Synthesis of glutamate can be limited in bacterial strains carrying mutations to loss of function of glutamate synthase (2-oxoglutarate.glutamine aminotransferase) by using low concentrations of NH4' in the growth medium. By using such gtBID mutant strains of Sabnonella typhimnurum, we demonstrated that: (i) a large glutamate pool, previously observed to correlate with growth at high external osmolality, is actually required for optimal growth under these conditions; (ii) the osmoprotectant glycine betaine (NNN-trimethylglycine) apparently cannot substitute for glutamate; and (iii) accumulation of glutamate is not necessary for high levels of induction of the proU operon in vivo. Expression of the proU operon, which encodes a transport system for the osmoprotectants proline and glycine betaine, is induced >100-fold in the wild-type strain under conditions of high external osmolality. Intracellular osmolality of organisms is determined by a few species of solutes known as compatible solutes, whose concentrations vary in parallel with the osmolality of the environment (3). Glutamate, trehalose, K+, glycine betaine, and proline are some of the important compatible solutes in members of the family Enterobacteriaceae (reviewed in reference 7). In these organisms, glutamate and trehalose are synthesized internally, but the other three are taken up from the medium under conditions of high external osmolality. It is well documented that K+ is an essential nutrient in all environments (12,13), that trehalose is required for growth in media of high osmolality (15), and that exogenous proline and glycine betaine can alleviate the inhibitory effects of high osmotic stress (7). However, a requirement for high internal concentrations of glutamate to counter high external osmolality has not been demonstrated.In bacteria, glutamate is synthesized by two pathways: biosynthetic glutamate dehydrogenase (GDH) and glutamate synthase (2-oxoglutarate:glutamine aminotransferase [GOGATI) in conjunction with glutamine sythetase (Fig. 1) (49). Since GDH has a relatively poor affinity for NH4' (Kin, -1 to 5 mM), glutamate is synthesized primarily via the glutanine synthetase-GOGAT cycle in media containing low concentrations of NH4' (2,34,49). In gitB or gltD mutant strains, which lack a functional GOGAT, glutamate can be synthesized only by GDH, and therefore it is possible to limit glutamate synthesis in such strains by growing them in media containing suboptimal concentrations of NH4+. We exploited this property of glutamate synthase * Corresponding author. Phone: (317) 496-1496. mutants of Salnonella typhimnuium to study the role of glutamate in osmotic adaptation and found that high concentrations of this solute are necessary for optimal growth in media of high osmolality.In S. typhimurium and Escherichia coli, proline and glycine betaine are accumulated to high concentrations under conditions of high osmolality by two permeases: the ProP system, which is synthesized constitutively, and the ProU system, which is induced >100-f...

In a genetic analysis of the determinants of thermotolerance in S. enterica serovar Typhimurium, we isolated the chr-1 mutation that increased the resistance of exponential phase cells to killing by high temperature. This mutation is a single base change in the mgtA riboswitch that causes high-level constitutive expression of mgtA. We showed that another mgtA riboswitch mutation, ⌬UTR re-100 , which had been constructed by Cromie et al., also confers similar increased thermotolerance. Surprisingly, the chr-1 mutation is located at a position that would not be predicted to be important for the regulatory function of the riboswitch. We obtained physiological evidence suggesting that the chr-1 mutation increases the cytosolic free Mg 2؉ concentration. High-level expression of the heterologous MgtE Mg 2؉ transport protein of Bacillus subtilis also enhanced the thermotolerance of S. enterica. We hypothesize that increased Mg 2؉ accumulation might enhance thermotolerance by protecting the integrity of proteins or membranes, by mitigating oxidative damage or acting as an inducer of thermoprotective functions.heat tolerance ͉ riboswitch ͉ Mg 2ϩ homeostasis ͉ heat shock A s a pathogen that can grow in a free living state in various environments and in hosts ranging from ectothermic reptiles to warm-blooded mammals and birds whose temperature may be elevated by fever (1), Salmonella possesses sophisticated mechanisms for thermal adaptation. Salmonella is the most frequent causative agent of foodborne bacterial infections in the United States (2), and because heat treatment is the most common and economical means for inactivating food pathogens better understanding of the regulation of thermotolerance in this organism has important applications in food safety.The best-studied mechanism of thermal adaptation is the heat shock response. This response involves the transient induction of thermoprotective proteins by exposure to sublethal high temperatures, which enables organisms to survive subsequent treatments with higher temperatures that would be otherwise lethal (3). Thermotolerance can be regulated by a number of other factors, including stationary phase (4) and acidic or alkaline pH (5, 6).Another environmental condition that regulates thermoresistance is water activity (a w ). Exposure to elevated osmolality enhances the thermotolerance of bacteria in two ways: by elevating their upper limit of growth temperature (7,8) and increasing their viability at lethal high temperatures (9, 10). Our laboratory observed that the thermoprotective effect of high osmolality on the survival of Salmonella enterica serovar Typhimurium is reversed by glycine betaine (11). Glycine betaine is an osmoprotectant, which can alleviate the growth inhibitory effects of high osmolality (12). Along with ameliorating the osmotic inhibition of growth, this compound suppresses a number of other responses brought about by osmotic stress, such as the accumulation of K ϩ , the synthesis of trehalose, and the shrinkage of the cytoplasmic volume (13).To ...

The transcriptional control of the kdpFABC (K ؉ transport) operon of Salmonella typhimurium was characterized with a lacZ fusion. The kdpFABC operon of this organism was induced by K ؉ limitation and high osmolality, and osmotic induction was antagonized by a high concentration of K ؉ . In the trkA (sapG) kdp ؉ mutant background, high concentrations of K ؉ inhibited growth, along with repressing the kdp operon. This result, which has not been reported for Escherichia coli, is inconsistent with the model in which the signal for the induction of the kdp operon is turgor loss.In enteric bacteria, K ϩ is taken up by two major permeases, Trk and Kdp, and by a minor permease, Kup (13,18). In Escherichia coli, the activity of these three systems has been shown to be stimulated by osmotic stress (2). Stimulation of Kdp is effected largely by transcriptional induction of the kdpFABC operon. The kdpFABC operon is also induced by K ϩ limitation. Transcriptional control of the kdpFABC operon is mediated by the KdpD and KdpE proteins (2), which belong to the family of two-component regulatory proteins. It has been proposed that the regulatory signal for the expression of the kdp operon is turgor (4), but whether the osmotic-stress-and the K ϩ -dependent regulations are mediated by a single mechanism or by two distinct mechanisms is controversial (1, 2, 6, 7).The regulation of the kdp operon in Salmonella typhimurium has not been studied. Because of difficulties in obtaining kdp mutations in this organism, it has been suggested that there may be an important difference between the properties of the Kdp system in E. coli and in S. typhimurium (17). In this report, we describe the isolation of a kdp-lacZ fusion in S. typhimurium.Strain construction. The medium used for the isolation of the kdp-lacZ mutations was K0 medium (5), which has a K ϩ concentration of 0.1 mM (introduced as a trace contaminant in other chemicals). Approximately 5,000 derivatives of the wildtype S. typhimurium strain LT2 carrying random MudI1734 (Km lac) insertions (10) were replica plated to K0 plates containing 10 mM glucose, kanamycin, and 40 mg of X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) per liter (12) and to plates containing the same ingredients plus 10 mM KCl. We identified one derivative, strain TL2626, which formed a very small, dark blue colony on the former medium and a normal-sized, white colony on the latter; the mutation in this strain was designated kdp-101::MudI1734.P22 transductions (3) demonstrated that the kdp-101:: MudI1734 insertion was 96% linked and was located upstream of a kdp::Tn10 insertion (obtained from E. Groisman) (data not shown). In Southern blot analyses, a fragment containing the kdpFABC operon of E. coli exhibited different patterns of hybridization to EcoRI, EcoRV, PvuII, and HincII fragments of the DNA from strain TL2626 than to those from strain LT2 (data not shown). The kdp-101::MudI1734 insertion was complemented by a plasmid which expressed the kdpB ϩ C ϩ genes of E. coli (obtained from W. Epstein)...

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of Salmonella enterica serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45°C. One of these mutations proved to be in the seqA gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with pgm (which encodes phosphoglucomutase), it is likely to be polar on the expression of the pgm gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the seqA::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an otsA mutation, which inactivates the first step of the trehalose biosynthetic pathway. The otsA mutation caused a growth defect at 45°C in minimal medium containing 0.2 M NaCl that was similar to that caused by the pgm mutation, but otsA did not affect growth rate in this medium at 37°C. These results suggest that the growth defect of the seqA-pgm mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45°C had a fivefold higher trehalose pool size than cells grown at 30°C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.Although high osmolality is generally regarded as a source of "stress" (3), this is not necessarily always the case, because raising the osmotic strength of the medium can increase the thermotolerance of bacteria (5,16,19,32).In bacteria, thermotolerance can be quantified by assays of at least two responses: viability at lethal high temperatures and growth rate at nonlethal but inhibitory high temperatures. Moderate or high osmolality enhances both of these aspects of thermotolerance in Escherichia coli and Salmonella enterica serovar Typhimurium. It is not clear whether these two osmoregulated responses, namely enhancement of viability at lethal high temperatures and stimulation of growth near the upper limit of nonlethal temperatures, are different manifestations of a single osmotically controlled thermotolerance mechanism or they represent two independent responses. Hengge-Aronis et al. (19) observed that the high-osmolality-dependent acquisition of increased viability at high temperatures in ex...

Transcriptional control of the osmotically regulated proU operon of Salmonella typhimurium is mediated in part by a transcriptional silencer downstream from the promoter (D. G. Overdier and L. N. Csonka, Proc. Natl. Acad. Sci. USA 89:3140-3144, 1992). We carried out a fine-structure deletion analysis to determine the structure and the position of the silencer, which demonstrated that this regulatory element is located between nucleotide positions ؉73 to ؉274 downstream from the transcription start site. The silencer appears to be made up of a number of components which have cumulative negative regulatory effects. Deletions or insertions of short nucleotide sequences (<40 bp) between the proU promoter and the silencer do not disrupt repression exerted by the silencer, but long insertions (>0.8 kbp) result in a high level of expression from the proU promoter, similar to that imparted by deletion of the entire silencer. The general DNA-binding protein H-NS is required for the full range of repression of the proU operon in media of low osmolality. Although in the presence of the silencer hns mutations increased basal expression from the proU promoter three-to sixfold, in the absence of the silencer they did not result in a substantial increase in basal expression from the proU promoter. Furthermore, deletion of the silencer in hns ؉ background was up to 10-fold more effective in increasing basal expression from the proU promoter than the hns mutations. These results indicate that osmotic control of the proU operon is dependent of some factor besides H-NS. We propose that the transcriptional regulation of this operon is effected in media of low osmolality by a protein which makes the promoter inaccessible to RNA polymerase by forming a complex containing the proU promoter and silencer.The proU operon, which specifies the three constituent proteins of a transport system for osmoprotectants such as glycinebetaine and proline (5), is induced Ͼ100-fold by high osmolality in Salmonella typhimurium and Escherichia coli. Transcriptional control of the proU operon is mediated at least in part by a cis-acting negative element, or silencer, that is located ϳ100 to 200 bp downstream of the promoter (7, 18). In S. typhimurium, deletion of this element resulted in up to a 40-fold increase in expression from the proU promoter in media of low osmolality (18).Thus far, attempts to isolate mutations in regulatory genes that are unique to the proU operon have not been successful. This impasse has led to the formulation of two models which propose that the transcriptional control mechanism of the proU operon is unusual in that it does not involve any specific regulatory proteins. Both models are based on the assumption that the proU promoter, which does not have a good match to the consensus optimal promoter (Fig. 1), is inherently weak and needs some activating factor for high-level expression in media of high osmolality. According to one model, potassium glutamate, which accumulates to high concentrations in media of high osmolality ...