Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe−4S Cluster

Abstract

In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S] 2+ cluster which reacts with NO to form dinitrosyl iron complexes. This method of NO sensing is analogous to that of the FNR protein of Escherichia coli. The Fe-S cluster of NsrR is also reactive toward other exogenous ligands such as cyanide, dithiothreitol, and O 2 . These results, together with the presence of only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a non-cysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and crosslinking experiments show that NsrR adopts a dimeric structure in its [4Fe-4S] 2+ holo form as well as in the apo form. These findings provide a first steppingstone to investigate the mechanism of NO sensing in NsrR.The ability to adapt to anaerobic conditions is vital for a great diversity of microorganisms. It is particularly true of pathogenic organisms which may encounter oxygen limitation within their hosts. Such bacteria also commonly face other stresses such as nitric oxide (NO) produced by phagocytic cells as part of the immune response of the host (1). Thus, an understanding of how bacteria sense and respond to these conditions is of considerable importance.An example of the bacterial response to oxygen limitation comes from Bacillus subtilis which, in the absence of oxygen, can grow by nitrate respiration (2). The ResDE two-component regulatory system is required for the induction of genes involved in nitrate respiration (3). These genes include fnr, (the gene encoding anaerobic transcription factor) (4), nasDEF (nitrite reductase genes) (5), and hmp (flavohemoglobin gene) (6). However, anaerobic conditions alone are not sufficient for the full induction of the ResDE-controlled genes, in particular hmp and nasDEF, and the presence of NO is required to attain the full induction of these genes (7). The effect of NO is abrogated in an nsrR mutant strain, indicating a role for NsrR in NOmediated control of the ResDE regulon (8).NsrR belongs to the Rrf2 family of transcription regulators and is widely found in various bacteria (9). Sequence analyses predict that NsrR contains a helix-turn-helix which likely binds to the promoter region of its target genes (9). In vivo studies of nsrR mutants and the effect of * To whom correspondence should be addressed. Tel: 503-748-167, Fax: 503-7481464, Email: ploccoz@ebs.ogi Materials & Methods Purification of N-terminal and C-terminal His 6 -tagged NsrR proteins (NH-NsrR & CH-NsrR)The various strains and plasmids used in this study are listed in Table S1 (see Supporting Information). NH-NsrR expression plasmid, pMMN648, was previously cons...

NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility

“…In sedimentation equilibrium experiments, the Streptomyces coelicolor NsrR formed a sequence-specific complex with DNA with a molecular weight consistent with the protein being dimeric (Tucker et al, 2008). However, these experiments were done with protein containing a [2Fe-2S] cluster; the physiologically relevant form of NsrR may contain a [4Fe-4S] cluster (Yukl et al, 2008). The [4Fe-4S] NsrR from Bacillus subtilis is dimeric in solution, its oligomeric state in the presence of a DNA target has not been examined (Yukl et al, 2008).…”

“…However, these experiments were done with protein containing a [2Fe-2S] cluster; the physiologically relevant form of NsrR may contain a [4Fe-4S] cluster (Yukl et al, 2008). The [4Fe-4S] NsrR from Bacillus subtilis is dimeric in solution, its oligomeric state in the presence of a DNA target has not been examined (Yukl et al, 2008). Interestingly, the NsrR homologue IscR also binds to two types of site (Type 1 and Type 2), though in this case they are unrelated sequences.…”

“…4), suggesting that inactivation of NsrR alleviates negative control of motility. The NsrR protein contains three conserved cysteine residues thought to be involved in the co-ordination of an [Fe-S] cluster, which is the likely site of NO sensing (Isabella et al, 2008;Tucker et al, 2008;Yukl et al, 2008). We have substituted cysteine 96 with serine, and found that the NsrR-C96S variant is unable to repress fully the NsrR targets ytfE, hmp, hcp and ygbA (J. Partridge and S. Spiro, unpubl.…”

“…The s 54 -dependent transcriptional activator NorR is stimulated by the formation of a nitrosyl species at a mono-nuclear iron site in its signalling domain (D′Autréaux et al, 2005). In the case of NsrR, the binding site for NO is likely to be an [Fe-S] cluster (Isabella et al, 2008;Tucker et al, 2008;Yukl et al, 2008).…”

Inefficient translation of nsrR constrains behaviour of the NsrR regulon in Escherichia coli

“…NsrR is an NO-sensitive repressor from the Rrf2 family that contains an [Fe-S] cluster. The cluster is likely to be [4Fe-4S], and reaction with NO is accompanied by a decrease in DNA binding affinity and de-repression of NsrR-regulated promoters (Bodenmiller & Spiro, 2006;Tucker et al, 2008;Yukl et al, 2008;Crack et al, 2015). The previously characterized NsrR regulon includes genes encoding the NO-scavenging flavohaemoglobin (hmp), the RIC (repair of iron centres) protein that participates in the repair of NO-damaged [Fe-S] clusters (ytfE), the respiratory nitrite reductase Nrf that is also an NO reductase (nrfABCDEFG), genes involved in motility ( fliA) and aromatic amine metabolism (tynA, feaB), and genes of unknown function, for example ygbA, yccM and yeaR-yoaG (Bodenmiller & Spiro, 2006;Filenko et al, 2007;Lin et al, 2007;Rankin et al, 2008;Partridge et al, 2009).…”