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
We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Indometacina/farmacologia , Glicoproteínas de Membrana/biossíntese , Monócitos/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Fosfoproteínas/biossíntese , Células Cultivadas , Grupo dos Citocromos b/biossíntese , Grupo dos Citocromos b/genética , Humanos , Interferon gama/farmacologia , Leucemia Monocítica Aguda/patologia , Glicoproteínas de Membrana/genética , Monócitos/enzimologia , NADPH Oxidase 2 , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The nucleotide changes present in a group of five cytochrome b mit- mutants were analyzed at the sequence level. Two single-base changes were found: one (M10-152) generated a nonsense codon in the first exon while the other (M8-181) created a missense substitution in the second exon. The other mutants all have multiple (three) substitutions that either resulted in a missense mutation in a coding region (M17-162) or else changed nucleotides in the last intron of the gene, so blocking its excision (M6-200 and M8-53). The synthesis of mitochondrial polypeptides and the steady state concentration of the complex-III subunits were examined. The Rieske protein and the core-4 and core-5 subunits were much reduced in all mutants. Consequently the overall stability of complex III is very sensitive even to amino-acid substitutions in the cytochrome b protein. Mutant M8-53 provides direct evidence for the proposed role of the P9.1 stem in the core structure of the group-I type last intron of this gene.