Two Types of Bacillus subtilis tetA (L) Deletion Strains Reveal the Physiological Importance of TetA(L) in K + Acquisition as well as in Na + , Alkali, and Tetracycline Resistance

The chromosomally encoded TetA(L) protein ofBacillus subtilis is a multifunctional tetracycline-metal/H+ antiporter that also exhibits monovalent cation/H+ antiport activity and a net K+ uptake mode. In this study,B. subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types oftetA (L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K+ , indicating that the net K+ uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na+ - or K+ -dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co+2 as well as Na+ sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression ofczcD-trkA , a locus with a proposed involvement in K+ uptake, occurred in both mutants. The increase was highest in the presence of Co2+ and was higher in JC112 than in JC112C. Deletion ofczcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low [K+ ], supporting a significant role for this locus in K+ uptake. Expression ofyheL , which is a homologue of the Na+ /H+ antiporter-encodingnhaC gene fromBacillus firmus OF4, was also increased in bothtetA (L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion ofyheL were consistent with a modest role for YheL in Na+ -dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na+ , and alkali resistance and K+ acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.