SummaryIn many streptococci, competence for natural DNA transformation is regulated by the Rgg-type regulator ComR and the pheromone ComS, which is sensed intracellularly. We compared the ComRS systems of four model streptococcal species using in vitro and in silico approaches, to determine the mechanism of the ComRS-dependent regulation of competence. In all systems investigated, ComR was shown to be the proximal transcriptional activator of the expression of key competence genes. Efficient binding of ComR to DNA is strictly dependent on the presence of the pheromone (C-terminal ComS octapeptide), in contrast with other streptococcal Rgg-type regulators. The 20 bp palindromic ComR-box is the minimal genetic requirement for binding of ComR, and its sequence directly determines the expression level of genes under its control. Despite the apparent speciesspecific specialization of the ComR-ComS interaction, mutagenesis of ComS residues from Streptococcus thermophilus highlighted an unexpected permissiveness with respect to its biological activity. In agreement, heterologous ComS, and even primary sequence-unrelated, casein-derived octapeptides, were able to induce competence development in S. thermophilus. The lack of stringency of ComS sequence suggests that competence of a specific Streptococcus species may be modulated by other streptococci or by non-specific nutritive oligopeptides present in its environment.

The pyruvate oxidase gene (poxB) from Lactobacillus plantarum Lp80 was cloned and characterized. Northern blot and primer extension analyses revealed that transcription of poxB is monocistronic and under the control of a vegetative promoter. poxB mRNA expression was strongly induced by aeration and was repressed by glucose. Moreover, Northern blotting performed at different stages of growth showed that poxB expression is maximal in the early stationary phase when glucose is exhausted. Primer extension and in vivo footprint analyses revealed that glucose repression of poxB is mediated by CcpA binding to the cre site identified in the promoter region. The functional role of the PoxB enzyme was studied by using gene overexpression and knockout in order to evaluate its implications for acetate production. Constitutive overproduction of PoxB in L. plantarum revealed the predominant role of pyruvate oxidase in the control of acetate production under aerobic conditions. The ⌬poxB mutant strain exhibited a moderate (20 to 25%) decrease in acetate production when it was grown on glucose as the carbon source, and residual pyruvate oxidase activity that was between 20 and 85% of the wild-type activity was observed with glucose limitation (0.2% glucose). In contrast, when the organism was grown on maltose, the poxB mutation resulted in a large (60 to 80%) decrease in acetate production. In agreement with the latter observation, the level of residual pyruvate oxidase activity with maltose limitation (0.2% maltose) was less than 10% of the wild-type level of activity.

Heterogeneity or segregation of microbial populations has been the subject of much research, but the real impact of this phenomenon on bioprocesses remains poorly understood. The main reason for this lack of knowledge is the difficulty in monitoring microbial population heterogeneity under dynamic process conditions. The main concepts resulting in microbial population heterogeneity in the context of bioprocesses have been summarized by two distinct hypotheses. The first involves the individual history of microbial cells or the "path" followed during their residence time inside the process equipment. The second hypothesis involves a coordinated response by the microbial population as a bet-hedging strategy, in order to cope with process-related stresses. The respective contribution of each hypothesis to microbial heterogeneity in bioprocesses is still unclear. This illustrates the fact that, although microbial phenotypic heterogeneity has been thoroughly investigated at a fundamental level, the implications of this phenomenon in the context of microbial bioprocesses are still subject to debate. At this time, automated flow cytometry is the best technique for investigating microbial heterogeneity under process conditions. However, dedicated software and relevant biomarkers are needed for the proper integration of flow cytometry as a bioprocess control tool.

Racemases catalyze the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/β fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes.