Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose biosynthesis

I.C. Boels, M.M. Beerthuyzen, M.H. Kosters, M.P.W. van Kaauwen, M. Kleerebezem, W.M. de Vos

Research output: Contribution to journalArticleAcademicpeer-review

28 Citations (Scopus)

Abstract

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.
Original languageEnglish
Pages (from-to)1239-1248
JournalJournal of Bacteriology
Volume186
Issue number5
DOIs
Publication statusPublished - 2004

Keywords

  • controlled gene-expression
  • gram-positive bacteria
  • subsp cremoris
  • capsular polysaccharide
  • streptococcus-pneumoniae
  • acid bacteria
  • cell-wall
  • o-antigen
  • exopolysaccharide production
  • escherichia-coli

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