotted arrows. The multiple reactions involved in synthesis of sugar polymers are indicated by diamonds.
FIG. 2
FIG. 2
Coomassie blue-stained gel after SDS-PAGE of CE of L. lactis subsp. cremoris NZ9000 (lane 2) and NZ9000 harboring pNZ4102 grown in the absence (lane 3) or in the presence (lane 4) of nisin. Lane 1 contained a molecular mass marker (molecular masses [in kilodaltons] are indicated on the left). The arrow indicates the position of the overproduced GalU protein.
See this image and copyright information in PMC

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