The K+ regulatory systems Trk and Kup Pictilisib in vivo are active at physiological K+ concentrations [15]. The expression of KdpD and consequently of the KdpABC system in E. coli is induced at low potassium concentrations (<60 mM) [25]. In E. coli KdpD is not essential at a potassium concentration >115 mM, as mutants with truncated forms of KdpD are viable under these conditions, but in media with <15 mM K+ those strains do not grow [25]. V. cholerae also possesses these three potassium regulatory systems for the adaptation to changing osmotic conditions [26, 27]. The V. cholerae mutant strain T283M grows well in media with high

and low K+ and Na+ concentrations in absence of vz0825 as shown in Figure  4. Even at 4 mM K+

growth is not diminished. This figure also shows the difference between the tolerance of the wild type and the T283M strain against vz0825. Our findings that T283M grows well in K+ reduced medium indicates that the inhibition of KdpD may have profound influence on some other, hitherto undefined, regulatory function of this protein in V. cholerae. The influence of vz0825 on KdpD may appear in different ways, e.g. reducing the binding of ATP to the histidine kinase, inhibiting the transfer of gamma-phosphate to the histidine residue, or to the asparagine residue of the response regulator. Like other histidine kinases KdpD also has phosphatase activity selleck kinase inhibitor [28], which may be disturbed by vz0825. The mutated amino acid on position 283 is located between the H-region and N-region. Mutations that alter this motif, which is termed the X-region, have been shown to alter the conformation of the histidine kinase EnvZ and significantly reduce its phosphatase activity [29]. EnvZ is a membrane receptor kinase-phosphatase, which modulates porin expression in E. coli in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases [29]. If KdpD is the major target of compound vz0825, the

deletion construct ΔkdpD should be insensitive to the LY2874455 supplier substance in media with physiological K+ concentration – provided that it is still viable. The construction of the required Methamphetamine plasmid for the generation of this construct, its transformation into E. coli S17-1 and the conjugation from E. coli into V. cholerae were successful in this study, but several attempts to induce the homolog recombination within V. cholerae NM06-058 failed. None of the analyzed clones showed a loss of the kdpD gene. The apparent growth reducing effect of vz0825 and its targeting of KdpD in V. cholerae suggests a more important role of KdpD in V. cholerae than in E. coli. Further experiments are required in order to corroborate the effect of vz0825 on KdpD, like functional assays with the expressed protein, in which the kinase- and phosphatase activities of the wild type and mutated forms in the presence of vz0825 are compared.