Anaerobically induced expression of the nitrite reductase cytochrome c-551 operon from Pseudomonas aeruginosa.

The nitrite reductase gene (denA) and the cytochrome c-551 gene (denB) are located only 50 bp apart from each other in the Pseudomonas aeruginosa chromosome. We report evidence that these two genes are co-transcribed as an operon only under anaerobic (denitrifying) conditions. The nucleotide sequence of the promoter (regulatory) region of the operon is highly AT-rich and contains a sequence closely resembling the consensus FNR binding site in E. coli.     

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.     

Regulation of denitrification genes in Neisseria meningitidis by nitric oxide and the repressor NsrR

The human pathogen Neisseria meningitidis is capable of growth using the denitrification of nitrite to nitrous oxide under microaerobic conditions. This process is catalyzed by two reductases: nitrite reductase (encoded by aniA) and nitric oxide (NO) reductase (encoded by norB). Here, we show that in N. meningitidis MC58 norB is regulated by nitric oxide via the product of gene NMB0437 which encodes NsrR. NsrR is a repressor in the absence of NO, but norB expression is derepressed by NO in an NsrR-dependent manner. nsrR-deficient mutants grow by denitrification more rapidly than wild-type N. meningitidis, and this is coincident with the upregulation of both NO reductase and nitrite reductase even under aerobic conditions in the absence of nitrite or NO. The NsrR-dependent repression of aniA (unlike that of norB) is not lifted in the presence of NO. The role of NsrR in the control of expression of aniA is linked to the function of the anaerobic activator protein FNR: analysis of nsrR and fnr single and nsrR fnr double mutants carrying an aniA promoter lacZ fusion indicates that the role of NsrR is to prevent FNR-dependent aniA expression under aerobic conditions, indicating that FNR in N. meningitidis retains considerable activity aerobically.     

Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function

The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and Deltafnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.     

Reversible interconversion of the functional state of the gene regulator FNR from Escherichia coli in vivo by O2 and iron availability

FNR, the gene regulator of anaerobic respiratory genes of Escherichia coli is converted in vivo by O₂ and by chelating agents to an inactive state. The interconversion process was studied in vivo in a strain with temperature controlled synthesis of FNR by measuring the expression of the frd (fumarate reductase) operon and the reactivity of FNR with the alkylating agent iodoacetic acid. FNR from aerobic bacteria is, after arresting FNR synthesis and shifting to anaerobic conditions, able to activate frd expression and behaves in the alkylation assay like anaerobic FNR. After shift from anaerobic to aerobic conditions, FNR no longer activates the expression of frd and reacts similar to aerobic FNR in the alkylation assay. The conversion of aerobic (inactive) to anaerobic (active) FNR occurs in the presence of chloramphenicol, an inhibitor of protein synthesis. Anaerobic FNR can also be converted post-translationally to inactive, metal-depleted FNR by growing the bacteria in the presence of chelating agents. The reverse is also possible by incubating metal-depleted bacteria with Fe²⁺. From the experiments it is concluded that the aerobic and the metal-depleted form of FNR can be transferred post-translationally and reversibly to the anaerobic (active) form. The response of FNR to changes in O₂ supply therefore occurs at the FNR protein level in a reversible mode.Abbreviation BV_red = reduced benzyl viologen     

The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc ⁻ strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O₂-stable [4Fe-4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by ⁵⁵Fe-labeling experiments. Using [³⁵S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O₂-inactivated apo-FNR into [4Fe-4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.     

Reduced apo-fumarate nitrate reductase regulator (apoFNR) as the major form of FNR in aerobically growing Escherichia coli

Under anoxic conditions, the Escherichia coli oxygen sensor FNR (fumarate nitrate reductase regulator) is in the active state and contains a [4Fe-4S] cluster. Oxygen converts [4Fe-4S]FNR to inactive [2Fe-2S]FNR. After prolonged exposure to air in vitro, apoFNR lacking a Fe-S cluster is formed. ApoFNR can be differentiated from Fe-S-containing forms by the accessibility of the five Cys thiol residues, four of which serve as ligands for the Fe-S cluster. The presence of apoFNR in aerobically and anaerobically grown E. coli was analyzed in situ using thiol reagents. In anaerobically and aerobically grown cells, the membrane-permeable monobromobimane labeled one to two and four Cys residues, respectively; the same labeling pattern was found with impermeable thiol reagents after cell permeabilization. Alkylation of FNR in aerobic bacteria and counting the labeled residues by mass spectrometry showed a form of FNR with five accessible Cys residues, corresponding to apoFNR with all Cys residues in the thiol state. Therefore, aerobically growing cells contain apoFNR, whereas a significant amount of Fe-S-containing FNR was not detected under these conditions. Exposure of anaerobic bacteria to oxygen caused conversion of Fe-S-containing FNR to apoFNR within 6 min. ApoFNR from aerobic bacteria contained no disulfide, in contrast to apoFNR formed in vitro by air inactivation, and all Cys residues were in the thiol form.     

Regulation of the ribonucleotide reductases in Lactococcus lactis subsp. cremoris

Lactococcus lactis has two essential ribonucleotide reductases for DNA biosynthesis and repair which are affected in the presence or absence of oxygen. Expression of glutaredoxin like protein (NrdH), the hydrogen donor for ribonucleotide reductase, was found to be regulated by the FNR like proteins (FlpA and FlpB). Proteomics study demonstrated that expression level of NrdH significantly decreased in the flpA and flpAB deletion mutants. The nrdH gene is located in an nrdHIEF operon and encoding the NrdEF ribonucleotide reductase, which is active under aerobic and anaerobic conditions. Regulation of expression of the nrdHIEF operons was investigated using beta-galactosidase as a reporter gene. The 588 bp fragment containing the nrdH promoter and gene cloned into the pORI vector immediately upstream of a promoterless lacZ gene. Constructed plasmid was transferred into wild type (MG1363), single mutant (flpA orflpB) and double mutant (flpAB). Aerobically, nrdH promoter activity is 15-fold higher than anaerobic expression.     

A role for rubredoxin in oxidative stress protection in Desulfovibrio vulgaris: catalytic electron transfer to rubrerythrin and two-iron superoxide reductase

Desulfovibrio vulgaris rubredoxin, which contains a single [Fe(SCys)4] site, is shown to be a catalytically competent electron donor to two enzymes from the same organism, namely, rubrerythrin and two-iron superoxide reductase (a.k.a. rubredoxin oxidoreductase or desulfoferrodoxin). These two enzymes have been implicated in catalytic reduction of hydrogen peroxide and superoxide, respectively, during periods of oxidative stress in D. vulgaris, but their proximal electron donors had not been characterized. We further demonstrate the incorrectness of a previous report that rubredoxin is not an electron donor to the superoxide reductase and describe convenient assays for demonstrating the catalytic competence of all three proteins in their respective functions. Rubrerythrin is shown to be an efficient rubredoxin peroxidase in which the rubedoxin:hydrogen peroxide redox stoichiometry is 2:1 mol:mol. Using spinach ferredoxin-NADP+ oxidoreductase (FNR) as an artificial, but proficient, NADPH:rubredoxin reductase, rubredoxin was further found to catalyze rapid and complete reduction of all Fe3+ to Fe2+ in rubrerythrin by NADPH under anaerobic conditions. The combined system, FNR/rubredoxin/rubrerythrin, was shown to function as a catalytically competent NADPH peroxidase. Another small rubredoxin-like D. vulgaris protein, Rdl, could not substitute for rubredoxin as a peroxidase substrate of rubrerythrin. Similarly, D. vulgaris rubredoxin was demonstrated to efficiently catalyze reduction of D. vulgaris two-iron superoxide reductase and, when combined with FNR, to function as an NADPH:superoxide oxidoreductase. We suggest that, during periods of oxidative stress, rubredoxin could divert electron flow from the electron transport chain of D. vulgaris to rubrerythrin and superoxide reductase, thereby simultaneously protecting autoxidizable redox enzymes and lowering intracellular hydrogen peroxide and superoxide levels.