Sensing iron availability <i>via</i> the fragile [4Fe–4S] cluster of the bacterial transcriptional repressor RirA

Martinez, Ma Teresa Pellicer; Martínez, Ana Bermejo; Crack, Jason C.; Holmes, John; Svistunenko, Dimitri A.; Johnston, Andrew; Cheesman, Myles R.; Todd, Jonathan D.; Brun, Nick E. Le

doi:10.1039/c7sc02801f

Cited by 31 publications

(71 citation statements)

References 65 publications

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“…Nondenaturing ESI-MS, in which noncovalently bound cofactors are retained upon ionization, has been shown to be extremely useful for determining the nature of the cluster in [Fe-S] proteins ( 22 , 24 – 27 ). Application here gave a deconvoluted spectrum containing a small peak at 15,919 Da corresponding to the apoprotein with all four cysteines in disulfide bridges.…”

Section: Resultsmentioning

confidence: 99%

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

Stewart

Bush

Crack

et al. 2020

Journal of Biological Chemistry

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The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd) for the SvWhiD-σHrdB4 complex as ∼0.7 μm, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8–10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.

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Section: Resultsmentioning

confidence: 99%

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

Stewart

Bush

Crack

et al. 2020

Journal of Biological Chemistry

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“…The predicted protein, which is identical to one encoded by S. fredii NGR234, exhibits high sequence similarity to the RirA proteins of S. meliloti , A. tumefaciens and R. leguminosarum bv. viciae (93.5%, 83.4%, and 79.4% identity, respectively), and conserves the three Cys residues predicted to ligate iron–sulfur clusters in Rrf2 family regulators [20] (Figure S1). The genetic context of the rirA gene is similar in HH103 and in S. meliloti .…”

Section: Resultsmentioning

confidence: 99%

“…Under iron-replete conditions, this regulator acts as a repressor of many iron-responsive genes by binding to Iron-Responsive Operator sequences (5’-TGA-(N9)-TCA-3’) known as IRO boxes [14,19]. A recent study has demonstrated that R. leguminosarum RirA contains a [4Fe–4S] cluster and that the holoprotein ([4Fe–4S]RirA) binds to an IRO box sequence [20]. Interestingly, the [4Fe–4S] cluster is sensitive to iron levels but also to O 2 , with low iron concentrations and aerobic conditions promoting the conversion of [4Fe–4S] cluster to [2Fe–2S] and subsequent cluster loss, which relieves gene repression [20].…”

Section: Introductionmentioning

confidence: 99%

“…A recent study has demonstrated that R. leguminosarum RirA contains a [4Fe–4S] cluster and that the holoprotein ([4Fe–4S]RirA) binds to an IRO box sequence [20]. Interestingly, the [4Fe–4S] cluster is sensitive to iron levels but also to O 2 , with low iron concentrations and aerobic conditions promoting the conversion of [4Fe–4S] cluster to [2Fe–2S] and subsequent cluster loss, which relieves gene repression [20]. Therefore, RirA seems to act as an iron sensor via iron–sulfur cluster availability.…”

Section: Introductionmentioning

confidence: 99%

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Sinorhizobium fredii HH103 RirA Is Required for Oxidative Stress Resistance and Efficient Symbiosis with Soybean

Crespo‐Rivas

Navarro‐Gómez

Alias‐Villegas

et al. 2019

IJMS

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Members of Rhizobiaceae contain a homologue of the iron-responsive regulatory protein RirA. In different bacteria, RirA acts as a repressor of iron uptake systems under iron-replete conditions and contributes to ameliorate cell damage during oxidative stress. In Rhizobium leguminosarum and Sinorhizobium meliloti, mutations in rirA do not impair symbiotic nitrogen fixation. In this study, a rirA mutant of broad host range S. fredii HH103 has been constructed (SVQ780) and its free-living and symbiotic phenotypes evaluated. No production of siderophores could be detected in either the wild-type or SVQ780. The rirA mutant exhibited a growth advantage under iron-deficient conditions and hypersensitivity to hydrogen peroxide in iron-rich medium. Transcription of rirA in HH103 is subject to autoregulation and inactivation of the gene upregulates fbpA, a gene putatively involved in iron transport. The S. fredii rirA mutant was able to nodulate soybean plants, but symbiotic nitrogen fixation was impaired. Nodules induced by the mutant were poorly infected compared to those induced by the wild-type. Genetic complementation reversed the mutant’s hypersensitivity to H2O2, expression of fbpA, and symbiotic deficiency in soybean plants. This is the first report that demonstrates a role for RirA in the Rhizobium-legume symbiosis.

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“…In Sinorhizobium meliloti , deletion of grx2 , coding for the sole class II Grx, leads to an impaired activity of the Fe–S containing aconitase and succinate dehydrogenase, to an increased intracellular iron content and to the deregulation of some genes controlled by the major transcriptional regulator of iron homeostasis RirA (rhizobial iron regulator) [ 40 ]. This suggests a direct role in the maturation of Fe–S proteins, but possibly other functions such as the regulation of the RirA regulator that senses the cellular iron and O 2 status through the conversion of a fragile [4Fe–4S] cluster into a [3Fe–4S] cluster [ 41–43 ]. One of the consequences of grx2 deletion was a defect in the development of symbiotic nodules with Medicago truncatula , thus impairing nitrogen fixation [ 40 ].…”

Section: Introductionmentioning

confidence: 99%

Sinorhizobium melilotiYrbA binds divalent metal cations using two conserved histidines

et al. 2020

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Sinorhizobium meliloti is a nitrogen-fixing bacterium forming symbiotic nodules with the legume Medicago truncatula. S. meliloti possesses two BolA-like proteins (BolA and YrbA), the function of which is unknown. In organisms where BolA proteins and monothiol glutaredoxins (Grxs) are present, they contribute to the regulation of iron homeostasis by bridging a [2Fe-2S] cluster into heterodimers. A role in the maturation of iron-sulfur (Fe-S) proteins is also attributed to both proteins. In this study, we have performed a structure-function analysis of SmYrbA showing that it coordinates diverse divalent metal ions (Fe2+, Co2+, Ni2+, Cu2+ and Zn2+) using His32 and His67 residues, that are also used for Fe-S cluster binding in BolA-Grx heterodimers. It also possesses the capacity to form heterodimers with the sole monothiol glutaredoxin (SmGrx2) present in this species. Using cellular approaches analyzing the metal tolerance of S. meliloti mutant strains inactivated in the yrbA and/or bolA genes, we provide evidence for a connection of YrbA with the regulation of iron homeostasis. The mild defects in M. truncatula nodulation reported for the yrbAbolA mutant as compared to the stronger defects in nodule development previously observed for a grx2 mutant suggest functions independent of SmGrx2. These results help clarifying the physiological role of BolA-type proteins in bacteria.

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Sensing iron availability via the fragile [4Fe–4S] cluster of the bacterial transcriptional repressor RirA

Abstract: The global iron regulator RirA controls transcription of iron metabolism genes via the binding of a fragile [4Fe–4S] cluster.

Cited by 31 publications

References 65 publications

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

Sinorhizobium fredii HH103 RirA Is Required for Oxidative Stress Resistance and Efficient Symbiosis with Soybean

Sinorhizobium melilotiYrbA binds divalent metal cations using two conserved histidines

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