RESUMO
In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.IMPORTANCEE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/genética , Ferritinas/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Reguladoras de Ferro/metabolismo , Ferro/metabolismo , RNA Bacteriano/metabolismo , Sinorhizobium meliloti/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/biossíntese , Grupo dos Citocromos b/biossíntese , Ferritinas/biossíntese , Proteínas Reguladoras de Ferro/genética , Mutação , RNA Bacteriano/genética , Sinorhizobium meliloti/genética , Fatores de Transcrição/genéticaRESUMO
Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a Kd value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-ß and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.
Assuntos
Proteínas de Bactérias/química , Heme/química , Oxigenases de Função Mista/química , Sinorhizobium meliloti/enzimologia , Proteínas de Bactérias/fisiologia , Biliverdina/química , Biocatálise , Indução Enzimática , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Hemina/farmacologia , Ferro/farmacologia , Cinética , Oxigenases de Função Mista/fisiologiaRESUMO
Sinorhizobium meliloti has multiple systems for iron acquisition, including the use of haem as an iron source. Haem internalization involves the ShmR haem outer membrane receptor and the hmuTUV locus, which participates in haem transport across the cytoplasmic membrane. Previous studies have demonstrated that expression of the shmR gene is negatively regulated by iron through RirA. Here, we identify hmuP in a genetic screen for mutants that displayed aberrant control of shmR. The normal induction of shmR in response to iron limitation was lost in the hmuP mutant, showing that this gene positively affects shmR expression. Moreover, the HmuP protein is not part of the haemin transporter system. Analysis of gene expression and siderophore production indicates that disruption of hmuP does not affect other genes related to the iron-restriction response. Our results strongly indicate that the main function of HmuP is the transcriptional regulation of shmR. Sequence alignment of HmuP homologues and comparison with the NMR structure of Rhodopseudomonas palustris CGA009 HmuP protein revealed that certain amino acids localized within predicted beta-sheets are well conserved. Our data indicate that at least one of the beta-sheets is important for HmuP activity.
Assuntos
Proteínas de Bactérias/metabolismo , Hemina/metabolismo , Sinorhizobium meliloti/metabolismo , Fatores de Transcrição/metabolismo , Transporte Biológico , Proteínas de Membrana Transportadoras/genética , Mutagênese , Sinorhizobium meliloti/genéticaRESUMO
The bacterium Sinorhizobium meliloti is able to use heme as a nutritional iron source. Here, we show that the iron-regulated shmR gene encodes an outer membrane protein required for growth on heme. Furthermore, an shmR mutant is resistant to the toxic heme analog gallium protoporphyrin. Thus, the receptor protein of the heme transport system has been identified in S. meliloti.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Heme/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sinorhizobium meliloti/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Farmacorresistência Bacteriana , Proteínas de Membrana Transportadoras/genética , Mutação , Protoporfirinas/toxicidade , Sinorhizobium meliloti/genéticaRESUMO
Fur is a transcriptional regulator involved in iron-dependent control of gene expression in many bacteria. In this work we analyzed the phenotype of a fur mutant in Sinorhizobium meliloti, an alpha-proteobacterium that fixes N(2) in association with host plants. We demonstrated that some functions involved in high-affinity iron transport, siderophore production, and iron-regulated outer membrane protein expression respond to iron in a Fur-independent manner. However, manganese-dependent expression of the MntABCD manganese transport system was lost in a fur strain as discerned by constitutive expression of a mntA::gfp fusion reporter gene in the mutant. Thus, Fur directly or indirectly regulates a manganese-dependent function. The data indicate a novel function for a bacterial Fur protein in mediating manganese-dependent regulation of gene expression.