Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli

Microbial cells possess numerous sensing/regulator systems in order to respond rapidly to environmental changes. Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. A group of global regulators, which include the one component Fnr protein and the two-component Arc system, coordinate the adaptive responses. To quantitate the contribution of Arc and FNR-dependent regulation under microaerobic conditions, the gene expression pattern of the electron transfer chain genes and the TCA cycle genes in wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA, fnr mutant, in glucose limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state using QRT-PCR. It was found that the TCA cycle genes, icd, gltA, sucC, and sdhC are repressed by ArcA while Fnr has a minor or no effect on the expression of these genes under microaerobic conditions. The expression levels of the electron transfer chain genes, nuoA, ndh, and ubiE, were not significantly affected by either ArcA or Fnr regulation proteins, while a lower expression of cydA (up to 9-fold lower) and a higher expression of cyoA (up to 31-fold higher) were observed in cultures of the arcA mutant strain compared to those of the wild type. Since significantly higher NADH/NAD+ ratios were previously observed in cultures of the arcA mutant strain compared to the wild type it seems that the cytochrome o oxidase (the product of cyoABCDE) cannot efficiently support aerobic respiration when the cells are grown under microaerobic conditions.     

Bacillus cereus fnr binds a [4Fe-4 S] cluster and forms a ternary complex with ResD and PlcR

ABSTRACT: BACKGROUND: Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrhealsyndrome may result from the secretion of various virulence factors including hemolysin BLand nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulatedby the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr isa member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so farbeen studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genesis thus to obtain and characterize holoFnr. RESULTS: Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UVvisibleand EPR spectroscopic analyses together with the chemical estimation of the ironcontent indicated that Fnr binds one [4Fe-4 S]2+ cluster per monomer. Atmospheric O2 causesdisassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- andapoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has ahigher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnrinteract with ResD and PlcR to form a ternary complex. CONCLUSIONS: Overall, this work shows that incorporation of the [4Fe-4 S]2+ cluster is not required for DNAbinding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of aternary complex with ResD and PlcR. This points to some new unusual properties of Fnr thatmay have physiological relevance in the redox regulation of enterotoxin gene regulation.     

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.     

Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions

Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. The adaptive responses are coordinated by a group of global regulators, which include the one-component Fnr protein, and the two-component Arc system. To quantitate the contribution of Arc and FNR dependent regulation under microaerobic conditions, the gene expression pattern of the fnr the arcA and arcB regulator genes, and the glycolysis related genes in a wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA, fnr mutant, in glucose limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state using QRT-PCR. It was found that ArcA has a negative effect on fnr expression under microaerobic conditions. Moreover, the expression levels of the FNR regulated genes, yfiD and frdA, were higher in cultures of the arcA mutant strain compared to the wild-type. These imply that a higher level of the FNR regulator is in the activated form in cultures of the arcA mutant strain compared to the wild-type during the transition from aerobic to microanaerobic growth. The results also show that the highest expression level of aceE, pflB, and adhE were obtained in cultures of the arcA mutant strain under microaerobic growth while higher levels of ldhA expression were obtained in cultures of the arcA mutant strain and the arcA, fnr double mutant strain compared to the wild-type and the fnr mutant strain. While the highest expression of adhE and pflB in cultures of the arcA mutant strain can explain the previous report of high ethanol flux and flux through pyruvate formate lyase (PFL) in cultures of this strain, the higher level of ldhA expression was not sufficient to explain the trend in lactate fluxes. The results indicate that lower conversion of pyruvate to acetyl-CoA is the main reason for high fluxes through lactate dehydrogenase (LDH) in cultures of the arcA, fnr double mutant strain.     

Effect of oxygen on the Escherichia coli ArcA and FNR regulation systems and metabolic responses

Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. The adaptive responses are coordinated by a group of global regulators, which include the one-component Fnr protein, and the two-component Arc system. To quantitate the contribution of Arc and Fnr-dependent regulation in catabolism, arcA and fnr mutant strains were constructed using the recently developed lambda derived recombination system. The metabolic activity of wildtype E. coli, an arcA mutant, an fnr mutant, and a double arcA-fnr mutant, via the fermentative pathways in glucose-limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state. It was found that the most significant role of ArcA is under microaerobic conditions, while that of FNR is under more strictly anaerobic conditions. The FNR protein is normally inactive during microaerobic conditions. However, our results indicate that in the arcA mutant strain the cells behave as if a higher level of the FNR regulator is in the activated form compared to the wildtype strain during the transition from aerobic to microanaerobic growth. The results show a significant increase in the flux through pyruvate formate lyase (PFL) in the presence of oxygen. The activity of FNR-regulated pathways in the arcA mutant strain is correlated with the high redox potential obtained under microaerobic growth.