Catherine Fontenelle

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.

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.

Acylation and immunological properties of Mycoplasma gallisepticum membrane proteins

The acylation of Mycoplasma gallisepticum membrane proteins was studied by electrophoresis after in vivo labelling with different 14C-fatty acids and by chemical analysis. The immunological properties of these proteins were investigated by Western blotting and crossed immunoelectrophoresis. Among the ca. 200 membrane polypeptides resolved by two-dimensional electrophoresis, 35 components (including the major protein p67) were covalently modified with acyl chains. These acylated proteins displayed lower pls than average (5.0-7.4 vs. 5.0-9.0) and proved to be the major membrane protein antigens and immunogens of M. gallisepticum. The apparent selectivity of fatty acid incorporation into proteins was, as suggested by in vivo labelling: palmitic acid (16:0) > myristic acid (14:0) > oleic acid (18:1c) > stearic acid (18:0) > linoleic acid (18:2c). However, the true order of selectivity, as revealed by chemical analysis, proved to be 18:2c > 16:0 > 18:1c > 18:0 > 14:0. More specifically, palmitic acid was the major O-ester-bound fatty acid and linoleic acid the major amide-linked fatty acid. The observed average ratio [O-ester-bound + amide-linked acyl chains]/O-ester-bound chains approximately 1.4 and the presence of S-glycerylcysteine suggest that, in M. gallisepticum, membrane proteins are lipid-modified according to a mechanism identical to that depicted for lipoproteins of Gram-negative eubacteria.

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.

Inhibition of spiralin processing by the lipopeptide antibiotic globomycin.

Abstract. The cyclic lipopeptide globomycin, a specific inhibitor of signal-peptidase II (Lsp A), proved toxic for the mollicute Spiroplasma melliferum with a minimal inhibitory concentration (MIC) in the range 6.25–12.5 μM, about one order of magnitude higher (that is, less efficient) than bee-venom mellitin. SDS-PAGE analysis of cell proteins followed by immunolabeling (“Western blotting”) and by crossed immunoelectrophoresis demonstrated that the cleavage of the prespiralin leader peptide was prevented by globomycin. Cell fractionation experiments showed that prespiralin was membrane bound and did not accumulate in the cytoplasm or in the culture medium. Furthermore, the use of the potential-sensitive fluorescent dye 3,3′-dipropyl-2,2′-thiadicarbocyanine iodide (diS-C3-[5]) revealed that, in contrast to valinomycin and mellitin, globomycin up to 30 μM has no effect on the electrical transmembrane potential of S. melliferum. This indicates that the toxicity of globomycin for spiroplasma cells is mainly if not exclusively owing to the inhibition of spiralin processing. Added to previously published data, these results suggest that spiralin and probably other lipoproteins of mollicutes are acylated and membrane targeted by a mechanism involving notably the processing of the prelipoprotein precursor by a type II, globomycin-sensitive signal peptidase.

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

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.

Biochemical and antigenic characterisation of Mycoplasma gallisepticum membrane proteins P52 and P67 (pMGA)

Abstract. Two membrane proteins from the avian pathogen Mycoplasma gallisepticum have been previously purified using a simple, efficient and non-denaturing method: a lipoprotein P67 (pMGA) and P52. In the current study, the lipid part of P67 was chemically analysed. The molecular structure of the lipoprotein-lipid component was determined to be S-glyceryl cysteine with two O-ester-linked acyl chains. Fatty acid analysis of the purified P67 indicated a heterogeneous composition: palmitic acid (16:0)>stearic acid (18:0)>oleic acid (18:1c)>myristic acid (14:0), with 16:0 as the major component. These findings, along with previously published results, support the conclusion that P67 is pMGA1.2, a true membrane-associated lipoprotein although not N-acylated. In contrast to P67, P52 is not a lipoprotein. Topological experiments using in situ treatment with proteases and growth inhibition in the presence of anti-P52 serum provided evidence of the surface exposition of the polypeptide. The N-terminal sequence of P52 was found to be similar to the dihydrolipoamide acetyltransferase from several mollicutes; this enzyme is a membrane-associated component of the pyruvate dehydrogenase complex. Immunoblotting techniques revealed that the surface antigens P52 and P67 were specific to the species M. gallisepticum and the closely related species M. imitans. No antigenic difference was revealed within these species with the anti-P52 serum, while anti-P67 serum confirmed the antigenic variability of P67. The potential of P52 and P67 as antigens in serological diagnosis tests or as candidates for anti-mycoplasma subunit vaccines is discussed.

Selective Acylation of Plasma Membrane Proteins of Mycoplasma mycoides subsp. mycoides SC, the Contagious Bovine Pleuropneumonia Agent

Abstract. The plasma membrane of Mycoplasma mycoides subsp. mycoides SC (strain KH3J) contains over 160 polypeptides with apparent molecular masses ranging from 14 to 125 kDa and isoelectric point values (pIs) from 5 to 9. In vivo labeling with [14C]-fatty acids revealed about 35 acylated polypeptides including the two major components p42 and p65 and displaying pIs between 5.5 and 9.0, with a majority between 6.5 and 8. The amphiphilic nature of most of these acyl proteins was confirmed by Triton X-114 phase partitioning. Gas-liquid chromatography analyses showed that the order of preference for protein acylation was 16:0 > 18:2c > 18:1c > 18:0 > 14:0, with 16:0 being the major O-ester-bound fatty acyl chain and 18:2c the major N-linked chain. The presence of S-glycerylcysteine and a ratio of [O-ester-bound acyl chains + N-linked chains]/O-ester bound chains of ≈ 1.2 in M. mycoides subsp. mycoides SC membrane proteins are consistent with a lipid modification similar to that occurring in lipoproteins of Gram-negative eubacteria that contain an N-terminal acyl S-glycerylcysteine.

Selective Acylation of Plasma Membrane Proteins of Mycoplasma agalactiae: The Causal Agent of Agalactia

Abstract. Revealed by in vivo labeling with 14C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) ≈ palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria.

Chemical Analysis of Lipid-Modified Membrane Proteins in Acholeplasma laidlawii

The lipid modification of membrane proteins was investigated in Acholeplasma laidlawii by metabolic labeling and by chemical analysis. A S-glycerylcysteine residue was identified from membrane proteins and we reported the strong preference for saturated acyl chains into the lipid modification. Differential release of fatty acids revealed a ratio [(O-ester- + amide-bound acyl chains)/O-ester-linked chains] close to 1.1 which suggests the involvement of only two O-ester linked fatty acids in the acylation process. Present data indicate that acyl proteins in A. laidlawii are true lipoproteins (mainly diacylated) probably processed by a mechanism analogous to that described for eubacteria and other mycoplasmas.