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.
. 178:5431-5437, 1996). Production of D-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for D-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires L-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by L-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of L-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is D-lactate dependent. We also show that the growth defect can be partially restored by expression of the D-alanyl-D-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various D-2-hydroxy acids but not D-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in D-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in D-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for D-lactate production upon inactivation or inhibition of the LdhD enzyme.In lactic acid bacteria (LAB), the pyruvate formed by the Embden-Meyerhof-Parnas pathway is reduced to lactate by NAD-dependent lactate dehydrogenases (Ldh). These enzymes are stereospecific and produce D-lactate (LdhD, EC 1.1.1.28) or L-lactate (LdhL, EC 1.1.1.27). LAB can be classified on the basis of the lactate stereoisomer(s) produced during growth on glucose, which is thought to reflect the type of Ldh(s) present in a species. LAB are usually divided in three groups based on the ratio of isomers produced (21,31,39,45).Most lactobacilli are DL-lactate producers, but the ratio of the two isomers is highly variable. This has mainly been attributed to different activities of the LdhD and LdhL enzymes (21, 45). Some exceptions among lactobacilli are Lactobacillus delbrueckii subsp. bulgaricus, which produces mainly D-lactate, in agreement with the absence of LdhL activity, and Lactobacillus casei, where L-lactate is the major isomer formed (21,39). In this species, the pathway of D-lactate production has not been investigated.The presence of a lactate racemase (EC 5. (27), and several halophilic archaea (41). Very few biochemical studies on lactate racemase have been reported. This is mainly due to the fact that the enzyme seems to be highly sensitive to oxidation (13,45). The enzymes from L. sakei and C. beijerinckii have been purified, and basic biochemical properties have been determined (9,29). A catalytic mechanism has been proposed for the lactate racemase of...
In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant.Acetate is the major fermentation end product of the lactic acid bacterium Lactobacillus plantarum when cultivated under aerobic conditions and sugar limitation. It is produced at the expense of lactate as glucose becomes depleted and cells enter the stationary phase of growth. The pathway for lactate-toacetate conversion under these conditions has been shown to involve three enzymatic steps (2,6,12,20): oxidation of lactate to pyruvate by the NAD-dependent D-and L-lactate dehydrogenases (LDH), oxidative decarboxylation of pyruvate to acetyl-phosphate (acetylϳP) by pyruvate oxidase (POX), and dephosphorylation of acetylϳP to acetate by acetate kinase (ACK). This last step produces ATP, which is believed to provide the cells with the additional energy needed for survival in the stationary phase. Acetate itself could also be involved in increased survival by maintaining the pH homeostasis (12,20). Concerning applications, the maintenance of a high viability in the stationary phase under aerobic conditions could be relevant in the development of long-shelf-life probiotic dairy products containing L. plantarum (14,29). Besides its implication in cell survival, acetate is also an important flavor compound of fermented products (e.g., sourdoughs) in which L. plantarum plays a major role (4, 5). Therefore, a better understanding of the pathways involved in acetate production in this species could contribute to the improvement of fermentation processes and products.Previously, it has been established that the oxidative decarboxylation of pyruvate catalyzed by POX is a key step in the lactate-to-acetate conversion pathway (12,27). A null mutant for the gene encoding PoxB, the major POX of L. plantarum, shows a decrease in acetate production up to 80% compared to the parent strain, depending on the growth conditions (12).This LDH-POX-ACK pathway is under control of two environmental factors: sugar and oxygen availability. Regulation takes place essentially at the level of POX activity, which is induced by oxygen or hydrogen peroxide and repressed by glucose (12,19,20,27). In the presence of excess glucose, P...
The physiology of Lactobacillus plantarum at extremely low growth rates, through cultivation in retentostats, is much closer to carbon-limited growth than to stationary phase, as evidenced from transcriptomics data, metabolic fluxes, and biomass composition and viability.Using a genome-scale metabolic model and constraint-based computational analyses, amino-acid fluxes—in particular, the rather paradoxical excretion of Asp, Arg, Met, and Ala—could be rationalized as a means to allow extensive metabolism of other amino acids, that is, that of branched-chain and aromatic amino acids.Catabolic products from aromatic amino acids are known to have putative plant-hormone action. The metabolism of amino acids, as well as transcription data, strongly suggested a plant environment-like response in slow-growing L. plantarum, which was confirmed by significant effects of fermented medium on plant root formation.
NAD-independent lactate dehydrogenases are commonly thought to be responsible for lactate utilization during the stationary phase of aerobic growth in Lactobacillus plantarum. To substantiate this view, we constructed single and double knockout mutants for the corresponding genes, loxD and loxL. Lactate-to-acetate conversion was not impaired in these strains, while it was completely blocked in mutants deficient in NADdependent lactate dehydrogenase activities, encoded by the ldhD and ldhL genes. We conclude that NADdependent but not NAD-independent lactate dehydrogenases are involved in this process.
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