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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.

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.