Analysis of the ArcA regulon in anaerobically grown <Emphasis Type="Italic">Salmonella enterica sv</Emphasis>. Typhimurium

Background  

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that must successfully adapt to the broad fluctuations in the concentration of dissolved dioxygen encountered in the host. In Escherichia coli, ArcA (Aerobic Respiratory Control) helps the cells to sense and respond to the presence of dioxygen. The global role of ArcA in E. coli is well characterized; however, little is known about its role in anaerobically grown S. Typhimurium.     

Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.     

Microbial production of meso-2,3-butanediol by metabolically engineered Escherichia coli under low oxygen condition

A metabolically engineered Escherichia coli has been constructed for the production of meso-2,3-butanediol (2,3-BD) under low oxygen condition. Genes responsible for 2,3-BD formation from pyruvate were assembled together to generate a high-copy plasmid pEnBD, in which each gene was transcribed with a constitutive promoter. To eliminate by-product formation under low oxygen condition, genes including ldhA, pta, adhE, and poxB which functioned for the mixed acid fermentation pathways were deleted in E. coli JM109. Compared with the wild type, the quadruple gene deletion mutant produced smaller amounts of acetate, succinate, and ethanol from glucose when cultivated in LB medium in shake flasks under low-aeration. When 2,3-BD producing pathway was introduced via pEnBD into the mutant, higher glucose consumption and faster 2,3-BD production rate compared with that of the wild-type control were observed under aerobic condition in shake flasks. In a 6-L fermentor supplied with only 3% dissolved oxygen (DO), the mutant harboring pEnBD converted glucose to 2,3-BD much faster than the control did. When DO supply was further lowered to 1% DO, the recombinant mutant grew much slower but produced 2,3-BD as a major fermentation metabolic product. In addition, the 2,3-BD yield showed an increase from 0.20 g BD/g glucose for the control to 0.43 g BD/g glucose for the mixed acid pathway deleted mutant grown in fermentors under 1% DO. These results reveals the potential of production of enantiomerically pure 2,3-BD isomer by recombinant E. coli under low oxygen condition.     

Citrate utilization under anaerobic environment in Escherichia coli is under direct control of Fnr and indirect control of ArcA and Fnr via CitA-CitB system

Most Escherichia coli (E. coli) strains do not cause disease, naturally living in the lower intestine and is expelled into the environment within faecal matter. Escherichia coli can utilize citrate under anaerobic conditions but not aerobic conditions. However, the underlying regulatory mechanisms are poorly understood. In this study, we explored regulatory mechanisms of citrate fermentation genes by global regulators ArcA and Fnr under anaerobic conditions. A gel mobility shift assay showed that the regulator proteins ArcA and Fnr binded to the promoter region localized between the citAB and citCDEFXGT operons. Subsequent assays confirmed that ArcA indirectly controled the expression of citrate fermentation genes via regulating CitA-CitB system, while Fnr directly regulated but also indirectly modulated citrate fermentation genes via controling CitA-CitB system. Deletions of arcA and fnr significantly reduced the growth of Escherichia coli in M9 medium with a citrate carbon source. We conclude that both ArcA and Fnr can indirectly control the citrate utilization via CitA-CitB system, while Fnr can also directly regulate the expression of citrate fermentation genes in E. coli under anaerobic conditions.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress

Background  

The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions.     

Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors

Background  

Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation. Because there are greater than 10⁹ cells in a fermentor, one can outfit a vessel with more than 10⁹ sensors. Our concept was tested in high density, lab-scale (5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency – one verses four impellers.     

Evidence against the physiological role of acetyl phosphate in the phosphorylation of the ArcA response regulator in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Arc two-component signal transduction system of Escherichia coli comprises the ArcB sensor kinase and the ArcA response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphos-phorylates ArcA, which, in turn, represses or activates its target operons. ArcA has been shown to be able to autophosphorylate in vitro at the expense of acetyl-P. Here, the in vivo effect of acetyl phosphate on the redox signal transduction by the Arc system was assessed. Our results indicate that acetyl phosphate can modulate the expression of ArcA-P target genes only in the absence of ArcB. Therefore, the acetyl phosphate dependent ArcA phosphorylation route does not seem to play a significant role under physiological conditions.