Regulation of CcpA on the growth and organic acid production characteristics of ruminal Streptococcus bovis at different pH

Background Catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase and pyruvate formate-lyase in Streptococcus bovis, but knowledge of its role in response to different pH is still limited. In this study, a ccpA-knockout strain of S. bovis S1 was constructed and then used to examine the effects of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at pH 5.5 or 6.5. Results There was a significant interaction between strain and pH for the maximum specific growth rate (μmax) and growth lag period (λ), which caused a lowest μmax and a longest λ in ccpA-knockout strain at pH 5.5. Deletion of ccpA decreased the concentration and molar percentage of lactic acid, while increased those of formic acid. Strains at pH 5.5 had decreased concentrations of lactic acid and formic acid compared to pH 6.5. The significant interaction between strain and pH caused the highest production of total organic acids and acetic acid in ccpA-knockout strain at pH 6.5. The activities of α-amylase and lactate dehydrogenase decreased in ccpA-knockout strain compared to the wild-type strain, and increased at pH 5.5 compared to pH 6.5. There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-knockout strain at pH 6.5. The expression of pyruvate formate-lyase and acetate kinase was higher in the ccpA-knockout strain compared to wild-type strain. The lower pH improved the relative expression of pyruvate formate-lyase, while had no effect on the relative expression of acetate kinase. The strain × pH interaction was significant for the relative expression of lactate dehydrogenase and α-amylase, both of which were highest in the wild-type strain at pH 5.5 and lowest in the ccpA-knockout strain at pH 6.5. Conclusions Overall, low pH inhibited the growth of S. bovis S1, but did not affect the fermentation pattern. CcpA regulated S. bovis S1 growth and organic acid fermentation pattern. Moreover, there seemed to be an interaction effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1.

Noise in a metabolic pathway leads to persister formation in Mycobacterium tuberculosis

Tuberculosis is difficult to treat due to dormant cells in hypoxic granulomas, and stochastically-formed persisters tolerant of antibiotics. Bactericidal antibiotics kill by corrupting their energy-dependent targets. We reasoned that noise in the expression of an energy-generating component will produce rare persister cells. In sorted low ATP M. tuberculosis grown on acetate there is considerable cell-to-cell variation in the level of mRNA coding for AckA, the acetate kinase. Quenching the noise by overexpressing ackA sharply decreases persisters, showing that it acts as the main persister gene under these conditions. This demonstrates that a low energy mechanism is responsible for the formation of M. tuberculosis persisters and suggests that the mechanism of their antibiotic tolerance is similar to that of dormant cells in a granuloma. Entrance into a low energy state driven by stochastic variation in expression of energy-producing enzymes is likely a general mechanism by which bacteria produce persisters.

Acetate excretion by a methanotroph, Methylocaldum marinum S8, under aerobic conditions

ABSTRACT Methane-oxidizing bacteria (methanotrophs) often coexist with methylotrophs that utilize methanol excreted by methanotrophs. Recently, we found that a facultative methylotroph, Methyloceanibacter caenitepidi Gela4T, possibly utilizes acetate rather than methanol in the coculture with a methanotroph, Methylocaldum marinum S8. Here, we examined the effects of oxygen concentrations on growth of and acetate excretion by M. marinum S8 in pure culture and the coculture with M. caenitepidi Gela4T. M. marinum S8 excreted acetate during the exponential growth phase not only under microaerobic (O2 concentrations of 3.5%-6%) but also under aerobic (O2 concentrations of 20%-31%) conditions. RNA-Seq analyses of M. marinum S8 cells grown under aerobic conditions suggested that phosphoketolase and acetate kinase were candidate genes involved in acetate production. Nonmethylotrophic bacteria, Cupriavidus necator NBRC 102504, could grow when cocultured with M. marinum S8, also supporting the existence of methanol-independent cross-feeding from M. marinum S8 under aerobic conditions.

A new pathway for forming acetate and synthesizing ATP during fermentation in bacteria

ABSTRACTMany bacteria and other organisms form acetate during fermentation. Forming acetate from high energy-precursors (acetyl-CoA or acetyl phosphate) is one of the few ways that fermentative bacteria generate ATP. Here we found a biochemical pathway for forming acetate and synthesizing ATP that was unknown in bacteria. We found the bacterium Cutibacterium granulosum formed acetate during fermentation of glucose. With enzymatic assays, we showed it formed acetate using a pathway involving two enzymes. The first enzyme, succinyl-CoA:acetate CoA-transferase (SCACT), forms acetate from acetyl-CoA. The second enzyme, succinyl-CoA synthetase (SCS), synthesizes ATP. This pathway is common in eukaryotes, but it has not been found in bacteria or other organisms. We found two related bacteria (C. acnes and Acidipropionibacterium acidipropionici) also used this pathway. None used the most common pathway for forming acetate in bacteria (involving acetate kinase and phosphotransacetylase). The SCACT/SCS pathway may be used by many bacteria, not just C. granulosum and relatives. When we searched genomes for bacteria known to form acetate, we found over 1/6 encoded this pathway. These bacteria belong to 104 different species and subspecies in 12 different phyla. With this discovery, all five pathways known to form acetate and ATP during fermentation can be found in bacteria. This discovery is important to manipulating fermentation and to the evolution of biochemical pathways.

Control of acetyl phosphate-dependent phosphorylation of the response regulator CiaR by acetate kinase in Streptococcus pneumoniae

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.

Acetate kinase activity test - a new approach to biogas production monitoring in the presence of chlorophenols

The proper functioning of processes related to the use of alternative energy sources is one of the key aspects in the field of biotechnology. Therefore, there is a need to develop new techniques allowing the adequate monitoring of the biogas production, especially in the presence of potentially inhibiting substances. The negative impact of chlorophenols on methane fermentation proces was confirmed in numerous studies. The inhibition effect is usually monitored based on chemical and microbiological assessment. However, the results of the tests sometimes do not reflect adequately the actual process. In this research the acetate kinase activity test was proposed as a sensitive control parameter for the monitoring of biogas production in the presence of 2,4-dichlorophenol. Acetate kinase can be considered a key enzyme in methanogenesis. The determination of its activity is simpler and less time consuming compared to other monitoring methods. The amount of the iron acetyl hydroxyamine complex measured in the proposed assay corresponds to the amount of acetyl phosphate formed during the methane fermentation. It was revealed that the acetate kinase activity can better reflect the changes in biogas yield and in methane concentration compared with chemical and microbiological analyses as well as the total dehydrogenase activity tests. Keywords: 2,4-dichlorophenol; Methane fermentation; Biogas production monitoring; Acetate kinase