Characterization of transcriptional regulation of the kdp operon of Salmonella typhimurium

Abstract

The transcriptional control of the kdpFABC (K ؉ transport) operon of Salmonella typhimurium was characterized with a lacZ fusion. The kdpFABC operon of this organism was induced by K ؉ limitation and high osmolality, and osmotic induction was antagonized by a high concentration of K ؉ . In the trkA (sapG) kdp ؉ mutant background, high concentrations of K ؉ inhibited growth, along with repressing the kdp operon. This result, which has not been reported for Escherichia coli, is inconsistent with the model in which the signal for the induction of the kdp operon is turgor loss.In enteric bacteria, K ϩ is taken up by two major permeases, Trk and Kdp, and by a minor permease, Kup (13,18). In Escherichia coli, the activity of these three systems has been shown to be stimulated by osmotic stress (2). Stimulation of Kdp is effected largely by transcriptional induction of the kdpFABC operon. The kdpFABC operon is also induced by K ϩ limitation. Transcriptional control of the kdpFABC operon is mediated by the KdpD and KdpE proteins (2), which belong to the family of two-component regulatory proteins. It has been proposed that the regulatory signal for the expression of the kdp operon is turgor (4), but whether the osmotic-stress-and the K ϩ -dependent regulations are mediated by a single mechanism or by two distinct mechanisms is controversial (1, 2, 6, 7).The regulation of the kdp operon in Salmonella typhimurium has not been studied. Because of difficulties in obtaining kdp mutations in this organism, it has been suggested that there may be an important difference between the properties of the Kdp system in E. coli and in S. typhimurium (17). In this report, we describe the isolation of a kdp-lacZ fusion in S. typhimurium.Strain construction. The medium used for the isolation of the kdp-lacZ mutations was K0 medium (5), which has a K ϩ concentration of 0.1 mM (introduced as a trace contaminant in other chemicals). Approximately 5,000 derivatives of the wildtype S. typhimurium strain LT2 carrying random MudI1734 (Km lac) insertions (10) were replica plated to K0 plates containing 10 mM glucose, kanamycin, and 40 mg of X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) per liter (12) and to plates containing the same ingredients plus 10 mM KCl. We identified one derivative, strain TL2626, which formed a very small, dark blue colony on the former medium and a normal-sized, white colony on the latter; the mutation in this strain was designated kdp-101::MudI1734.P22 transductions (3) demonstrated that the kdp-101:: MudI1734 insertion was 96% linked and was located upstream of a kdp::Tn10 insertion (obtained from E. Groisman) (data not shown). In Southern blot analyses, a fragment containing the kdpFABC operon of E. coli exhibited different patterns of hybridization to EcoRI, EcoRV, PvuII, and HincII fragments of the DNA from strain TL2626 than to those from strain LT2 (data not shown). The kdp-101::MudI1734 insertion was complemented by a plasmid which expressed the kdpB ϩ C ϩ genes of E. coli (obtained from W. Epstein)...

Timing of Induction of Osmotically Controlled Genes in Salmonella enterica Serovar Typhimurium, Determined with Quantitative Real-Time Reverse Transcription-PCR

“…The experiments in this work were done with derivatives of S. enterica serovar Typhimurium LT2. Strain TL3025 (proX1917::TnphoA DUP [kdpA101::Mud1-8::nadA216]/kdp ϩ ) is a Kdp ϩ strain, which carries an intact copy of the kdpABCDE genes and a kdpAlacZ transcriptional fusion generated by phage Mud1-8 (14) in a chromosomal duplication of the kdpA-nadA segment of the chromosome. Strain TL3353 (DUP [proX(proU)1884::Mud1-8::pheA564]/proX1917::TnphoA) carries a chromosomal duplication of the pheA-to-proU region of the chromosome, in which one copy of the proU operon contains a proX-phoA translational fusion (obtained from E. Eisenstadt via J. Galán) and a second copy carries a proX-lacZ translational fusion (30).…”

The Staphylococcus aureus KdpDE Two-Component System Couples Extracellular K + Sensing and Agr Signaling to Infection Programming

“…In S. aureus, the organization of the kdpFABC operon is similar to that of E. coli, but the kdpDE genes are arranged in a reverse orientation upstream of the kdpA gene. Although ex-perimental evidence has shown that the Kdp-ATPase system in several bacteria also functions as a high-affinity K ϩ transporter (1,5,21,22,50), the role of the Kdp system in pathogenic bacteria has not been investigated in detail.…”

Cs + Induces the kdp Operon of Escherichia coli by Lowering the Intracellular K + Concentration

“…ϩ signal is related to the internal K ϩ level and/or the processes of K ϩ transport (3,9) or to the external K ϩ concentration (16). Based on the results obtained with right-side-out membrane vesicles, a new model has been established, according to which the intracellular K ϩ concentration and ionic strength directly influence KdpD autophosphorylation activity, whereby K ϩ has an inhibitory effect and ionic strength has a stimulatory effect (10).…”