Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.Water availability is primordial for life of all organisms. Bacteria submitted to a severe hyperosmotic stress instantaneously lose a large amount of their intracellular water to balance the osmotic strength between intracellular and extracellular spaces. The subsequent decrease of cellular water activity together with the loss of cell turgor lead to lessen the bacterial cell expansion rate (1). Surviving such injuring conditions implies the reversion of water flux across the cell membrane; this can be achieved by amassing highly soluble compounds termed osmolytes (2, 3). Thus, Escherichia coli cells rapidly take up high amounts of potassium ions (4, 5) and subsequently increase their glutamate content to balance electric charges. To avoid the perturbing effect of elevated ionic strength, K ϩ -glutamate can be progressively replaced by organic osmolytes that behave neutral at physiological pH (6). Such compounds, termed compatible solutes (7), may be endogenously synthesized or imported from the surrounding medium (3,8). Imported compatible solutes generally confer a high degree of osmotic tolerance to injured cells. Among these so-called osmoprotectants, glycine betaine (GB) 1 is by far the most effective and the most commonly assayed for hyperosmotic purposes.In addition to the obvious predominant role they play in cellular osmotic adjustment, internalized and accumulated osmoprotectants should directly participate in other intracellular processes. Protective as well as stabilizing effects of betaine and other solutes on proteins denaturation because of increased salinity or temperature have been reported (9 -12). It is tempting to extrapolate these results in vivo; however, bacteria submitte...

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...

Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA geneencoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/ betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.Microbial pathogens encounter extremely diverse environments both inside and outside their hosts. In response to these adverse conditions, they undergo striking adaptations in order to survive and retain virulence. The growth of Erwinia chrysanthemi, which is involved in a systemic soft rot disease on a variety of higher plants, is influenced by desiccation (18, 21). We have recently analyzed the influence of osmotic strength on E. chrysanthemi growth and pathogenicity in the absence and presence of osmoprotectants (10). The consequences for pathogenicity were estimated by the effect of osmotic pressure on transcription of pel genes and pectate lyase activity. The transcription of the pelE gene, encoding the major extracellular pectate lyase enzyme, is induced in medium of high osmolarity, whereas the cellular growth rate was reduced. Osmoprotectants such as glycine betaine, proline, ectoine, and pipecolic acid were shown to be accumulated in the cells through an osmoinducible mechanism and stimulated growth. However, pelE transcription was reduced to basal levels.Although uptake and accumulation of osmoprotectants have been observed in many bacteria, transporter structural genes have been characterized in only a few microorganisms. Considering the regulation of their transcription, a knowledge of which is essential to understanding osmoregulation, only the proP and proU operons of Escherichia coli and Salmonella typhimurium have been analyzed in depth (6-8, 13, 14, 20, 27, 28, 31, 32). The identification of structural domains involved in osmosensing and substrate binding could be improved by comparison of ProP and ProU with other osmoprotectant transporters in other bacteria. In this report, we characterize one of the osmoprotectant transporters of E. chrysanthemi, analogous to ProP of E. coli. MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. The E. coli and E. chrysanthemi strains and plasmids used are described in Table 1. Cells were grown in LB or M63 glucose (0.2%) medium (33). E. chrysanthemi cells were usually incubated at 30ЊC, and E. coli cells were incubated at 37ЊC. The osmoprotectants choline, glycine betaine, proline, ectoin...