With an oxystat, changes in the pattern of expression of FNR-dependent genes from Escherichia coli were studied as a function of the O 2 tension (pO 2 ) in the medium. Expression of all four tested genes was decreased by increasing O 2 . However, the pO 2 values that gave rise to half-maximal repression (pO 0.5 ) were dependent on the particular promoter and varied between 1 and 5 millibars (1 bar ؍ 10 5 Pa). The pO 0.5 value for the ArcA-regulated succinate dehydrogenase genes was in the same range (pO 0.5 ؍ 4.6 millibars). At these pO 2 values, the cytoplasm can be calculated to be well supplied with O 2 by diffusion. Therefore, intracellular O 2 could provide the signal to FNR, suggesting that there is no need for a signal transfer chain. Genetic inactivation of the enzymes and coenzymes of aerobic respiration had no or limited effects on the pO 0.5 of FNR-regulated genes. In response to O 2 availability, the transcriptional regulator FNR of Escherichia coli controls the expression of genes required for anaerobic metabolism, such as structural genes of anaerobic respiration, substrate transport, and biosyntheses of coenzymes for anaerobic metabolism (18,37,42,43). The Arc system on the other hand controls the expression of many genes of aerobic metabolism in response to O 2 (20, 22). The Arc system belongs to the two-component regulatory family, with ArcB as the membrane sensor protein and ArcA as the response regulator. FNR is in the regulatory competent state only under anaerobic conditions (12,15,25), although it is present in rather constant amounts in E. coli grown under either aerobic or anaerobic conditions (18,36,44). The O 2 -sensing mechanism has been attributed to an essential Fe cofactor (15,17,29,35,39), and according to recent experiments, this cofactor is an FeS cluster (1, 25). In vivo, FNR can switch reversibly from the inactive (aerobic) to the active (anaerobic) state (12). Apart from O 2 , FNR can also be inactivated in vivo by applying positive redox potential to the medium, e.g., by the addition of ferricyanide (45). In vitro, DNA binding of FNR and transcriptional activation were stimulated by applying reducing conditions (15,25). Therefore, a redox reaction at the FeS cofactor may trigger the functional switch of FNR.How O 2 is sensed by FNR is not well understood. It is not known whether O 2 itself or a product of aerobic metabolism reacts with FNR and whether other mediators are required. The failure to isolate mutations in other loci which cause defective FNR function suggests that there are no specific protein components required for signal transfer or reaction with O 2 . To further analyze the pathway by which O 2 affects FNR function, here the role of O 2 as the signal and effector was analyzed and quantified. The transition point of oxygen regulation (pO 0.5 ) was determined to obtain a quantitative measure for the effect of O 2 on FNR.By using this same approach, the aerobic respiratory chain was studied as a potential site for O 2 sensing or for providing a signal. Mutants...
Availability of O2 is one of the most important regulatory signals in facultatively anaerobic bacteria. Various two- or one-component sensor/regulator systems control the expression of aerobic and anaerobic metabolism in response to O2. Most of the sensor proteins contain heme or Fe as cofactors that interact with O2 either by binding or by a redox reaction. The ArcA/ArcB regulator of aerobic metabolism in Escherichia coli may use a different sensory mechanism. In two-component regulators, the sensor is located in the cytoplasmic membrane, whereas one-component regulators are located in the cytoplasm. Under most conditions, O2 can readily reach the cytoplasm and could provide the signal in the cytoplasm. The transcriptional regulator FNR of E. Coli controls the expression of many genes required for anaerobic metabolism in response to O2. Functional homologs of FNR are present in facultatively anaerobic Proteobacteria and presumably also in gram-positive bacteria. The target genes of FNR are mostly under multiple regulation by FNR and other regulators that respond to O2, nitrate, or glucose. FNR represents a 'one-component' sensor/regulator and contains Fe for signal perception. In response to O2 availability, FNR is converted reversibly from the aerobic (inactive) state to the anaerobic (active) state. Experiments suggest that the Fe cofactor is bound by four essential cysteine residues. The O2-triggered transformation between active and inactive FNR presumably is due to a redox reaction at the Fe cofactor, but other modes of interaction cannot be excluded. O2 seems to affect the site-specific DNA binding of FNR at target genes or the formation of an active transcriptional complex with RNA polymerase.
Availability of O2 is one of the most important regulatory signals in facultatively anaerobic bacteria. Various two- or one-component sensor/regulator systems control the expression of aerobic and anaerobic metabolism in response to O2. Most of the sensor proteins contain heme or Fe as cofactors that interact with O2 either by binding or by a redox reaction. The ArcA/ArcB regulator of aerobic metabolism in Escherichia coli may use a different sensory mechanism. In two-component regulators, the sensor is located in the cytoplasmic membrane, whereas one-component regulators are located in the cytoplasm. Under most conditions, O2 can readily reach the cytoplasm and could provide the signal in the cytoplasm. The transcriptional regulator FNR of E. Coli controls the expression of many genes required for anaerobic metabolism in response to O2. Functional homologs of FNR are present in facultatively anaerobic Proteobacteria and presumably also in gram-positive bacteria. The target genes of FNR are mostly under multiple regulation by FNR and other regulators that respond to O2, nitrate, or glucose. FNR represents a 'one-component' sensor/regulator and contains Fe for signal perception. In response to O2 availability, FNR is converted reversibly from the aerobic (inactive) state to the anaerobic (active) state. Experiments suggest that the Fe cofactor is bound by four essential cysteine residues. The O2-triggered transformation between active and inactive FNR presumably is due to a redox reaction at the Fe cofactor, but other modes of interaction cannot be excluded. O2 seems to affect the site-specific DNA binding of FNR at target genes or the formation of an active transcriptional complex with RNA polymerase.
The oxygen sensor regulator FNR (fumarate nitrate reductase regulator) of Escherichia coli is known to be inactivated by O 2 as the result of conversion of a [4Fe-4S] cluster of the protein into a [2Fe-2S] cluster. Further incubation with O 2 causes loss of the [2Fe-2S] cluster and production of apoFNR. The reactions involved in cluster assembly and reductive activation of apoFNR isolated under anaerobic or aerobic conditions were studied in vivo and in vitro. In a gshA mutant of E. coli that was completely devoid of glutathione, the O 2 tension for the regulatory switch for FNR-dependent gene regulation was decreased by a factor of 4±5 compared with the wildtype, suggesting a role for glutathione in FNR function. In isolated apoFNR, glutathione could be used as the reducing agent for HS 2 formation required for [4Fe-4S] assembly by cysteine desulfurase (NifS), and for the reduction of cysteine ligands of the FeS cluster in FNR. Air-inactivated FNR (apoFNR without FeS) could be reconstituted to [4Fe-4S]´FNR by the same reaction as used for apoFNR isolated under anaerobic conditions. The in vivo effects of glutathione on FNR function and the role of glutathione in the formation of active [4Fe-4S]´FNR in vitro suggest an important role for glutathione in the de novo assembly of FNR and in the reductive activation of air-oxidized FNR under anaerobic conditions. Keywords: anaerobic regulation; fumarate nitrate reductase regulator (FNR); glutathione; [4Fe-4S] assembly; O 2 regulation.In Escherichia coli, the fumarate nitrate reductase regulator (FNR) is one of the major regulators controlling the switch from aerobic to anaerobic metabolism at the transcriptional level [1±3]. Three functionally different forms of FNR have been described (see Fig. 5 . In previous studies, the O 2 -dependent inactivation of FNR was studied in vivo in an oxystat by measuring the expression of FNRdependent lacZ reporter gene fusions as a function of the O 2 tension [11,12]. The O 2 tensions that cause loss of FNRdependent gene activation, or, respectively, the switch from anaerobic (active) to aerobic (inactive) FNR, are in the range of 1±5 mbar O 2 in the medium (corresponding to about 0.5±2.5% air saturation) for most FNR-regulated genes.During isolation of FNR under anoxic conditions, most of the [4Fe-4S] cluster is lost, but [4Fe-4S]´FNR can be reconstituted in vitro using cysteine, cysteine desulfurase (NifS), Fe(II) ions, and dithiothreitol as reducing agent [4±6]. For reconstitution, mainly FNR isolated under anoxic conditions was used, but the procedure could also be used for O 2 -exposed FNR [7,8]. The desulfurase requires dithiothreitol for the reduction of elemental sulfur from cysteine (via persulfide) to sulfide [13,14]. The physiological reductant is not known. In vitro, NifS Av from Azotobacter vinelandii is generally used for the assembly of the FeS cluster in FNR because of its well-characterized reaction [14], but E. coli encodes proteins homologous to NifS Av .In contrast with the O 2 -dependent inactivation, little is ...
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