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...
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