Mutational analysis of exopolysaccharide biosynthesis by Lactobacillus sakei 0-1

Lactobacillus sakei strain 0-1 produces an exopolysaccharide (EPS) consisti ng of glucose and rhamnose in a ratio of 3:2. As part of a biochemical and molecular analysis of the EPS biosynthetic pathway in L. sakei strain 0-1, we have isolated a random set of EPS-negative mutants. Following treatment of cells with the mutagen ethylmethane sulfonic acid, a total of 10 mutants were identified that lacked the clear ropy appearance of wild-type colonie s on agar plates. Their characterization revealed that eight mutants had co mpletely lost the ability to synthesize the normal EPS. Six of these mutant s lacked activities of enzymes involved in the biosynthesis of dTDP-rhamnos e, required for EPS production. Only mutant strains 12 and 20 were directly affected in EPS synthesis. Strain 12 synthesized EPS with a different suga r composition, however. Interestingly, strain 12 showed temperature-depende nt EPS synthesis, with the highest amounts synthesized at 12 degrees C, and low amounts at the optimal temperature for growth (30 degrees C). Two muta nts were in fact EPS-positive, producing the normal EPS, but displayed a di fferent cell morphology (elongated cells), indicating a modification in cel l wall synthesis. (C) 1998 Federation of European Microbiological Societies . Published by Elsevier Science B.V. All rights reserved.