1887

Abstract

The two-component histidine kinase Chk1p of has been implicated in the regulation of cell wall biosynthesis. Deletion of results in avirulence that in part may be due to the increased sensitivity of mutant strains to polymorphonuclear leukocytes. The mutant also does not adhere to human oesophageal tissue , probably as a consequence of its altered cell wall. In the current study, a promoter- reporter () construct was expressed in wild-type strain CAI4 and in two-component signal transduction mutants to determine the effect of environmental stress conditions on the regulation of and the co-regulatory activities among these proteins. It is shown that expression varied according to the type of growth conditions and incubation time; expression was also influenced by the strain background. expression in CAI4 was greater at 37 °C and at a pH of 3·5 and in the presence of 4 mM HO, 0·1 mM menadione, 10 % serum or 1·5 M NaCl compared to cells grown at 30 or 42 °C. The increases in expression were time-dependent and not observed until cells were incubated for 120 min in these conditions (<0·05). As a correlate of the increase in transcription of - in the presence of HO, the mutant was more sensitive than wild-type and revertant cells to HO . In addition to strain CAI4, we also measured - reporter activity of mutants deleted in genes encoding other two-component proteins such as the response regulator gene , the histidine kinases, and , and the MAP kinase. Of these proteins, Ssk1p and Sln1p are presumed to mediate phosphotransfer to the HOG1 [ypersmotic lycerol] MAP kinase pathway during oxidative and perhaps osmotic stress in . Compared to strain CAI4, reporter activity increased significantly in the mutant under all growth conditions after a 10 and 120 min incubation (<0·0001). expression in the mutant was less at 42 °C compared to all other growth conditions (<0·05). Furthermore, reporter activity also increased in the mutant of . These data suggest that and indirectly or directly negatively regulate under most growth conditions tested. In the mutant, downregulation of was observed in all growth conditions compared to strain CAI4 (<0·05), while regulation of in the mutant was similar to strain CAI4 except when cells were incubated in the presence of 4 mM HO for 120 min (<0·05). Western blot analysis was used to determine the role of Chk1p in phosphorylation of Hog1p under oxidative or osmotic stress. It was found that Hog1p was phosphorylated in the mutant similar to wild-type CAF2-1 cells, although the temporal events of phosphorylation differed slightly in mutant cells. These results show that transcription of , as measured by the reporter assay, is statistically increased when cells are exposed to several types of stress or when incubated in 10 % serum in a mutant-specific background and at a specific time point. Of importance, our data also suggest that expression is indirectly or directly regulated by the HOG1 MAP kinase pathway, although a determination of its position in this pathway or in a cross-talking pathway awaits additional studies.

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2004-10-01
2019-10-16
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