The ubiquitous bacterial RNA-binding protein Hfq is involved in stress resistance and pathogenicity. In Sinorhizobium meliloti, Hfq is essential for the establishment of symbiosis with Medicago sativa and for nitrogen fixation. A proteomic analysis identifies 55 proteins with significantly affected expression in the hfq mutant; most of them are involved in cell metabolism or stress resistance. Important determinants of oxidative stress resistance, such as CysK, Gsh, Bfr, SodC, KatB, KatC, and a putative peroxiredoxine (SMc00072), are downregulated in the hfq mutant. The hfq mutant is affected for H 2 O 2 , menadione, and heat stress resistance. Part of these defects could result from the reductions of rpoE1, rpoE2, rpoE3, and rpoE4 expression levels in the hfq mutant. Some proteins required for efficient symbiosis are reduced in the hfq mutant, contributing to the drastic defect in nodulation observed in this mutant.Gene expression in bacteria is regulated by a wide diversity of mechanisms, including alternative sigma factors, transcriptional regulatory proteins, attenuation mechanisms (including riboswitches) (15), and translational and posttranslational regulations (37). The interplay of central regulatory proteins and alternative sigma factors allows the creation of complex regulatory networks modulating transcription (4).Compared to transcription regulation, the mechanisms affecting the regulation of translation are less understood. Studies dedicated to translation regulation have increased over the past few years (55,76,77). An important development has been the recognition of small regulatory RNAs (sRNAs) that have emerged as crucial actors of translation regulation. In enterobacteria, most sRNAs require Hfq to complex with their targets. Hfq is an RNA chaperone necessary for the pairing of sRNAs with mRNAs (40). Furthermore, Hfq affects translation efficiency by allowing the polyadenylation of specific mRNAs (44). Thus, Hfq is a central actor in translation regulation (72). Hfq is also able to affect transcription, directly by coupling with RNA polymerase (67) or indirectly via its action on sRNAs modulating translation of sigma factors (19,32,69).Due to its central role, hfq inactivation results in a pleiotropic phenotype in enterobacteria and Brucella abortus, including growth defects, stress susceptibility, and altered pathogenicity (56,65,76). Our accompanying study shows that loss of hfq impairs the ability of Sinorhizobium meliloti to establish a nitrogen-fixing symbiosis with its legume host, Medicago sativa. S. meliloti faces numerous stresses during the course of invading the developing root nodules and colonizing the plant cells (21,22,35,46,62). Bacterial abilities to resist and adapt to these stresses are of crucial importance for the symbiosis. Oxidative stress has been the most intensively investigated stress that S. meliloti must withstand and appears as a key factor for bacterium-plant cell interaction. To cope with oxidative stress, S. meliloti cells posses a detoxification system involvi...
BackgroundSinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H2O2 and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti 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.ResultsIn this study we report the characterisation of organic hydroperoxide resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione in vitro. The expression of ohr and ohrR genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes ohr and ohrR. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both ohr and ohrR are expressed in nodules suggesting that they play a role during nitrogen fixation.ConclusionsThis report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in S. meliloti. The ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation.
Methionine 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 B 12 -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 experiment.
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