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

BACKGROUND: Sinorhizobium 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. RESULTS: In 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. CONCLUSIONS: This 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.

Proteomic alterations explain phenotypic changes in Sinorhizobium meliloti lacking the RNA chaperone Hfq.

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.

RpoE2 of Sinorhizobium meliloti is necessary for trehalose synthesis and growth in hyperosmotic media.

Adaptation to osmotic stress can be achieved by the accumulation of compatible solutes that aid in turgor maintenance and macromolecule stabilization. The genetic regulation of solute accumulation is poorly understood, and has been described well at the molecular level only in enterobacteria. In this study, we show the importance of the alternative sigma factor RpoE2 in Sinorhizobium meliloti osmoadaptation. Construction and characterization of an S. meliloti rpoE2 mutant revealed compromised growth in hyperosmotic media. This defect was due to the lack of trehalose, a minor carbohydrate osmolyte normally produced in the initial stages of growth and in stationary phase. We demonstrate here that all three trehalose synthesis pathways are RpoE2 dependent, but only the OtsA pathway is important for osmoinducible trehalose synthesis. Furthermore, we confirm that the absence of RpoE2-dependent induction of otsA is the cause of the osmotic phenotype of the rpoE2 mutant. In conclusion, we have highlighted that, despite its low level, trehalose is a crucial compatible solute in S. meliloti, and the OtsA pathway induced by RpoE2 is needed for its accumulation under hyperosmotic conditions.

Sinorhizobium meliloti rpoE2 is necessary for H(2)O(2) stress resistance during the stationary growth phase.

RpoE2 is an extracytoplasmic sigma factor produced by Sinorhizobium meliloti during stationary growth phase. Its inactivation affected the synthesis of the superoxide dismutase, SodC, and catalase, KatC. The absence of SodC within the cell did not result in an increased sensitivity to extracellular superoxides. In contrast, the absence of KatC affected the resistance of S. meliloti to H(2)O(2) during the stationary growth phase. A katC strain behaved as an rpoE2 strain during an H(2)O(2) challenge, suggesting that the H(2)O(2) sensitivity of the rpoE2 strain resulted only from the lack of KatC in this strain.

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

tmRNA (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.

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

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.

Purification and characterization of the major lipoprotein (P28) of Spiroplasma apis.

The plasma membrane of Spiroplasma apis contains a 28-kDa major protein (P28), like other spiroplasmas which also possess a main 26- to 28-kDa membrane polypeptide, called spiralin. In the work described here, we have developed a simple and efficient method for the purification of P28 of this mollicute, a wall-less eubacteria. Proteins were first selectively extracted from the isolated membrane with the mild detergents (i) sodium N-lauroylsarcosinate (Sarkosyl) and (ii) 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (Chaps) and subjected to size-exclusion HPLC in the presence of Chaps. The P28-enriched fraction was thereafter subjected to the second chromatographic step involving cation exchange HPLC in the presence of the same detergent. P28 was purified at the milligram level (yield, 40%). Metabolite labeling with [14C]palmitic acid and chemical analysis of P28 indicated that it is covalently modified by two O-ester-bound fatty acids and one amide-linked chain and contains a S-glycerylcysteine at the N-terminus. By charge-shift electrophoresis, Triton X-114 phase separation, and growth inhibition tests it was shown that P28 is a typical amphiphilic protein exposed, at least partly, at the cell surface. Together, our data provided evidence that P28 is a "classical" lipoprotein (i.e., triacylated) like the members of the spiralin family.

Antibacterial activity of glycine betaine analogues: involvement of osmoporters.

Glycine betaine (GB) analogues were obtained using solid phase organic synthesis and assayed for their toxic activity against 15 Gram positive and Gram negative bacteria. Four benzyl derivatives of GB were selected to determine their effect on bacterial growth. Bacteriostatic and lethal effects were observed for compound 1 and compound 2, respectively. The importation of the two GB analogues into bacterial cells appeared strictly dependent on the presence of the powerful betaine membrane osmoporters; their capacity to be amassed intracellularly at molar levels from extremely dilute solutions might constitute a basis to design a new class of antimicrobial agents.