Regulation of Inducible Potassium Transporter KdpFABC by the KdpD/KdpE Two-Component System in Mycobacterium smegmatis

Abstract

Kdp-ATPase is an inducible high affinity potassium uptake system that is widely distributed in bacteria, and is generally regulated by the KdpD/KdpE two-component system (TCS). In this study, conducted on Mycobacterium smegmatis, the kdpFABC (encoding Kdp-ATPase) expression was found to be affected by low concentration of K⁺, high concentrations of Na⁺, and/or [Formula: see text] of the medium. The KdpE was found to be a transcriptional regulator that bound to a specific 22-bp sequence in the promoter region of kdpFABC operon to positively regulate kdpFABC expression. The KdpE binding motif was highly conserved in the promoters of kdpFABC among the mycobacterial species. 5'-RACE data indicated a transcriptional start site (TSS) of the kdpFABC operon within the coding sequence of MSMEG_5391, which comprised a 120-bp long 5'-UTR and an open reading frame of the 87-bp kdpF gene. The kdpE deletion resulted in altered growth rate under normal and low K⁺ conditions. Furthermore, under K⁺ limiting conditions, a single transcript (kdpFABCDE) spanning kdpFABC and kdpDE operons was observed. This study provided the first insight into the regulation of kdpFABC operon by the KdpD/KdpE TCS in M. smegmatis.

Keywords: K+ limitation; Kdp-ATPase; KdpD/KdpE; KdpFABC; Mycobacterium smegmatis; potassium transporter; two-component system (TCS).

Figure 1

Relative expression levels of kdp loci in response to growth in K⁺ limiting and non-limiting conditions. Cultures of wild type, as well as ΔkdpD and ΔkdpE mutants were grown to late exponential phase in the presence and absence of K⁺ in HdB medium. Total RNA was extracted and cDNA was used for quantitative analysis. Data represent the averages of biological triplicates. Error bars indicate standard deviation. sigA (MSMEG-2758) gene was used as reference gene for the determination of relative expression levels of kdpFABC and kdpDE genes in K⁺ limiting condition with respect to normal K⁺ condition (wild type strain). Normal K⁺ stands for HdB medium where K⁺ concentration was similar to that defined earlier (Smeulders et al., 1999), which was ~17.8 mM as measured by flame photometry, whereas 0 mM K⁺ stands for HdB medium in which K⁺ salt was replaced by similar concentration of Na⁺ salt and the residual K⁺ concentration was ~23 μM as measured by flame photometry.

Figure 2

Binding of purified KdpE protein to specific DNA motif in the PkdpF. (A) EMSA of KdpE with PkdpF. Left lane 1: 6-FAM-labeled probe of PkdpF, left lanes 2–5: complex formation with increasing concentrations of KdpE. Right lane 1: 6-FAM-labeled probe of PkdpF, right lanes 2–3: complex formation with increasing concentrations of KdpE, right lanes 4–5: competition with increasing concentration of unlabeled probe. (B) Electropherograms indicating protected DNA region in PkdpF after DNase-I digestion of 6-FAM-labeled probe in the absence of KdpE (upper panel) and in the presence of KdpE (lower panel). The fluorescence signals of 6-FAM-labeled fragments are plotted against probe length. The protected region in electropherograms and the 18-bp DNA motif of KdpE are boxed in red. The 300-bp DNA probe used in the assay is shown below with numerical values along the DNA sequence representing the positions of nucleotides with reference to the above graphical scale.

Figure 3

Determination of minimum DNA sequence required for successful binding of KdpE protein with PkdpF. (A) EMSA competition assay performed with labeled (300-bp 6-FAM-labeled probe) and unlabeled 33-bp DNA probes. With increasing concentration of unlabeled 33-bp probe, the band intensity of labeled probe with KdpE protein decreased while that of unlabeled probe increased, indicating that unlabeled 33-bp probes could effectively compete with the 300-bp 6-FAM-labeled probe in binding KdpE. (B) The sizes and sequences of different DNA probes used for the determination of exact foot print sequence. (C) Shorter double stranded DNA probes used (based on DNase-I foot printing results) in EMSA to determine the minimum DNA sequence required for KdpE binding with PkdpF. Sizes of DNA probes were decreased by removing few nucleotides on both ends of the 33-bp probe to determine minimum nucleotides required for binding. (D) Unnecessary nucleotides (based on DNA sequence homology with other mycobacterial species) were trimmed down from the 3′ end of the 28-bp probe to determine the minimum sequence required for KdpE-DNA interaction.

Figure 4

Multiple DNA sequences alignments of KdpE binding motifs from different mycobacterial species. (A) The homology of minimum 22-bp binding sequence was searched in different species of genus Mycobacterium. Highly similar nucleotides are shown in white letters highlighted in red background, less similar nucleotides in black bold letters highlighted in yellow, while dissimilar nucleotides in black letters in white background. (B) KdpE binding sequences found in different bacterial species; E. coli (Sugiura et al., 1992), C. acetobutylicum (Behrens and Duerre, 2000), S. aureus (Xue et al., 2011) and M. smegmatis (present study). (C) The DNA logo of DNA motifs required for efficient binding of KdpE protein to the PkdpF based on DNA homology among different mycobacterial species. The logo was prepared using WebLogo (Crooks et al., 2004).

Figure 5

Mapping of TSS and characterization of the PkdpF. (A) The 5′-RACE adaptor sequence (boxed in yellow) along with the transcript sequence after DNA sequencing. The TSS is marked with a bent arrow. (B) Fully characterized PkdpF sequence, where the KdpE binding sequence is shown in red nucleotides boxed in black, the SigF-dependent promoter consensus −10 and −35 regions shown in dark green, the TSS nucleotide shown with red letter marked by a bent arrow. All potential start codons are double underlined, with the newly identified start codon indicated at the +120 position. The candidate kdpF gene is shown in magenta letters. The sequence of MSMEG_5391 containing a 5′-UTR region (120 bp) of kdpFABC and the newly determined kdpF coding sequence (87-bp) is shown overlapped with the start codon of kdpA. The possible Shine-Dalgarno sequences in RBS are underlined with blue line.

Figure 6

Protein sequence homology of KdpF of M. smegmatis with other mycobacterial species. The DNA sequence of kdpF in M. smegmatis was determined based on 5′-RACE and subsequent β-galactosidase assays. Its homologs were searched in other mycobacterial species in the upstream region of kdpA. The KdpF protein sequences of high similarity are shown in white letters highlighted in red background, low similarity in black bold letters highlighted in yellow background, and dissimilar residues in black letters in white background.

Figure 7

Induction of kdpF under K⁺ limiting conditions. β-galactosidase assay was performed to determine functionality of PkdpF, translation start codon of MSMEG_5391 (TTG) as well as newly identified translation start codon of kdpF (GTG). Promoter regions were fused with promoter less-lacZ gene (in frame and out of frame) in pMV261 to generate the Pttg::lacZ, Pttga::lacZ, Pgtg::lacZ, and Pgtga::lacZ plasmids. Plasmids were separately transferred into wild type (A), ΔkdpD (B), and ΔkdpE (C) to determine regulation of PkdpF by KdpD/KdpE TCS. For positive control, Phsp was fused with the lacZ gene (Phsp::lacZ) and for the negative control promoter less-lacZ plasmid (Pnull::lacZ) was used. Strains were grown in HdB broth under K⁺ normal and limiting conditions (0 mM K⁺) and β-galactosidase activity was determined to investigate promoter functionality.

Figure 8

Promoter functionality and effect of different stimuli on kdpFABC transcription. The strains used are described in Figure 7. Strains were grown in 7H9 broth under different concentrations of NaCl (A–C), NH₄Cl (D–F), KCl (G–I), sucrose (J–L), and pH-values (M–O). β-galactosidase activities were determined to investigate promoter functionality and expression of kdpFABC.

Figure 9

Growth curves of wild type MC²155 and its derivative strains under different concentrations of K⁺ in HdB medium. Bacterial growth curves were generated by growing all bacterial strains including wild type MC²155, ΔkdpD, ΔkdpE, and CΔkdpE, in 7H9 and HdB broth (with and without K⁺). Overnight cultures of all the strains were used to inoculate fresh media at a starting OD₆₀₀ of 0.02. Each experiment was performed in triplicate with mean values shown in the graph. Cultures were incubated at 37°C under stirring condition for 40 h. OD₆₀₀ was determined by sampling cultures in every 3 h. (A) Growth experiment for M. smegmatis and its derivatives was studied in 7H9 broth. Similarly, growth curves of M. smegmatis and its derivatives were also generated in HdB medium under different concentrations of K⁺ (B = normal, C = 0 mM, D = 1 mM, E = 2 mM, F = 4 mM).

Figure 10

Proposed schematic representation of the induction and transcription of kdpFABC operon in M. smegmatis. Membrane bound KdpD senses stimuli such as low K⁺, high Na⁺, and ${\text{NH}}_4^{+}$ concentrations of the medium. KdpE acts as a transcriptional regulator and is phosphorylated by KdpD to positively regulate the transcription of kdpFABC operon along with SigF-dependent RNA polymerase in a cooperative way. The 22-bp KdpE binding sequence is shown in red and SigF-dependent promoter consensus −35 and −10 regions are shown in green. The TSS nucleotide is shown in red with bend arrow. The Trk K⁺ uptake system and other possible channels are also shown in the diagram.