Interaction of DNA-binding protein H-NS with the flagellar motor of E. coli

The histone-like nucleoid-structuring protein (H-NS) is well known as a global regulator of transcription. A number of studies have suggested that H-NS also positively influences the function of the flagellar motor, but the details of its motility-regulating action remain unclear. In an effort to characterize the actions of H-NS in the flagellar motor, we sought to test the effects of specific mutations in H-NS that are predicted to alter its state of multimerization. As a foundation for this work, we examined the effects of H-NS expression in strains that expressed the flagellar regulatory proteins FlhDC at various levels, from various plasmids. The results gave indications that certain plasmids previously used to provide FlhDC constitutively did not, in fact, express the proteins at levels sufficient to stimulate flagellar assembly. This complicates the interpretation of previous work, because the cells retained the chromosomal copies of the flhDC genes whose expression is known to be influenced by H-NS. Thus, effects in the previous experiments may have been the result of up-regulation of chromosomal flhDC rather than direct actions at the flagellar motor. To overcome this problem, I constructed new strains in which the chromosomal copies of flhDC were deleted, and revisited the question of HNS action in the motor. For these experiments, the flhDC genes were expressed from a regulatable plasmid that had been verified by complementation of the flhDC deletion strain, and H-NS was expressed from a second regulatable plasmid. The results indicate that H-NS contributes to flagellar motility in ways other than its stimulatory effect flhDC iv expression, as was suggested on the basis of the previous work. Details of its action are different from those reported previously. An analysis of mutants altered at interfaces needed for H-NS multimerization gives evidence that H-NS must act as a dimer or larger multimer, in both its gene-regulatory and motility regulating.