Functional analysis of glycosyltransferase genes from Lactococcus lactis and other gram-positive cocci: complementation, expression, and diversity

Abstract

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.

FIG. 1

(A) Genetic map of the eps gene cluster of L. lactis NIZO B40. The SstI recognition sites are indicated by downward-pointing arrows. The predicted functions of the gene products are depicted above the map (34). (B) DNA fragments of the NIZO B40 eps gene cluster used for hybridization. (C) PCR fragments generated by the primers indicated by the arrowheads used to determine the order of the conserved eps genes of various strains (see text). (D and E) Genetic maps of the eps gene cluster of L. lactis NIZO B891 and NIZO B35 based on DNA sequences of cloned fragments and on PCR analysis.

FIG. 2

TLC of ¹⁴C-labelled intermediates isolated from the lipid fraction of permeabilized E. coli cells. (A) E. coli expressing NIZO B35 epsD incubated with UDP-[¹⁴C]galactose. (B) E. coli expressing NIZO B891 epsD (1, 2) or NIZO B891 epsDEF incubated with a combination of UDP-[¹⁴C]glucose and UDP-[¹⁴C]galactose (3, 4). The positions of the standard sugars glucose (Glc), galactose (Gal), and lactose (Glc-Gal) are indicated on the right. The products which are nonspecific for lactococcal GTF activity are indicated by the asterisk on the left. C, complete acid hydrolysis; M, mild acid hydrolysis.

FIG. 3

Multiple-sequence alignment of priming GTFs with known sugar specificity from gram-positive bacteria. Cps14E, B40EpsD, and B891EpsD are glucosyltransferases from S. pneumoniae serotype 14 (11) and L. lactis NIZO B40 and NIZO B891, respectively. B35EpsD and Sfi6EpsE are galactosyltransferases from L. lactis NIZO B35 and S. thermophilus Sfi6 (30), respectively. Residues conserved in all five sequences, residues conserved only in glucosyltransferases, and residues conserved only in galactosyltransferases are shaded light grey, dark grey, and black, respectively. The three conserved blocks (A, B, and C) described by Wang et al. (40) are indicated.