RirA of Dinoroseobacter shibae senses iron via a [3Fe-4S]1+ cluster co-ordinated by three cysteine residues.

In the marine bacterium, Dinoroseobacter shibae the transcription factor rhizobial iron regulator A (RirA) is involved in the adaptation to iron-limited growth conditions. In vitro iron and sulfide content determinations in combination with UV/Vis and electron paramagnetic resonance (EPR) spectroscopic analyses using anaerobically purified, recombinant RirA protein suggested a [3Fe-4S]1+ cluster as a cofactor. In vivo Mössbauer spectroscopy also corroborated the presence of a [3Fe-4S]1+ cluster in RirA. Moreover, the cluster was found to be redox stable. Three out of four highly conserved cysteine residues of RirA (Cys 91, Cys 99, Cys 105) were found essential for the [3Fe-4S]1+ cluster coordination. The dimeric structure of the RirA protein was independent of the presence of the [3Fe-4S]1+ cluster. Electro mobility shift assays demonstrated the essential role of an intact [3Fe-4S]1+ cluster for promoter binding by RirA. The DNA binding site was identified by DNase I footprinting. Mutagenesis studies in combination with DNA binding assays confirmed the promoter binding site as 3'-TTAAN10AATT-5'. This work describes a novel mechanism for the direct sensing of cellular iron levels in bacteria by an iron-responsive transcriptional regulator using the integrity of a redox-inactive [3Fe-4S]1+ cluster, and further contributes to the general understanding of iron regulation in marine bacteria.

Functional definition of the two effector binding sites, the oligomerization and DNA binding domains of the Bacillus subtilis LysR-type transcriptional regulator AlsR.

The LysR-type transcriptional regulator (LTTR) AlsR from Bacillus subtilis activates the transcription of the alsSD operon encoding enzymes for acetoin formation in response to the presence of acetate. The structural basis for effector binding, oligomerization, DNA binding, higher ordered complex formation, DNA bending and transcriptional control by B. subtilis AlsR was functionally characterized. The binding of two molecules of acetate per molecule AlsR was determined. Acetate-dependent transcription complex formation was observed. A structural model of AlsR was used to identify the amino acid residues V98, S100, H147 of the binding site 1, which were experimentally verified. The second binding site formed by T193, V194, A196, T201 and L202 mediated high acetate responsive induction. Residues L124, E225 Q74, I79 and R111 contributed to dimerization of AlsR. A22, Q29, P30, S33, K37, L39, E46, R50 and R53 of the winged helix-turn-helix motif were important for promoter recognition. The DNA binding domain alone dimerized and effectively bound the promoter. The LTTR promoter elements RBS and ABS had to be localized on the same site of the DNA. Higher ordered complex formation resulted in bending of promoter DNA and transcriptional activation.

Heme and nitric oxide binding by the transcriptional regulator DnrF from the marine bacterium Dinoroseobacter shibae increases napD promoter affinity.

Under oxygen-limiting conditions, the marine bacterium Dinoroseobacter shibae DFL12T generates energy via denitrification, a respiratory process in which nitric oxide (NO) is an intermediate. Accumulation of NO may cause cytotoxic effects. The response to this nitrosative (NO-triggered) stress is controlled by the Crp/Fnr-type transcriptional regulator DnrF. We analyzed the response to NO and the mechanism of NO sensing by the DnrF regulator. Using reporter gene fusions and transcriptomics, here we report that DnrF selectively repressed nitrate reductase (nap) genes, preventing further NO formation. In addition, DnrF induced the expression of the NO reductase genes (norCB), which promote NO consumption. We used UV-visible and EPR spectroscopy to characterize heme binding to DnrF and subsequent NO coordination. DnrF detects NO via its bound heme cofactor. We found that the dimeric DnrF bound one molecule of heme per subunit. Purified recombinant apo-DnrF bound its target promoter sequences (napD, nosR2, norC, hemA, and dnrE) in electromobility shift assays, and we identified a specific palindromic DNA-binding site 5'-TTGATN4ATCAA-3' in these target sequences via mutagenesis studies. Most importantly, successive addition of heme as well as heme and NO to purified recombinant apo-DnrF protein increased affinity of the holo-DnrF for its specific binding motif in the napD promoter. On the basis of these results, we propose a model for the DnrF-mediated NO stress response of this marine bacterium.

The blue light-dependent LOV-protein LdaP of Dinoroseobacter shibae acts as antirepressor of the PpsR repressor, regulating photosynthetic gene cluster expression.

In the marine α-proteobacterium Dinoroseobacter shibae more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a D. shibae transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified in vivo using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated in vivo testing of a bchF-lacZ reporter gene fusion in a heterologous Escherichia coli-based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B12) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in D. shibae was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B12-dependent antirepressor PpaA.

Purification, crystallization and preliminary X-ray analysis of the effector domain of AlsR, an LysR-type transcriptional regulator from Bacillus subtilis.

AlsR from Bacillus subtilis, a member of the LysR-type transcriptional regulator (LTTR) family, regulates the transcription of the alsSD operon encoding enzymes involved in acetoin biosynthesis. LTTRs represent the largest known family of transcriptional regulators in bacteria. In this study, AlsR82-302S100A, representing the effector domain, was produced in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method in the presence of 2.1 M DL-malic acid pH 7.0 at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 142.91, b = 74.96, c = 94.39 Å, ß = 110.543°. X-ray data extending to a resolution of 2.6 Šwere collected.

The transcription factor AlsR binds and regulates the promoter of the alsSD operon responsible for acetoin formation in Bacillus subtilis.

Bacillus subtilis forms acetoin under anaerobic fermentative growth conditions and as a product of the aerobic carbon overflow metabolism. Acetoin formation from pyruvate requires α-acetolactate synthase and acetolactate decarboxylase, both encoded by the alsSD operon. The alsR gene, encoding the LysR-type transcriptional regulator AlsR, was found to be essential for the in vivo expression of alsSD in response to anaerobic acetate accumulation, the addition of acetate, low pH, and the aerobic stationary phase. The expressions of the alsSD operon and the alsR regulatory gene were independent of other regulators of the anaerobic regulatory network, including ResDE, Fnr, and ArfM. A negative autoregulation of alsR was observed. In vitro transcription from the alsSD promoter using purified B. subtilis RNA polymerase required AlsR. DNA binding studies with purified recombinant AlsR in combination with promoter mutagenesis experiments identified a 19-bp high-affinity palindromic binding site (TAAT-N(11)-ATTA) at positions -76 to -58 (regulatory binding site [RBS]) and a low-affinity site (AT-N(11)-AT) at positions -41 to -27 (activator binding site [ABS]) upstream of the transcriptional start site of alsSD. The RBS and ABS were found to be essential for in vivo alsSD transcription. AlsR binding to both sites induced the formation of higher-order, transcription-competent complexes. The AlsR protein carrying the S100A substitution at the potential coinducer binding site still bound to the RBS and ABS. However, AlsR(S100A) failed to form the higher-order complex and to initiate in vivo and in vitro transcription. A model for AlsR promoter binding and transcriptional activation was deduced.

Aspartate 141 is the fourth ligand of the oxygen-sensing [4Fe-4S]2+ cluster of Bacillus subtilis transcriptional regulator Fnr.

The Bacillus subtilis redox regulator Fnr controls genes of the anaerobic metabolism in response to low oxygen tension. An unusual structure for the oxygen-sensing [4Fe-4S](2+) cluster was detected by a combination of genetic experiments with UV-visible and Mössbauer spectroscopy. Asp-141 was identified as the fourth iron-sulfur cluster ligand besides three Cys residues. Exchange of Asp-141 with Ala abolished functional in vivo complementation of an fnr knock-out strain by the mutagenized fnr gene and in vitro DNA binding of the recombinant regulator FnrD141A. In contrast, substitution of Asp-141 with Cys preserved [4Fe-4S](2+) structure and regulator function.

The Influence of Genes on the "Killer Plasmid" of Dinoroseobacter shibae on Its Symbiosis With the Dinoflagellate Prorocentrum minimum.

The marine bacterium Dinoroseobacter shibae shows a Jekyll-and-Hyde behavior in co-culture with the dinoflagellate Prorocentrum minimum: In the initial symbiotic phase it provides the essential vitamins B12 (cobalamin) and B1 (thiamine) to the algae. In the later pathogenic phase it kills the dinoflagellate. The killing phenotype is determined by the 191 kb plasmid and can be conjugated into other Roseobacters. From a transposon-library of D. shibae we retrieved 28 mutants whose insertion sites were located on the 191 kb plasmid. We co-cultivated each of them with P. minimum in L1 medium lacking vitamin B12. With 20 mutant strains no algal growth beyond the axenic control lacking B12 occurred. Several of these genes were predicted to encode proteins from the type IV secretion system (T4SS). They are apparently essential for establishing the symbiosis. With five transposon mutant strains, the initial symbiotic phase was intact but the later pathogenic phase was lost in co-culture. In three of them the insertion sites were located in an operon predicted to encode genes for biotin (B7) uptake. Both P. minimum and D. shibae are auxotrophic for biotin. We hypothesize that the bacterium depletes the medium from biotin resulting in apoptosis of the dinoflagellate.

Adaptation of Dinoroseobacter shibae to oxidative stress and the specific role of RirA.

Dinoroseobacter shibae living in the photic zone of marine ecosystems is frequently exposed to oxygen that forms highly reactive species. Here, we analysed the adaptation of D. shibae to different kinds of oxidative stress using a GeLC-MS/MS approach. D. shibae was grown in artificial seawater medium in the dark with succinate as sole carbon source and exposed to hydrogen peroxide, paraquat or diamide. We quantified 2580 D. shibae proteins. 75 proteins changed significantly in response to peroxide stress, while 220 and 207 proteins were differently regulated by superoxide stress and thiol stress. As expected, proteins like thioredoxin and peroxiredoxin were among these proteins. In addition, proteins involved in bacteriochlophyll biosynthesis were repressed under disulfide and superoxide stress but not under peroxide stress. In contrast, proteins associated with iron transport accumulated in response to peroxide and superoxide stress. Interestingly, the iron-responsive regulator RirA in D. shibae was downregulated by all stressors. A rirA deletion mutant showed an improved adaptation to peroxide stress suggesting that RirA dependent proteins are associated with oxidative stress resistance. Altogether, 139 proteins were upregulated in the mutant strain. Among them are proteins associated with protection and repair of DNA and proteins (e. g. ClpB, Hsp20, RecA, and a thioredoxin like protein). Strikingly, most of the proteins involved in iron metabolism such as iron binding proteins and transporters were not part of the upregulated proteins. In fact, rirA deficient cells were lacking a peroxide dependent induction of these proteins that may also contribute to a higher cell viability under these conditions.

Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor.

The Gram-negative rod Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract, and HlyX, the A. pleuropneumoniae homologue of fumarate and nitrate reduction regulator (FNR), has been shown to be important for persistence. An A. pleuropneumoniae hlyX deletion mutant has a decreased generation time but highly prolonged survival in comparison to its wild type parent strain when grown anaerobically in glucose-supplemented medium. Applying a combination of proteomic and transcriptomic approaches as well as in silico analyses, we identified 23 different proteins and 418 genes to be modulated by HlyX (> or = twofold up- or down-regulated). A putative HlyX-box was identified upstream of 54 of these genes implying direct control by HlyX. Consistent with its role as a strong positive regulator, HlyX induced the expression of genes for anaerobic metabolism encoding alternative terminal reductases and hydrogenases. In addition, expression of virulence-associated genes encoding iron uptake systems, a putative DNA adenine modification system, and an autotransporter serine protease were induced by HlyX under anaerobic growth conditions. With respect to virulence-associated genes, we focused on the iron-regulated protein B (FrpB) as it is the outer membrane protein most strongly up-regulated by HlyX. An frpB deletion mutant of A. pleuropneumoniae had the same growth characteristics as wild type grown aerobically and anaerobically. In contrast, A. pleuropneumoniae DeltafrpB did not cause any disease and could not be re-isolated from experimentally infected pigs, thereby identifying FrpB as a previously unknown virulence factor.

JProGO: a novel tool for the functional interpretation of prokaryotic microarray data using Gene Ontology information.

A novel program suite was implemented for the functional interpretation of high-throughput gene expression data based on the identification of Gene Ontology (GO) nodes. The focus of the analysis lies on the interpretation of microarray data from prokaryotes. The three well established statistical methods of the threshold value-based Fisher's exact test, as well as the threshold value-independent Kolmogorov-Smirnov and Student's t-test were employed in order to identify the groups of genes with a significantly altered expression profile. Furthermore, we provide the application of the rank-based unpaired Wilcoxon's test for a GO-based microarray data interpretation. Further features of the program include recognition of the alternative gene names and the correction for multiple testing. Obtained results are visualized interactively both as a table and as a GO subgraph including all significant nodes. Currently, JProGO enables the analysis of microarray data from more than 20 different prokaryotic species, including all important model organisms, and thus constitutes a useful web service for the microbial research community. JProGO is freely accessible via the web at the following address: http://www.jprogo.de.

FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae.

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated.

Thioredoxin can influence gene expression by affecting gyrase activity.

The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension. Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus. Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression. Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity. While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity. Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression. Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes.

Regulation of the anaerobic metabolism in Bacillus subtilis.

The Gram-positive soil bacterium Bacillus subtilis encounters changing environmental conditions in its habitat. The access to oxygen determines the mode of energy generation. A complex regulatory network is employed to switch from oxygen respiration to nitrate respiration and various fermentative processes. During adaptation, oxygen depletion is sensed by the [4Fe-4S](2+) cluster containing Fnr and the two-component regulatory system ResDE consisting of the membrane-bound histidine kinase ResE and the cytoplasmic ResD regulator. Nitric oxide is the signal recognized by NsrR. Acetate formation and decreasing pH are measured via AlsR. Finally, Rex is responding to changes in the cellular NAD(+)/NADH ration. The fine-tuned interplay of these regulators at approximately 400 target gene promoters ensures efficient adaptation of the B. subtilis physiology.

Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis.

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis.

NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low-GC Gram-positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe-4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe-4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species-specific between B. subtilis and Escherichia coli.

The Pseudomonas aeruginosa universal stress protein PA4352 is essential for surviving anaerobic energy stress.

During infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa microcolonies are embedded in the anaerobic CF mucus. This anaerobic environment seems to contribute to the formation of more robust P. aeruginosa biofilms and to an increased antibiotic tolerance and therefore promotes persistent infection. This study characterizes the P. aeruginosa protein PA4352, which is important for survival under anaerobic energy stress conditions. PA4352 belongs to the universal stress protein (Usp) superfamily and harbors two Usp domains in tandem. In Escherichia coli, Usp-type stress proteins are involved in survival during aerobic growth arrest and under various other stresses. A P. aeruginosa PA4352 knockout mutant was tested for survival under several stress conditions. We found a decrease in viability of this mutant compared to the P. aeruginosa wild type during anaerobic energy starvation caused by the missing electron acceptors oxygen and nitrate. Consistent with this phenotype under anaerobic conditions, the PA4352 knockout mutant was also highly sensitive to carbonyl cyanide m-chlorophenylhydrazone, the chemical uncoupler of the electron transport chain. Primer extension experiments identified two promoters upstream of the PA4352 gene. One promoter is activated in response to oxygen limitation by the oxygen-sensing regulatory protein Anr. The center of a putative Anr binding site was identified 41.5 bp upstream of the transcriptional start site. The second promoter is active only in the stationary phase, however, independently of RpoS, RelA, or quorum sensing. This is the second P. aeruginosa Usp-type stress protein that we have identified as important for survival under anaerobic conditions, which resembles the environment during persistent infection.

The Fnr regulon of Bacillus subtilis.

The Bacillus subtilis transcriptional regulator Fnr is an integral part of the regulatory cascade required for the adaptation of the bacterium to low oxygen tension. The B. subtilis Fnr regulon was defined via transcriptomic analysis in combination with bioinformatic-based binding site prediction. Four distinct groups of Fnr-dependent genes were observed. Group 1 genes (narKfnr, narGHJI, and arfM) are generally induced by Fnr under anaerobic conditions. All corresponding promoters contain an essential Fnr-binding site centered -41.5/-40.5 bp upstream of the transcriptional start point, suggesting their induction by direct Fnr interaction. Group 2 genes (alsSD, ldh lctP, ywcJ, and cydABCD) are characterized by anaerobic repression in the presence of nitrate. Mutational analysis of the Fnr-binding sites found in three of the corresponding promoters excluded their function in Fnr-mediated repression. Genetic evidence showing that group 2 genes are anaerobically repressed by nitrate reductase formation was accumulated. A possible role of the redox regulator YdiH in the regulation of group 2 genes was initially investigated. Group 3 genes are characterized by their Fnr-dependent activation in the presence of nitrate and the lack of an Fnr-binding site in their promoters. The analysis of Group 3 gene transcription (ykuNOP and ydbN) indicated that Fnr induces nitrate reductase production, which leads to the formation of the regulatory compound nitrite from nitrate. Finally, the group 4 operon acoABCL, lacking an Fnr-binding site, requires Fnr-dependent nitrate reductase formation for its general anaerobic induction. A regulatory model for the observed complex Fnr-mediated gene expression was deduced.

The Bacillus subtilis nrdEF genes, encoding a class Ib ribonucleotide reductase, are essential for aerobic and anaerobic growth.

Ribonucleotide reductases (RNRs) are essential for the biosynthesis of the deoxyribonucleoside triphosphates of DNA. Recently, it was proposed that externally supplied deoxyribonucleosides or DNA is required for the growth of Bacillus subtilis under strict anaerobic conditions (M. J. Folmsbee, M. J. McInerney, and D. P. Nagle, Appl. Environ. Microbiol. 70:5252-5257, 2004). Cultivation of B. subtilis on minimal medium in the presence of oxygen indicators in combination with oxygen electrode measurements and viable cell counting demonstrated that growth occurred under strict anaerobic conditions in the absence of externally supplied deoxyribonucleosides. The nrdEF genes encode the only obvious RNR in B. subtilis. A temperature-sensitive nrdE mutant failed to grow under aerobic and anaerobic conditions, indicating that this oxygen-dependent class I RNR has an essential role under both growth conditions. Aerobic growth and anaerobic growth of the nrdE mutant were rescued by addition of deoxynucleotides. The nrd locus consists of an nrdI-nrdE-nrdF-ymaB operon. The 5' end of the corresponding mRNA revealed transcriptional start sites 45 and 48 bp upstream of the translational start of nrdI. Anaerobic transcription of the operon was found to be dependent on the presence of intact genes for the ResDE two-component redox regulatory system. Two potential ResD binding sites were identified approximately 62 bp (site A) and 50 bp (site B) upstream of the transcriptional start sites by a bioinformatic approach. Only mutation of site B eliminated nrd expression. Aerobic transcription was ResDE independent but required additional promoter elements localized between 88 and 275 bp upstream of the transcriptional start.

Bacillus subtilis Fnr senses oxygen via a [4Fe-4S] cluster coordinated by three cysteine residues without change in the oligomeric state.

The oxygen regulator Fnr is part of the regulatory cascade in Bacillus subtilis for the adaptation to anaerobic growth conditions. In vivo complementation experiments revealed the essential role of only three cysteine residues (C227, C230, C235) at the C-terminus of B. subtilis Fnr for the transcriptional activation of the nitrate reductase operon (narGHJI) and nitrite extrusion protein gene (narK) promoters. UV/VIS, electron paramagnetic spin resonance (EPR) and Mössbauer spectroscopy experiments in combination with iron and sulphide content determinations using anaerobically purified recombinant B. subtilis Fnr identified the role of these three cysteine residues in the formation of one [4Fe-4S]2+ cluster per Fnr molecule. The obtained Mössbauer parameters are supportive for a [4Fe-4S]2+ cluster with three cysteine ligated iron sites and one non-cysteine ligated iron site. Gel filtration experiments revealed a stable dimeric structure for B. subtilis Fnr which is independent of the presence of the [4Fe-4S]2+ cluster. Gel mobility shift and in vitro transcription assays demonstrated the essential role of an intact [4Fe-4S]2+ cluster for promoter binding and transcriptional activation. An amino acid exchange introduced in the proposed alphaD-helix of B. subtilis Fnr (G149S) abolished its in vivo and in vitro activities indicating its importance for intramolecular signal transduction. The clear differences in the localization and coordination of the [4Fe-4S] cluster and in the organization of the oligomeric state between Escherichia coli and B. subtilis Fnr indicate differences in their mode of action.