Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti

<p>Abstract</p> <p>Background</p> <p><it>Sinorhizobium meliloti </it>is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants <it>Medicago sativa</it>. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H₂O₂and superoxides has been extensively studied in <it>S. meliloti</it>. In contrast resistance to organic peroxides has not been investigated while <it>S. meliloti </it>genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria.</p> <p>Results</p> <p>In this study we report the characterisation of organic hydroperoxide resistance gene <it>ohr </it>and its regulator <it>ohrR </it>in <it>S. meliloti</it>. The inactivation of <it>ohr </it>affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione <it>in vitro</it>. The expression of <it>ohr </it>and <it>ohrR </it>genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes <it>ohr </it>and <it>ohrR</it>. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of <it>ohr </it>did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both <it>ohr </it>and <it>ohrR </it>are expressed in nodules suggesting that they play a role during nitrogen fixation.</p> <p>Conclusions</p> <p>This report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in <it>S. meliloti</it>. The <it>ohr </it>and <it>ohrR </it>genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation.</p

Tolérance acquise aux stress en phase stationnaire chez Sinorhizobium meliloti (rôle du gène rpoE2)

Pour résister aux fluctuations de l environnement, les bactéries ont développé différents mécanismes qui sont finement régulés. Les facteurs sigma extracytoplasmiques sont des régulateurs qui redirigent la transcription lors de stress. Sinorhizobium meliloti, bactérie de la rhizosphère, possède 11 homologues de gènes rpoE alors qu aucun facteur sigma général de stress n a été identifié. Nous avons caractérisé le rôle du facteur RpoE2 dans la réponse au stress. RpoE2 est impliqué dans la tolérance acquise en phase stationnaire à un choc thermique et à l H2O2. La résistance acquise à l H2O2 est due à l induction de la catalase KatC sous la dépendance de RpoE2. Le mutant rpoE2 est aussi affecté lors de la croissance en conditions hyperosmotiques. Ce défaut est corrélé à une absence d accumulation de tréhalose. RpoE2 contrôle l expression des gènes treY et otsA qui sont respectivement impliqués dans la synthèse du tréhalose en phase stationnaire et lors d un stress hyperosmotique.In order to cope with physio-chemical fluctuations of the environment, bacteria have developed many mechanisms of adaptation. They must be acutely regulated. Extra-cytoplasmic sigma factors (ECF) are regulators binding RNA polymerase to adjust transcription during a stress. Sinorhizobium meliloti is a rhizospheric bacterium able to manage numerous stresses. While no general stress sigma factor (rpoS) is annotated, its genome encodes 11 rpoE homologs. We characterised in this study, RpoE2, an ECF of S. meliloti. RpoE2 is involved during stationary phase in resistance to heat choc and H2O2. This resistance to H2O2 results of a RpoE2 dependant induction of KatC. RpoE2 defective strains are also affected for growth under hyperosmotic conditions. This osmosensitivity is related to the absence of trehalose synthesis in the rpoE2 mutant. This defect was linked to the control of treY, treS and otsA by RpoE2. In fact, otsA mutant is also osmosensitive.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Characterization of HicAB toxin-antitoxin module of Sinorhizobium meliloti

Abstract Background Toxin-antitoxin (TA) systems are little genetic units generally composed of two genes encoding antitoxin and toxin. These systems are known to be involved in many functions that can lead to growth arrest and cell death. Among the different types of TA systems, the type II gathers together systems where the antitoxin directly binds and inhibits the toxin. Among these type II TA systems, the HicAB module is widely distributed in free-living Bacteria and Archaea and the toxin HicA functions via RNA binding and cleavage. The genome of the symbiotic Sinorhizobium meliloti encodes numerous TA systems and only a few of them are functional. Among the predicted TA systems, there is one homologous to HicAB modules. Results In this study, we characterize the HicAB toxin-antitoxin module of S. meliloti. The production of the HicA of S. meliloti in Escherichia coli cells abolishes growth and decreases cell viability. We show that expression of the HicB of S. meliloti counteracts HicA toxicity. The results of double hybrid assays and co-purification experiments allow demonstrating the interaction of HicB with the toxin HicA. Purified HicA, but not HicAB complex, is able to degrade ribosomal RNA in vitro. The analysis of separated domains of HicB protein permits us to define the antitoxin activity and the operator-binding domain. Conclusions This study points out the first characterization of the HicAB system of the symbiotic S. meliloti whereas HicA is a toxin with ribonuclease activity and HicB has two domains: the COOH-terminal one that binds the operator and the NH2-terminal one that inhibits the toxin

pBLA8, from Brevibacterium linens, belongs to a Gram-positive subfamily of ColE2-related plasmids

International audienceSUMMARY: A 3.1 kb DNA fragment from pBLA8, a Brevibacterium linens cryptic plasmid, containing all the information required for autonomous replication was cloned and sequenced. Using deletion analysis, the fragment essential and sufficient for autonomous replication was delimited to 1.5 kb. This fragment is characterized by the presence of an ori site located upstream of an operon encoding two proteins, RepA and RepB, both essential for replication. Based on structural similarities and a strong conservation of ori, RepA and RepB, pBLA8 was assigned to a new subfamily of the ColE2 plasmid family. This subfamily is distinguished by the requirement for two Rep proteins and the location of an ori site upstream of the mpAB operon. RepA is thought to encode primase activity, whereas RepB could be a DNA-binding protein. An Escherichia coli-B. linens shuttle vector, derived from pBLA8, was constructed. Its host spectrum was extended to Arthrobacter species

Interrelations between glycine betaine catabolism and methionine biosynthesis in Sinorhizobium meliloti strain 102F34

International audienceMethionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B12-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experimen

Characterization and expression patterns of Sinorhizobium meliloti tmRNA (ssrA).

International audiencetmRNA (ssrA) in Sinorhizobium meliloti is a small RNA annotated by homology with the Bradyrhizobium japonicum sra molecule. Here, this molecule is described in Sinorhizobium meliloti as a model for such molecules in Alphaproteobacteria subgroup-2. Northern blot analysis and mapping of both 5' and 3' ends of this tmRNA allow the identification of two pieces: a 214 nt mRNA-like domain and an 82 nt tRNA-like domain, both highly stable, whereas the premature form is unstable. Transcriptional studies reveal that Sinorhizobium meliloti tmRNA is mainly expressed during growth resumption, replication initiation and various stress responses

Glucose 6-phosphate dehydrogenase is required for sucrose and trehalose to be efficient osmoprotectants in Sinorhizobium meliloti.

Equipe Osmorégulation chez les Bactéries (OB) devenue équipe Dualité ou Universalité de l'Adaptation lors d'un Stress (DAULS)International audienceInactivation of the zwf gene in Sinorhizobium meliloti induces an osmosensitive phenotype and the loss of osmoprotection by trehalose and sucrose, but not by ectoine and glycine betaine. This phenotype is not linked to a defect in the biosynthesis of endogenous solutes. zwf expression is induced by high osmolarity, sucrose and trehalose, but is repressed by betaine. A zwf mutant is more sensitive than its parental strain to superoxide ions, suggesting that glucose 6-phosphate dehydrogenase involvement in the osmotic response most likely results from the production of reactive oxygen species during osmotic stress