A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium. The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels. A genetic fusion to osmY was mapped to 99.3 min on the E. coli chromosome. The gene was cloned and sequenced, and an open reading frame was identified. The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage. Sequencing of the periplasmic OsmY protein confirmed the open reading frame and defined the signal peptide cleavage site as Ala-Glu. A mutation caused by the osmY::TnphoA genetic fusion resulted in slightly increased sensitivity to hyperosmotic stress.
The osmY gene, which encodes a periplasmic protein with an apparent Mr of 22,000, is induced by both osmotic and growth phase signals. We demonstrate here that osmY expression is regulated at the level of transcription and that transcription initiates 242 nucleotides upstream Bacteria in nature are exposed to a variety of environmental stresses such as heat, UV radiation, toxic chemicals, nutrient deprivation, and fluctuations in osmolarity. Cells respond to these conditions by inducing the synthesis of specific sets of proteins that are characteristic of each stress (13). These proteins are thought to be important for adaptation to the new environment as well as for protection against future potentially lethal exposures to that stress. The enteric bacterium Escherichia coli is normally found in the gut of vertebrate animals but can also survive and grow in seawater. Both are environments where nutrients are scarce and the osmolarity is high.Exposure to high osmolarity induces the expression of many genes or operons (7) and more than 20 proteins (as determined by two-dimensional gel electrophoresis) (4,18,22). Blocking expression of the osmotically induced proteins demonstrates their importance to hyperosmotic stress protection (22). Normally, E. coli becomes significantly more resistant to extreme osmotic challenge if it is preexposed to a moderate osmotic stress (22). If protein synthesis is prevented by adding chloramphenicol during the preexposure, the cells do not develop resistance to extreme challenge (22).Many osmotically inducible genes have been identified by transposon mutagenesis to create reporter gene fusions. Early studies generated fusions to 3-galactosidase (lacZ) (5,14), while recent work has used protein fusions to phoA which are specific for proteins exported to the cell envelope (15,46). Genetic loci responsive to hyperosmotic stress are distributed throughout the E. coli genome (7,15,46), raising the question of how these genes are regulated. As yet, no central regulatory protein for the hyperosmotic stress stimulon has been found. Some of the loci within the stimulon are known to be controlled by specific regulatory factors. For example, the kdpABC operon and the ompC/ompF genes are positively regulated by two-component regulatory systems (12, 20, 32-34, 36, 41, 45).
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