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Acid resistance is an intrinsic characteristic of intestinal bacteria in order to survive passage through the stomach. Adenosine triphosphate (ATP), the ubiquitous chemical used to power metabolic reactions, activate signaling cascades, and form precursors of nucleic acids, was also found to be associated with the survival of Escherichia coli (E. coli) in acidic environments. The metabolic pathway responsible for elevating the level of ATP inside these bacteria during acid adaptation has been unclear. E. coli uses several mechanisms of ATP production, including oxidative phosphorylation, glycolysis and the oxidation of organic compounds. To uncover which is primarily used during adaptation to acidic conditions, we broadly analyzed the levels of gene transcription of multiple E. coli metabolic pathway components. Our findings confirmed that the primary producers of ATP in E. coli undergoing mild acidic stress are the glycolytic enzymes Glk, PykF and Pgk, which are also essential for survival under markedly acidic conditions. By contrast, the transcription of genes related to oxidative phosphorylation was downregulated, despite it being the major producer of ATP in neutral pH environments.

Background and purpose Increasing energy expenditure through adipocyte thermogenesis is generally accepted as a promising strategy to mitigate obesity and its related diseases. However, few clinically effective and safe agents are known to promote adipocyte thermogenesis. In this study, 20 traditional Chinese herbal medicines were screened to examine whether they induced adipocyte thermogenesis. Experimental approach The effects of Chinese herbal medicines or components isolated from extracts of A. membranaceus, on adipocyte thermogenesis were analysed by assessing expression of uncoupling protein 1 (UCP1) by qPCR. Eight-week-old C57BL6/J male mice were fed a high-fat diet for 8 weeks and then randomized to two groups treated with vehicle or formononetin for another 8 weeks. Glucose tolerance tests and staining of adipose tissue with haematoxylin and eosin were carried out. Whole-body oxygen consumption was measured with an open-circuit indirect calorimetry system. Key results Extracts of A. membranaceus increased expression of Ucp1 in primary cultures of mouse adipocytes. Formononetin was the only known component of A. membranaceus extracts to increase adipocyte Ucp1 expression. Diet-induced obese mice treated with formononetin gained less weight and showed higher energy expenditure than untreated mice. In addition, formononetin binds directly with PPARγ. Conclusions and implication Taken together, our study demonstrates that the Chinese herbal medicine from A. membranaceus and its constituent formononetin have the potential to reduce obesity and associated metabolic disorders. Our results suggest that formononetin regulates adipocyte thermogenesis as a non-classical PPARγ agonist.

ATP participates in many cellular metabolic processes as a major substrate to supply energy. Many systems for acidic resistance (AR) under extremely acidic conditions have been reported, but the role of ATP has not been examined. To clarify whether or not ATP is necessary for the AR in Escherichia coli , the AR of mutants deficient in genes for ATP biosynthesis was investigated in this study. The deletion of purA or purB , each of which encodes enzymes to produce AMP from inosinate (IMP), markedly decreased the AR. The content of ATP in these mutants decreased rapidly at pH 2.5 compared to that of the wild type. The AR was again decreased significantly by the mutation of adk , which encoded an enzyme to produce ADP from AMP. The DNA damage in the purA and purB mutants was higher than that in the wild type. These results demonstrated that metabolic processes that require ATP participate in survival under extremely acidic conditions, and that one such system is the ATP-dependent DNA repair system.

Background FabG is the only known enzyme that catalyzes reduction of the 3-ketoacyl-ACP intermediates of bacterial fatty acid synthetic pathways. However, there are two Ralstonia solanacearum genes, RSc1052 (fabG1) and RSp0359 (fabG2), annotated as encoding putative 3-ketoacyl-ACP reductases. Both FabG homologues possess the conserved catalytic triad and the N-terminal cofactor binding sequence of the short chain dehydrogenase/reductase (SDR) family. Thus, it seems reasonable to hypothesize that RsfabG1 and RsfabG2 both encode functional 3-ketoacyl-ACP reductases and play important roles in R. solanacearum fatty acid synthesis and growth. Methods Complementation of Escherichia colifabG temperature-sensitive mutant with R. solanacearum fabGs encoded plasmids was carried out to test the function of RsfabGs in fatty acid biosynthesis. RsFabGs proteins were purified by nickel chelate chromatography and fatty acid biosynthetic reaction was reconstituted to investigate the 3-ketoacyl-ACP reductase activity of RsFabGs in vitro. Disruption of both RsfabG genes was done via DNA homologous recombination to test the function of both RsfabG in vivo. And more we also carried out pathogenicity tests on tomato plants using RsfabG mutant strains. Results We report that expression of either of the two proteins (RsFabG1 and RsFabG2) restores growth of the E. coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrate that both proteins restore fatty acid synthetic ability to extracts of the E. coli strain. The RsfabG1 gene carried on the R. solanacearum chromosome is essential for growth of the bacterium, as is the case for fabG in E. coli. In contrast, the null mutant strain with the megaplasmid-encoded RsfabG2 gene is viable but has a fatty acid composition that differs significantly from that of the wild type strain. Our study also shows that RsFabG2 plays a role in adaptation to high salt concentration and low pH, and in pathogenesis of disease in tomato plants. Conclusion R. solanacearum encodes two 3-ketoacyl-ACP reductases that both have functions in fatty acid synthesis. We supply the first evidence that, like other enzymes in the bacterial fatty acid biosynthetic pathway, one bacterium may simultaneously possess two or more 3-oxoacyl-ACP reductase isozymes. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0554-x) contains supplementary material, which is available to authorized users.

Although the pH values of blood and tissues are usually maintained in a narrow range around 7.4, some diseased areas, such as cancer nests, inflammatory loci, and infarction areas, are acidified. In the present study, the effect of extracellular acidic pH on TCR signaling was examined with human acute leukemia T cell line Jurkat cells because T cell infiltration is often observed in acidic diseased areas. The phosphorylation levels of CD3-ξ ZAP-70, and PLC-γ1 induced by OKT-3, anti-CD3 antibody, were higher at pH 6.3 than those at pH 7.6. The activation of PLC-γ1 induced by OKT-3 was further increased by the co-stimulation with CD28.6, anti- CD28 antibody, at pH 7.6, but not at pH 6.3. The level of cytosolic free calcium ions was increased to a higher level by the addition of OKT-3 at pH 6.3, compared with that by the addition of OKT-3 plus CD28.6 at pH 7.6. Further addition of CD28.6 decreased the level of cytosolic free calcium ions induced by OKT-3 at pH 6.3. The Ca2+ mobilization was strongly inhibited by BTP2, a potent inhibitor of Ca2+ channels in the plasma membrane, at pH 7.6, while the inhibition was weak at pH 6.3. The Ca2+ mobilization at pH 6.3 was dependent on ZAP-70 and LAT, but not SLP-76. The activation of ERK and p38 increased as pH decreased. No activation of ERK2 in the presence of OKT-3 was observed in the Jurkat mutant deficient in ZAP-70 at pH 6.3, while ERK1 was activated by the addition of OKT-3 in this mutant. The expression of IL-2 was not induced by OKT-3 or OKT-3 plus CD28.6 at pH 6.3. These results suggest that the TCR signaling initiated by CD3 stimulation is more active at acidic pH in Jurkat cells and its pathway is different in parts under different pH conditions.

The Escherichia coli strain DH42 is sensitive to high osmolarity in an alkaline medium. Using mini-Tn5 mutagenesis, construction of mutant strains by homologous recombination and subcloning of DNA fragment techniques, gene ompC was identified as the key factor that, once disrupted, caused osmosis-sensitivity of E. coli strain DH42 grown in an alkaline medium. Through P1 transduction, a mutant strain, D9 (W3110 ompC:kan), was constructed and growth comparison was performed between DH42 and D9 under different pHs and salt concentrations. The result showed that ompC was necessarily required for hyperosmotic adaptation of E. coli in the alkaline medium.