EFFECTS OF HIGH PRESSURE ON THE ROTATION OF THE ESCHERICHIA COLI FLAGELLAR MOTOR

The flagellar motor of Escherichia coli is a molecular machine that rotates the helical filaments. The nucleoid protein H-NS is known as a global transcriptional regulator that silence many genes but it activates the flagellar regulon by silencing the transcription of the hdfR gene encoding the repressor of the flhDC operon encoding the flagellar master regulator. Previous reports showed that an hns mutant remains poorly motile even in the presence of FlhDC . In this study, an hns hdfR deletion strain was constructed and was shown to swim efficiently at prolonged incubation, arguing against that H-NS directly modulates the flagellar rotation speed. Next, I aimed at the directional control of the motor rotation. The flagellar motor rotates both counterclockwise (CCW) and clockwise (CW). CW rotation is triggered by binding of the phosphorylated form of the response egulator CheY (CheY-P). The detailed mechanism of switching remains unresolved. In this study, I demonstrate that high hydrostatic pressure can induce CCW rotation even in the presence of CheY-P. I then constructed CheY mutants that are constitutively active with the phosphorylation site substituted. The CW rotation bias of the strain overexpressing the CheY mutants was about 1 at normal pressure, and was lowered to almost 0 at about 80 MPa, suggesting that high ydrostatic pressure affects binding of CheY-P to the motor rather than phosphorylation of CheY. Furthermore, I constructed and characterize CheY-independent CW-locked mutants of the flagellar motor protein FliM, the target of CheY-P. Motors with those FliM mutants rotated CW even at 80 MPa. These results suggest that high pressure affects the interaction of CheY-P and FliM