A two‐gene ABC‐type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore

Abstract

A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na at elevated pH, was deficient in energy‐dependent Na extrusion. The capacity of the mutant JC901 for Na ‐dependent pH homeostasis was unaffected relative to the wild‐type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3 ‐terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane‐spanning regions at its C‐terminus, and exhibits modest sequence similarity to regions of eukaryotic Na⁺/H⁺ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP‐binding proteins that are components of transport systems of the ATP‐binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natAB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p‐chlorophenylhydrazone (CCCP), and, more modestly, by Na⁺, and K⁺, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na⁺‐sensitive phe‐notype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol‐resistance does not result from NatAB‐dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na⁺