Spx mediates oxidative stress regulation of the methionine sulfoxide reductases operon in Bacillus subtilis

<p>Abstract</p> <p>Background</p> <p>All aerobically grown living cells are exposed to oxidative damage by reactive oxygen species (ROS). A major damage by ROS to proteins is caused by covalent modifications of methionine residues giving methionine sulfoxide (Met-SO). Methionine sulfoxide reductases are enzymes able to regenerate methionine and restore protein function after oxidative damage.</p> <p>Results</p> <p>We characterized the methionine sulfoxide reductase genes <it>msrA </it>and <it>msrB </it>in <it>Bacillus subtilis</it>, forming an operon transcribed from a single sigma A-dependent promoter. The <it>msrAB </it>operon was specifically induced by oxidative stress caused by paraquat (PQ) but not by H₂O₂. Spx, a global oxidative stress regulator in <it>B. subtilis</it>, is primarily responsible for this PQ-specific induction of <it>msrAB </it>expression. In support of this finding, an <it>spx </it>deletion mutant is extremely sensitive to PQ, and increased expression of <it>msrA </it>was identified in a <it>clpX </it>mutant in which Spx accumulated. However, the Spx effect was also visible under conditions where the protein did not accumulate (PQ treatment), suggesting a specific molecular effect at the level of the Spx protein. Indeed, the CXXC motif of Spx was found essential for its function in the PQ-specific induction of <it>msrAB </it>expression. PQ caused a modification of Spx requiring at least one of the cysteines of the CXXC motif of Spx. The PQ modified form of Spx showed a dynamic change <it>in vivo</it>.</p> <p>Conclusion</p> <p>The Spx mediated PQ-specific regulation pathway of the <it>msrAB </it>operon in <it>B. subtilis </it>is reported. Our results suggest that PQ induced the expression of <it>msrAB </it>partially through an oxidation on Spx via modification of its CXXC motif.</p

The two authentic methionine aminopeptidase genes are differentially expressed in Bacillus subtilis

BACKGROUND: Two putative methionine aminopeptidase genes, map (essential) and yflG (non-essential), were identified in the genome sequence of Bacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in B. subtilis. RESULTS: In silico analysis of MAP evolution uncovered a coordinated pattern of MAP and deformylase that did not correlate with the pattern of 16S RNA evolution. Biochemical assays showed that both MAP (MAP_Bs) and YflG (YflG_Bs) from B. subtilis overproduced in Escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. Compared with MAP_Bs, YflG_Bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates. Both map and yflG genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both E. coli and B. subtilis. In contrast, lacZ gene transcriptional fusions showed that the promoter activity of map was 50 to 100-fold higher than that of yflG. Primer extension analysis detected the transcription start site of the yflG promoter. Further work identified that YvoA acted as a possible weak repressor of yflG expression in B. subtilis in vivo. CONCLUSION: Both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vitro and in vivo. The high expression level of map and low expression level of yflG may account for their essentiality and dispensality in B. subtilis, respectively, when cells are grown under laboratory conditions. Their difference in activity on synthetic substrates suggests that they have different protein targets in vivo

Studies of methionine recycling and repair in Bacillus subtilis

In Bacillus subtilis, methionine aminopeptidases (MAP) and methionine sulfoxide reductases (MSR) maintain the methionine pool. Recycling of the initiation methionine of proteins is essential. Whereas most prokaryotes have a single MAP gene, B. subtilis has two, mapA and mapB (yflG). MapB was 100-fold more efficient than MapA when assayed on some synthetic peptide substrates. Unlike MapA, MapB showed a strong preference for cobalt. They are both functional in vivo, and their gene products distribute evenly in the cell. The expression of mapA is 50 to 100-fold higher than that of mapB in vivo, which could explain why only mapA is the essential gene. Further phylogeny analysis suggests that the function of MapB might be associated to pathogenicity related activities. MSRs are widely studied for their repair role but not for their expression regulation. In B. subtilis, MSR-coding genes (msrA and msrB) constitute an operon with a unique sigma A-dependent promoter. Their expression was induced specifically by paraquat (PQ) but not by H2O2. Spx, a global oxidative stress regulator, was implicated in the above process but partially. The function of Spx in the regulation depended on its CXXC motif. A genome-wide mutagenesis analysis identified a second gene, yjbH, participating in the expression regulation of this operon, possibly through controlling the amount of Spx in vivo. YjbH and Spx were shown to display direct protein-protein interaction. For the first time, our work uncovered several elements controlling the expression regulatory pathway of the msrAB operon in B. subtilis, especially the PQ-specific oxidative induction pathway.Les méthionine aminopeptidases (MAP) et les méthionine sulfoxide réductases (MSR) maintiennent un stock disponible de méthionine chez Bacillus subtilis. Recyclant la méthionine du début des protéines, les premières sont essentielles. La plupart des procaryotes ont un seul gène map: nous avons démontré que B. subtilis en a deux, mapA and mapB (yflG). MapB est 100 fois plus efficace que MapA avec des substrats synthétiques. A la différence de MapA, MapB requiert le cobalt pour son action. Toutes deux sont actives in vivo, et se répartissent uniformément dans la cellule. L expression de mapA est 50 à 100 fois supérieure à celle de mapB in vivo, en accord avec son rôle essentiel. Notre analyse phylogénétique associe l activité de MapB à l apparition de la pathogénicité. Les MSR ont été très étudiées pour leur rôle réparateur mais on ne sait rien de la régulation de leur expression. Nous avons montré que msrA et msrB forment un opéron partant d un promoteur sigma A. Son expression est induite spécifiquement par le paraquat (PQ) et pas par H2O2. Nous avons découvert que Spx, régulateur du stress oxydant, est partiellement impliqué. Cette fonction passe par l intégrité d un motif CXXC. La mutagenèse du génome par transposons nous a permis d identifier un second gène de régulation, yjbH, dont le produit contrôle la disponibilité de Spx. Nous avons mis au jour des interactions protéine/protéine entre YjbH et Spx. Nos travaux ont découvert, pour la première fois, un ensemble d éléments de contrôle de l expression de l opéron msrAB chez B. subtilis, et ont montré que ce contrôle résulte d un effet spécifique de la catégorie des stress oxydants induite par le PQ.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

One‐carbon metabolism, folate, zinc and translation

Summary The translation process, central to life, is tightly connected to the one‐carbon (1‐C) metabolism via a plethora of macromolecule modifications and specific effectors. Using manual genome annotations and putting together a variety of experimental studies, we explore here the possible reasons of this critical interaction, likely to have originated during the earliest steps of the birth of the first cells. Methionine, S‐adenosylmethionine and tetrahydrofolate dominate this interaction. Yet, 1‐C metabolism is unlikely to be a simple frozen accident of primaeval conditions. Reactive 1‐C species (ROCS) are buffered by the translation machinery in a way tightly associated with the metabolism of iron–sulfur clusters, zinc and potassium availability, possibly coupling carbon metabolism to nitrogen metabolism. In this process, the highly modified position 34 of tRNA molecules plays a critical role. Overall, this metabolic integration may serve both as a protection against the deleterious formation of excess carbon under various growth transitions or environmental unbalanced conditions and as a regulator of zinc homeostasis, while regulating input of prosthetic groups into nascent proteins. This knowledge should be taken into account in metabolic engineering

A growth-rate composition formula for the growth of E.coli on co-utilized carbon substrates

When bacteria are cultured in medium with multiple carbon substrates, they frequently consume these substrates simultaneously. Building on recent advances in the understanding of metabolic coordination exhibited by Escherichia coli cells through cAMP-Crp signaling, we show that this signaling system responds to the total carbon-uptake flux when substrates are co-utilized and derive a mathematical formula that accurately predicts the resulting growth rate, based only on the growth rates on individual substrates

Small RNA GcvB Regulates Oxidative Stress Response of Escherichia coli

Small non-translated regulatory RNAs control plenty of bacterial vital activities. The small RNA GcvB has been extensively studied, indicating the multifaceted roles of GcvB beyond amino acid metabolism. However, few reported GcvB-dependent regulation in minimal medium. Here, by applying a high-resolution RNA-seq assay, we compared the transcriptomes of a wild-type Escherichia coli K-12 strain and its gcvB deletion derivative grown in minimal medium and identified putative targets responding to GcvB, including flu, a determinant gene of auto-aggregation. The following molecular studies and the enhanced auto-aggregation ability of the gcvB knockout strain further substantiated the induced expression of these genes. Intriguingly, the reduced expression of OxyR (the oxidative stress regulator) in the gcvB knockout strain was identified to account for the increased expression of flu. Additionally, GcvB was characterized to up-regulate the expression of OxyR at the translational level. Accordingly, compared to the wild type, the GcvB deletion strain was more sensitive to oxidative stress and lost some its ability to eliminate endogenous reactive oxygen species. Taken together, we reveal that GcvB regulates oxidative stress response by up-regulating OxyR expression. Our findings provide an insight into the diversity of GcvB regulation and add an additional layer to the regulation of OxyR

The two authentic methionine aminopeptidase genes are differentially expressed in <it>Bacillus subtilis</it>

Abstract Background Two putative methionine aminopeptidase genes, map (essential) and yflG (non-essential), were identified in the genome sequence of Bacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in B. subtilis. Results In silico analysis of MAP evolution uncovered a coordinated pattern of MAP and deformylase that did not correlate with the pattern of 16S RNA evolution. Biochemical assays showed that both MAP (MAP_Bs) and YflG (YflG_Bs) from B. subtilis overproduced in Escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. Compared with MAP_Bs, YflG_Bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates. Both map and yflG genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both E. coli and B. subtilis. In contrast, lacZ gene transcriptional fusions showed that the promoter activity of map was 50 to 100-fold higher than that of yflG. Primer extension analysis detected the transcription start site of the yflG promoter. Further work identified that YvoA acted as a possible weak repressor of yflG expression in B. subtilis in vivo. Conclusion Both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vitro and in vivo. The high expression level of map and low expression level of yflG may account for their essentiality and dispensality in B. subtilis, respectively, when cells are grown under laboratory conditions. Their difference in activity on synthetic substrates suggests that they have different protein targets in vivo.</p