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

Abstract

SummaryA transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Nat at elevated pH, was deficient in energydependent 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 3'4erminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative mernbranespanning 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 Kf, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJYl), complemented the Na'-sensitive phenotype 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 NatABdependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na'

A Comparative Analysis of ABC Transporters in Complete Microbial Genomes

“…Group 7, as well as probably, group 11, corresponds to the ABC-2 type transporters that include the capsular polysaccharide export system in Gramnegative bacteria (Reizer et al 1992) and NatB for sodium export in Bacillus subtilis (Cheng et al 1997). There is no substrate-binding protein in this group.…”

Transcriptome Analysis of Alkali Shock and Alkali Adaptation inListeria monocytogenes10403S

“…For example, the Na þ (K þ )=H þ antiporter (and to a lesser degree ABC type exporters) is suggested to be the most important system in maintaining pH homeostasis and Na þ extrusion in B. subtilis (Cheng et al, 1997). In this study, AS repressed the potassium transporting operon Kdp.…”

The Bacillus subtilis NatK–NatR two-component system regulates expression of the natAB operon encoding an ABC transporter for sodium ion extrusion

“…One-step competence medium (MC) was used for the determination of b-galactosidase activity (Ogura & Tanaka, 1996;Ogura et al, 1997). TKM medium was used for growth tests in the presence of NaCl or ethanol (Cheng et al, 1997). Escherichia coli JM103 cells for DNA manipulation were grown in Luria-Bertani (LB) medium.…”

“…In those studies, the NatR (YccH) response regulator was identified as an activator for the natAB operon encoding a two-gene ABC transporter involved in Na + extrusion (Cheng et al, 1997;Kobayashi et al, 2001). This ABC transporter is a member of a small subfamily named ABC-2, which includes the two-component daunomycin-and doxorubicin-efflux system in Streptomyces peucetius Drr (Guilfoile & Hutchinson, 1991;Reizer et al, 1992).…”

“…This ABC transporter is a member of a small subfamily named ABC-2, which includes the two-component daunomycin-and doxorubicin-efflux system in Streptomyces peucetius Drr (Guilfoile & Hutchinson, 1991;Reizer et al, 1992). It has been reported that NatAB has a physiological role in Na + resistance at alkaline pH, since a high concentration of cellular Na + is toxic to the cell, and plays a role in ethanol resistance at neutral pH (Cheng et al, 1997;Padan & Schuldiner, 1994).…”