Role of glutathione in the formation of the active form of the oxygen sensor FNR ([4Fe-4S].FNR) and in the control of FNR function.

The oxygen sensor regulator FNR (fumarate nitrate reductase regulator) of Escherichia coli is known to be inactivated by O2 as the result of conversion of a [4Fe-4S] cluster of the protein into a [2Fe-2S] cluster. Further incubation with O2 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 O2 tension for the regulatory switch for FNR-dependent gene regulation was decreased by a factor of 4-5 compared with the wild-type, suggesting a role for glutathione in FNR function. In isolated apoFNR, glutathione could be used as the reducing agent for HS- 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.     

Purification of the FLP site-specific recombinase by affinity chromatography and re-examination of basic properties of the system.

The FLP protein, a site-specific recombinase encoded by the 2 micron plasmid of yeast, has been purified to near homogeneity from extracts of E. coli cells in which the protein has been expressed. The purification is a three column procedure, the final step employing affinity chromatography. The affinity ligand consists of a DNA polymer with multiple FLP protein binding sites arranged in tandem repeats. This protocol yields 2 mg of FLP protein which is 85% pure. The purified protein is highly active, stable for several months at -70 degrees C and free of detectable nucleases. The molecular weight and N-terminal sequence are identical to that predicted for the FLP protein by the DNA sequence of the gene. Purified FLP protein primarily, but not exclusively, promotes intramolecular recombination. Intermolecular recombination becomes the dominant reaction when E. coli extracts containing no FLP protein are added to the reaction mixture. These extracts are not specifically required for recombination, but demonstrate that some properties previously attributed to FLP protein can be assigned to contaminating proteins present in E. coli.     

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa was characterized which could not grow anaerobically with nitrate as the terminal electron acceptor or with arginine as the sole energy source. In this anr mutant, nitrate reductase and arginine deiminase were not induced by oxygen limitation. The anr mutation was mapped in the 60-min region of the P. aeruginosa chromosome. A 1.3-kb chromosomal fragment from P. aeruginosa complemented the anr mutation and also restored anaerobic growth of an Escherichia coli fnr deletion mutant on nitrate medium, indicating that the 1.3-kb fragment specifies an FNR-like regulatory protein. The arcDABC operon, which encodes the arginine deiminase pathway enzymes of P. aeruginosa, was rendered virtually noninducible by a deletion or an insertion in the -40 region of the arc promoter. This -40 sequence (TTGAC....ATCAG) strongly resembled the consensus FNR-binding site (TTGAT....ATCAA) of E. coli. The cloned arc operon was expressed at low levels in E. coli; nevertheless, some FNR-dependent anaerobic induction could be observed. An FNR-dependent E. coli promoter containing the consensus FNR-binding site was expressed well in P. aeruginosa and was regulated by oxygen limitation. These findings suggest that P. aeruginosa and E. coli have similar mechanisms of anaerobic control.     

Control of gene expression by FNR-like proteins in facultatively anaerobic bacteria

Facultatively anaerobic bacteria are able to adapt to many different growth conditions. Their capability to change their metabolism optimally is often ensured by FNR-like proteins. The FNR protein ofEscherichia coli functions as the main regulator during the aerobic-to-anaerobic switch. Low oxygen tensions activate this protein which is expressed constitutively and is inactive under aerobic conditions. The active form is dimeric and contains a [4Fe−4S]²⁺ cluster. The direct dissociation of the cluster to the [2Fe−2S]²⁺ cluster by the effect of oxygen leads to destabilization of the FNR dimer and to loss of its activity. The active FNR induces the expression of many anaerobic genes; the set comprises over 100 of controlled genes. Many other bacteria contain one or more FNR analogues. All these proteins form the FNR family of regulatory proteins. Properties of these proteins are very distinct, sometimes even among representatives of different strains of the same bacterial species. FNR-like proteins together with other regulators (e.g., two-component system ArcBA, nitrate-sensing system NarXL,etc.) control a complicated network of modulons that is characteristic for every species or even strain and enables fine tuning of gene expression.