Genetics of osmoregulation in Eschericia coli K12 : molecular organisaton and regulation of proU Locus

Abstract

The pro U locus has been identified in both Escherichia coli and Salmonella newlinetyphimurium as an osmoresponsive locus which is induced several hundred-fold, at the level newlineof transcription, in media of elevated osmolarity. ProU is involved in the transport of Lproline newlineand glycine betaine: both substrates, when provided exogenously, are known to exert an newlineosmoprotective effect (consequent to their accumulation inside the cell) on the growth of the newlinebacteria in media of low water activity. The structural organisation of proU in E.coli, both at newlinegenetic and molecular levels, and the regulation of proU expression were investigated in this newlinestudy. newlineUsing the bacteriophage Mu dll(lac Amp), of Casada ban and Chou, several newlineosmoresponsive proU::lac gene fusions were isolated. All of them were induced about 100-fold newlinein media of high osmolarity. When these fusion strains were tested for complementa~ion using newlinethe plasmids carrying different functional regions of the proU locus, all insertions mapped to newlineonly one of the two complementation groups that had been identified ealier by Gowrishankar newlineet a[ (1986). newlineIn order to characterise the other complementation group, additional lac fusions were newlineisolated using A. placMu55, which creates operon fusions. Characterisation of about 80 newlineosmoresponsive lac fusions in proU unexpectedly revealed the presence of/ not one but two newlinecomplementation groups in this region, that is, a total of three complementation groups at the newlineproU locus. The three cistrons representing the three complementation groups were designated newlineas pro V, proW, and pro X, and the designation proU was retained to represent the entire locus. newlineEach of the Mu dii (lac Amp) insertions was replaced by the mini-Mu lac fusion newlinephage, Mu dll1734 (also called Mu dK) that encodes Kanr, by a process of gene conversion. newlineDetailed complementation analysis of the Mu dK-converted strains indicated that the gene newlinefusions characterised earlier in this study were in· fact distributed into two of the three newlinecomplementation groups above