No abstract

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...

In Pseudomonas aeruginosa, SigX is an extra-cytoplasmic function σ factor that belongs to the cell wall stress response network. In previous studies, we made the puzzling observation that sigX mutant growth was severely affected in rich lysogeny broth (LB) but not in minimal medium. Here, through comparative transcriptomic and proteomic analysis, we show that the absence of SigX results in dysregulation of genes, whose products are mainly involved in transport, carbon and energy metabolisms. Production of most of these genes is controlled by carbon catabolite repression (CCR), a key regulatory system than ensures preferential carbon source uptake and utilization, substrate prioritization and metabolism. The strong CCR response elicited in LB was lowered in a sigX mutant, suggesting altered nutrient uptake. Since the absence of SigX affects membrane composition and fluidity, we suspected membrane changes to cause such phenotype. The detergent polysorbate 80 (PS80) can moderately destabilize the envelope resulting in non-specific increased nutrient intake. Remarkably, growth, membrane fluidity and expression of dysregulated genes in the sigX mutant strain were restored in LB supplemented with PS80. Altogether, these data suggest that SigX is indirectly involved in CCR regulation, possibly via its effects on membrane integrity and fluidity.

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.

No abstract

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.

The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.

The prevalence of the insertion sequence IS1548 is strongly linked to clonal complex 19 Streptococcus agalactiae strains associated with neonatal meningitis and endocarditis. We previously reported that IS1548 insertion upstream of lmb is involved in stronger binding of a S. agalactiae meningitic strain to laminin. A few other IS1548 insertion sites were also identified by others. In this study, we analyzed IS1548 described target sites in S. agalactiae and showed that most of them are linked to zinc-responsive genes. Moreover, we identified two not yet described IS1548 insertion sites in the adcRCB operon encoding the main regulator of zinc homeostasis and subunits of a zinc ABC transporter. We also identified two conserved motifs of 8 and 10 bp close to IS1548 insertion sites. These motifs representing potential IS1548 targets were found upstream of several S. agalactiae ORFs. One of these predicted IS1548 targets was validated experimentally, allowing the identification of an IS1548 insertion site upstream of murB in all of the clonal complex 19 strains tested. The possible effects of these insertions on the virulence of the strains are discussed.