Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations in putative channel components

The rotation of the Na+-driven flagellar motor is specifically and strongly inhibited by phenamil, an amiloride analog. Here, we provide the first evi dence that phenamil interacts directly with the Na+-channel components (Pom A and PomB) of the motor. The alterations in Mpa(r) ((m) under bar otility (r) under bar esistant to (p) under bar hen (a) under bar mil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pom B from two Mpa(r) strains, NMB205 and NM8201, and found a substitution in p omA (Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Both residu es are predicted to be near the cytoplasmic ends of the putative transmembr ane segments. Mutational analyses at PomA-Asp148 and PomB-Pro16 suggest tha t a certain structural change around these residues affects the sensitivity of the motor to phenamil. Go-expression of the PomA D148Y and PomB P16S pr oteins resulted in an Mpa(r) phenotype which seemed to be less sensitive to phenamil than either of the single mutants, although motility was more sev erely impaired in the absence oi inhibitors. These results support the idea that PomA and PomB interact with each other and suggest that multiple resi dues, including Asp148 of PomA and Pro16 of PomB, constitute a high-affinit y phenamil-binding site at the inner face of the PomA/PomB channel complex. : (C) 1999 Academic Press.