Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain

Abstract

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...

Alkalitolerance: A biological function for a multidrug transporter in pH homeostasis

“…Interestingly, in this regard, H ϩ ͞K ϩ (Na ϩ ) antiport activities have been described for the tetracycline-specific transporters from B. subtilis (e.g., TetL) (38), which play a primary role in pH homeostasis of this bacterium. As shown here with MdfA, (i) a TetLdeleted strain exhibited substantial pH sensitivity, and (ii) complementing growth at alkaline pH (8.3) by a plasmid harboring the tetL gene also required monovalent cations (39). These results suggest that MdfA in E. coli and TetL in B. subtilis might perform similar pH homeostasis-related functions, although the magnitude of the MdfA-mediated alkaline pH tolerance is extraordinary.…”

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4

“…The major roles attributed to these gene products in eubacteria are in Na ϩ resistance and the capacity to maintain a cytoplasmic pH that is below the external pH during growth at, or a sudden shift to, an alkaline pH (22,31,41). The importance of successfully meeting these physiological challenges is supported by the fragility and pleiotropy of mutants lacking one or more functional antiporter genes (7,30,33,40) and by the likelihood that the genes encoding structurally distinct Na ϩ /H ϩ antiporters evolved at separate times (31), resulting in the presence of multiple genes of potentially overlapping function in each individual eubacterial species thus far examined. There are several examples of apparent compensation for mutational loss or insufficiency of one antiporter by up-regulation of a gene for a different Na ϩ /H ϩ antiporter, sometimes facilitated by a mutational change in a regulatory gene, or introduction of a heterologous regulatory gene (7,13,33,36).…”

“…The importance of successfully meeting these physiological challenges is supported by the fragility and pleiotropy of mutants lacking one or more functional antiporter genes (7,30,33,40) and by the likelihood that the genes encoding structurally distinct Na ϩ /H ϩ antiporters evolved at separate times (31), resulting in the presence of multiple genes of potentially overlapping function in each individual eubacterial species thus far examined. There are several examples of apparent compensation for mutational loss or insufficiency of one antiporter by up-regulation of a gene for a different Na ϩ /H ϩ antiporter, sometimes facilitated by a mutational change in a regulatory gene, or introduction of a heterologous regulatory gene (7,13,33,36). In addition, many bacteria probably use distinct K ϩ /H ϩ antiporters for the pH homeostasis function when Na ϩ is scarce or the Na ϩ /H ϩ antiporters are mutationally inactivated (7,29,35).…”

“…There are several examples of apparent compensation for mutational loss or insufficiency of one antiporter by up-regulation of a gene for a different Na ϩ /H ϩ antiporter, sometimes facilitated by a mutational change in a regulatory gene, or introduction of a heterologous regulatory gene (7,13,33,36). In addition, many bacteria probably use distinct K ϩ /H ϩ antiporters for the pH homeostasis function when Na ϩ is scarce or the Na ϩ /H ϩ antiporters are mutationally inactivated (7,29,35). However, extremely alkaliphilic Bacillus species exhibit Na ϩ specificity for this particularly crucial physiological process (23).…”

“…The rates of growth, in doublings per hour, were calculated from the rates of change in A 600 during the logarithmic phase of growth. Growth studies on B. firmus OF4811M DA78 were conducted with the same media, and those on B. subtilis strains were conducted in malate-containing media at either pH 7 or pH 8.3, as described earlier (7).…”