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

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were a nalyzed for the chemical compositions of their EPSs and the locations, sequ ences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representat ives 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 clon ed, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase i nvolved in the second step of repeating-unit synthesis. The NIZO B40 primin g glucosyltransferase gene epsD was replaced with an erythromycin resistanc e gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with know n function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus th ermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could c ompletely restore or even increase EPS production in L. lactis.