Involvement of ArcA and Fnr in expression of Escherichia coli thiol peroxidase gene

To explore the oxygen response regulators involved in thiol peroxidase gene (tpx) expression in Escherichia coli, we constructed a single-copy tpx-lacZ operon fusion and monitored tpx-lacZ expression in various genetic backgrounds. Expression of the tpx-lacZ fusion was increased 4-fold by aerobic growth. Anaerobic expression of tpx-lacZ in either (delta)arcA or delta(fnr) strains was 2.5-fold depressed compared with that of the wild-type strain. The results of immunoblotting experiments also demonstrated that ArcA and Fnr regulatory proteins repressed thiol peroxidase gene expression during anaerobic growth. Inspection of the tpx promoter region revealed putative binding sites for ArcA and Fnr. It thus appears that ArcA and Fnr function as repressors by blocking the binding of RNA polymerase to the tpx promoter in E. coli under anaerobic growth conditions.     

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.     

Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr

Succinate dehydrogenase (SDH) of Escherichia coli, the sole membrane-bound enzyme of the tricarboxylic acid cycle, participates in the aerobic electron-transport pathway to generate energy via oxidative phosphorylation reactions. Previous studies have established that succinate dehydrogenase (SDiH) synthesis is elevated by aerobiosis and supressed during growth with glucose. To examine how the sdhCDAB genes that encode SDH are regulated by changes in the environment, sdh–lacZ fusions were constructed and analysed in vivo following cell growth under a variety of alternative culture conditions. Expression of sdh–lacZ was highest under aerobic conditions and was decreased 10-foid in the absence of oxygen. The fnr and arcA gene products are required for this oxygen control and each acts to repress sdhC–lacZ expression. Expression of sdh–lacZ also varied 10- to 14-foid depending on the type of carbon substrate used or the medium richness. This control was shown to be independent of the crp and fruR gene products, and indicates that some other regulatory element exists in the ceil to adjust SDH enzyme levels accordingly. Iron and haem availability affected sdhC–lacZ expression by two- to threefold. Lastly, fold. Lastly, sdhC–lacZ expression was shown to vary with the cell growth rate during aerobic and anaerobic conditions.     

arcA基因提高大肠杆菌对有机溶剂的耐受性

【目的】将来源于恶臭假单胞菌(Pseudomonas putida JUCT1)的基因arc A(编码精氨酸脱亚胺酶)整合到Escherichia coli JM109(DE3)基因组中,以提高该菌对有机溶剂的耐受性。【方法】以P.putida JUCT1的基因组为模板扩增基因arc A,并与p ET-20b(+)连接后导入E.coli JM109(DE3)中,验证该基因提高E.coli JM109(DE3)对有机溶剂的耐受性。利用Red同源重组的方法将arc A整合到E.coli JM109(DE3)基因组中。【结果】E.coli JM109(DE3)/p ET-20b(+)-arc A在添加了2.0%(体积比)环己烷、0.1%(体积比)甲苯、4.0%(体积比)萘烷和0.1%(体积比)丁醇的培养基中培养8 h后,其OD660由初始的0.2分别上升到0.8、0.9、1.8和1.3。将arc A成功整合到E.coli JM109(DE3)基因组中,获得了具有较好遗传稳定性的溶剂耐受E.coli JM109(DE3)宿主菌株。【结论】外源基因arc A能提高大肠杆菌菌株的有机溶剂耐受性,为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。     

Global gene expression profiling in Escherichia coli K12: effects of oxygen availability and ArcA

The ArcAB two-component system of Escherichia coli regulates the aerobic/anaerobic expression of genes that encode respiratory proteins whose synthesis is coordinated during aerobic/anaerobic cell growth. A genomic study of E. coli was undertaken to identify other potential targets of oxygen and ArcA regulation. A group of 175 genes generated from this study and our previous study on oxygen regulation (Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield, G. W., and Gunsalus, R. P. (2003) J. Biol. Chem. 278, 29837-29855), called our gold standard gene set, have p values <0.00013 and a posterior probability of differential expression value of 0.99. These 175 genes clustered into eight expression patterns and represent genes involved in a large number of cell processes, including small molecule biosynthesis, macromolecular synthesis, and aerobic/anaerobic respiration and fermentation. In addition, 119 of these 175 genes were also identified in our previous study of the fnr allele. A MEME/weight matrix method was used to identify a new putative ArcA-binding site for all genes of the E. coli genome. 16 new sites were identified upstream of genes in our gold standard set. The strict statistical analyses that we have performed on our data allow us to predict that 1139 genes in the E. coli genome are regulated either directly or indirectly by the ArcA protein with a 99% confidence level.     

Erratum to: Deletion of arcA increased the production of acetyl-CoA-derived chemicals in recombinant Escherichia coli

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