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Challenge of Salmonella typhimurium with high osmolality enhances its thermotolerance, manifested as faster growth rate at non-lethal high temperatures and enhanced resistance to killing at lethal high temperatures. Previously, it has been shown that the potent osmoprotectant glycine betaine blocks the latter response. In this work, we tested the effects of ten other osmoprotectants on the induction of thermotolerance by high osmolality. The high osmolality-dependent growth stimulation at 45°C was decreased partially by γ -aminobutyrate betaine, 3,4-dehydroproline betaine, and blocked completely by pipecolate betaine and homarine. Osmotic enhancement of survival at 53°C was greatly impaired by dehydroproline betaine, pipecolate betaine, and proline betaine. Our observations point to the need for further research to discover additional betaine analogs, which might act as even more potent antagonists of the induction of thermotolerance, for possible use in food products to enhance the susceptibility of Salmonella to thermal injury or killing.

Factors limiting the growth ofSalmonellaat high temperature were analyzed in minimal medium. The growth ofS. typhimuriumwas curtailed at 44°C by high temperature-dependent block in methionine synthesis. At 45°C, the organism could not grow in glucose-mini- mal medium even with methionine, but growth could be restored at this temperature by the addition of NaCl in the final concentration range of 0.15–0.3m. Similar results were obtained with twoS. enteritidisstrains. The growth stimulatory effect of high osmolarity at 45°C was not altered by the osmoprotectant compound glycine betaine. Exposure to 0.3mNaCl also increased the resistance ofS. typhimuriumto thermal death at 50°C and to the oxidizing agent H2O2. However, the high osmolarity-dependent induction of enhanced resistance to 50°C and to H2O2was completely abolished by glycine betaine. The finding that glycine betaine blocks the induction of resistance to lethal high temperature by high osmolarity could provide a means for enhancing the bactericidal efficacy of heating, because it may be possible to increase the heat sensitivity of contaminating bacteria by supplementing food products with this naturally occurring plant metabolite, prior to thermal processing.

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 (> or = 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.

Mg 2+ homeostasis is important for Salmonella pathogenesis. In Salmonella enterica , the transcription of the mgtA gene, which encodes a Mg 2+ transporter, is regulated by a Mg 2+ -sensing riboswitch [Cromie MJ, Shi Y, Latifi T, Groisman EA (2006) Cell 125:71–84]. 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, Δ 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.

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.

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.

We carried out PCR mutagenesis of the proU promoter of Salmonella typhimurium, in order to identify sequences important for its osmotic control. We obtained five mutations in the -35 element: two decreased the promoter strength, one increased it, and the others had no effect. However, none abolished osmotic control, suggesting that the sequence of the -35 element is not crucial for osmotic control.