Deletion of the tetA(L) chromosomal region of Bacillus subtilis in a strain designated JC112 increased the strain's sensitivity to low tetracycline concentrations. It also resulted in phenotypic changes that correlate with the previously found role of TetA(L) in mediating electrogenic Na ؉ /H ؉ antiport. Growth of JC112 was impaired relative to that of the wild type at both pH 7.0 and 8.3; Na ؉ -and K ؉ -dependent pH homeostases were impaired at alkaline pH. The phenotype of JC112 was complemented by plasmid-borne tetA(L) and related tet(K) genes; the antiport activity conferred by the tet(K) gene had an apparently higher preference for K ؉ over Na ؉ than that conferred by tetA(L). The data were consistent with TetA(L) being the major Naantiporter involved in pH homeostasis in B. subtilis as well as a significant Na ؉ extrusion system. The phenotype of JC112 was much more pronounced than that of an earlier transposition mutant, JC111, with a disruption in the putative tetA(L) promoter region. Northern (RNA) blot analysis of tetA(L) RNA from wild-type and JC111 strains revealed the same patterns. That JC111 nevertheless exhibited some Na ؉ and alkali sensitivity may be accounted for by disruption of regulatory features that, in the wild type, allow increased tetA(L) expression under specific conditions of pH and monovalent cation concentration. Evidence for several different regulatory effects emerged from studies of lacZ expression from the transposon of JC111 and from a tetA(L)-lacZ translational fusion introduced into the amyE locus of wild-type and JC112 strains.The chromosomal tetA(L) locus of Bacillus subtilis is very similar in sequence and apparent organization to tet loci of certain plasmids of gram-positive bacteria (20,24,25,34,37). It is composed of a short open reading frame (ORF) encoding the tetA(L) leader peptide, which is thought to be involved in translational regulation, and a second ORF, tetA(L), which encodes a porter that catalyzes efflux of a divalent cationtetracycline complex in coupled exchange for protons (i.e., metal-tetracycline/H ϩ antiport) (17,40). Each of these ORFs is preceded by a putative Shine-Dalgarno sequence. On the basis of the nucleotide sequence upstream of the leader region, a single promoter in a position analogous to that of the promoter of plasmid-borne tet genes was identified (20,24,34). The presence of the tetA(L) gene in a B. subtilis strain does not confer resistance to the usual challenge concentrations of the antibiotic (2), and yet attempts to delete the porter-encoding gene were unsuccessful (22). This result suggested that an important physiological role might exist for this gene. Recently, it was found that the tetA(L)-encoded porter does catalyze Co 2ϩ -tetracycline/H ϩ antiport, but the porter also enhances Na ϩ /H ϩ antiport that is physiologically important during the growth of B. subtilis at elevated Na ϩ and pH (8, 17). A Na ϩ -and alkaline pH-sensitive transpositional mutant of B. subtilis, JC111, was disrupted between the putative Ϫ35 and Ϫ10 regio...

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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'