Specific inhibition of the Na(+)-driven flagellar motors of alkalophilic Bacillus strains by the amiloride analog phenamil

Abstract

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus strains, was found to cause growth inhibition; therefore, the use of amiloride for the isolation of motility mutants was difficult. On the other hand, phenamil, an amiloride analog, inhibited motor rotation without affecting cell growth. A concentration of 50 microM phenamil completely inhibited the motility of strain RA-1 but showed no effect on the membrane potential, the intracellular pH, or Na(+)-coupled amino acid transport, which was consistent with the fact that there was no effect on cell growth. Kinetic analysis of the inhibition of motility by phenamil indicated that the inhibition was noncompetitive with Na+ in the medium. A motility mutant was isolated as a swarmer on a swarm agar plate containing 50 microM phenamil. The motility of the mutant showed an increased resistance to phenamil but normal sensitivity to amiloride. These results suggest that phenamil and amiloride interact at different sites on the motor. By examining various bacterial species, phenamil was found to be a specific and potent inhibitor for the Na(+)-driven flaggellar motors not only in various strains of alkalophilic Bacillus spp. but also in a marine Vibrio sp.