Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amounts of a glucan (d-glucose) and a fructan (d-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and 13C or1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted α-glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2→6)-β-d-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan.
Fructosyltransferase (FTF) enzymes have been characterized from various Gram‐positive bacteria, but not from Lactobacillus sp. In a screening of 182 lactobacilli for polysaccharide production only one strain, Lactobacillus reuteri strain 121, was found to produce a fructan being a levan. Here we report the first‐time identification and biochemical characterization of a Lactobacillus FTF enzyme. When incubated with sucrose the enzyme produced a levan that is identical to that produced by Lb. reuteri strain 121 cells.
Lactobacillus sakei strain 0-1 produces an exopolysaccharide (EPS) consisting 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 colonies on agar plates. Their characterization revealed that eight mutants had completely lost the ability to synthesize the normal EPS. Six of these mutants lacked activities of enzymes involved in the biosynthesis of dTDP-rhamnose, required for EPS production. Only mutant strains 12 and 20 were directly affected in EPS synthesis. Strain 12 synthesized EPS with a different sugar composition, however. Interestingly, strain 12 showed temperature-dependent EPS synthesis, with the highest amounts synthesized at 12³C, and low amounts at the optimal temperature for growth (30³C). Two mutants were in fact EPS-positive, producing the normal EPS, but displayed a different cell morphology (elongated cells), indicating a modification in cell wall synthesis. z
Contrary to expectation, a mutant of Hansenula polymorpha blocked in dihydroxyacetone (DHA) synthase was able to assimilate methanol‐carbon when grown in chemostat culture on mixtures of xylose and methanol. Incubation of a DHA synthase‐ and DHA kinase‐negative double mutant resulted in DHA accumulation, indicating that a DHA synthase‐type of reaction was involved. Low residual DHA synthase activity subsequently was shown to be present when using an assay with improved sensitivity. This activity was not associated with the (mutated) DHA synthase protein, which was still present in the peroxisomes, but with the enzyme transketolase. Transketolase from methanol grown cells was purified (525‐fold) to homogeneity in 9% yield. The native enzyme was dimeric, as has been reported fro other transketolases, with a subunit molecular weight of 74000. The affinity of the purified enzyme for formaldehyde was low (Km = 5 mM), but high for xylulose‐5‐phosphate (ca. 10 μM). The in vivo functioning of transketolase in formaldehyde assimilation, and the influence of the hydration state of formaldehyde is discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.