Functional study of an Escherichia coli protease

摘要

Escherichia coli are one of the most common gram-negative bacteria that cause bacteremia and urinary tract infections Antibiotic treatment is the traditional way to manage the E coli-caused infections Due to the rapid emergence of antibiotic-resistant strains developing novel strategies against such diseases becomes critical The E coli protease Rep is a potential target for developing the new antimicrobial strategies because deletion of rep increased the sensitivity to multiple antibiotics and decreased pathogenic E coli’s ability to survive in the bloodstream Also the E coli rep mutant exhibit a growth defect under the combined low osmotic and high temperature stresses Characterizing the function of Rep would facilitate the development of alternative antibacterial strategies In this study we utilize E coli proteome chip assay to screen for the potential Rep substrates The E coli proteome chip assay revealed that 100 E coli proteins might be the substrate of Rep Among these 100 possible substrates 13 of them could be cleaved by the Rep protease in vitro Further one of them ECK1083 was shown to contribute to the growth defect of the E coli rep mutant under the combined osmotic and high temperature stresses because the eck1083 deletion in the rep mutant partially rescued the growth defect To assess whether ECK1083 accumulates in the rep mutant we constructed the strains with eck1083-3xFlag at the chromosomal locus of eck1083 to allow the detection of the ECK1083 protein with the anti-Flag antibody Since ECK1083 has been identified as an inner membrane protein the membrane fraction from the wild-type and rep mutant strains were isolated under the combined osmotic and high temperature stresses condition The level of ECK1083 in the membrane fraction was measured by western blot analysis The level of ECK1083 in the rep mutant was significantly higher than that in the wild-type strain Also the mRNA level of eck1083 in the rep mutant was similar to that of the wild-type strain These results suggested that the lack of Rep-mediated degradation cause the accumulation of ECK1083 in the E coli rep mutant and that the accumulation may contribute the growth defect under the combined osmotic and high temperature stresses

獎項日期	2016 3月 30
原文	English
監督員	Ching-Hao Teng (Supervisor)