KtrAB and KtrCD: Two K + Uptake Systems in Bacillus subtilis and Their Role in Adaptation to Hypertonicity

Abstract

ABSTRACT Recently, a new type of K + transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T. Nakamura, R. Yuda, T. Unemoto, and E. P. Bakker, J. Bacteriol. 180: 3491-3494, 1998). The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane. The genome sequence of Bacillus subtilis contains two genes for each of these subunits: yuaA ( ktrA ) and ykqB ( ktrC ) encode homologues to the V. alginolyticus KtrA protein, and yubG ( ktrB ) and ykrM ( ktrD ) encode homologues to the V. alginolyticus KtrB protein. We constructed gene disruption mutations in each of the four B. subtilis ktr genes and used this isogenic set of mutants for K + uptake experiments. Preliminary K + transport assays revealed that the KtrAB system has a moderate affinity with a K m value of approximately 1 mM for K + , while KtrCD has a low affinity with a K m value of approximately 10 mM for this ion. A strain defective in both KtrAB and KtrCD exhibited only a residual K + uptake activity, demonstrating that KtrAB and KtrCD systems are the major K + transporters of B. subtilis . Northern blot analyses revealed that ktrA and ktrB are cotranscribed as an operon, whereas ktrC and ktrD , which occupy different locations on the B. subtilis chromosome, are expressed as single transcriptional units. The amount of K + in the environment or the salinity of the growth medium did not influence the amounts of the various ktr transcripts. A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective. In the ktrAB strain, the osmotically mediated growth defect was associated with a rapid loss of K + ions from the cells. Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically induced proHJ , opuA , and gsiB genes was not impaired, demonstrating that a high cytoplasmic K + concentration is not essential for the transcriptional activation of these genes at high osmolarity. Taken together, our data suggest that K + uptake via KtrAB and KtrCD is an important facet in the cellular defense of B. subtilis against both suddenly imposed and prolonged osmotic stress.