Inhibition of the DevSR Two-Component System by Overexpression of Mycobacterium tuberculosis PknB in Mycobacterium smegmatis

Abstract

The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.

Keywords: DevSR; Ser/Thr protein kinase; mycobacterium; two-component system.

Fig. 1

In vitro phosphorylation of Mtb RRs by the KD of Mtb PknB. Phosphorylation reactions of the RRs (0.2 nmol each) were conducted in the presence (+PknB) and absence (-PknB) of 0.2 nmol of purified PknB KD. Reactions were started by adding 100 μM cold ATP and 1,000 Ci/mole [γ-³²P] ATP and incubated at 30°C for 30 min. After reactions were terminated, proteins were resolved by SDS-PAGE. Phosphorylated proteins were detected by autoradiography. 0.2 nmol each of purified RRs and 0.2 nmol of PknB KD were subjected to SDS-PAGE and the gel was stained by Coomassie brilliant blue (CBB). The stained gel is presented below the autoradiogram to compare the size and amounts of the purified proteins used in the experiment.

Fig. 2

Multiple alignment analysis of the effector domains of Mtb RRs and comparison of the amino acid sequence of Mtb DosR with that of M. smegmatis DevR. (A) Multiple alignment was generated using ClustalW2. The Thr residues, which are located within the DNA-binding domains (helix-turn-helix motifs) and predicted to be phosphorylated by PknB, are indicated by the gray background. The RRs of the OmpR family are aligned separately from those of the NarL family. The relevant secondary structures are shown above the aligned sequences. The position of the secondary structures is based on the determined three-dimensional structures of PhoP and DosR (Menon and Wang, 2011; Wisedchaisri et al., 2008). (B) Sequence alignment indicates 85% identity between DevR and DosR. The Thr and Ser residues, which were subjected to site-directed mutagenesis, are indicated by the gray background (Thr180, Thr198, and Ser205).

Fig. 3

Determination of protein-protein interactions between DosR and STPKs of Mtb in Y2H assay. The gene portions encoding the KDs of Mtb STPKs, DosS, and DosT were cloned into pGBKT7 (encoding the GAL4 DNA-binding domain). The dosR gene was cloned into pGADT7linker (encoding the GAL4 activation domain), resulting in pPLDosR. The yeast strains cotransformed with both pGBKT7 derivatives and pPLDosR were used for Y2H assay. To discriminate false-positive interactions, the empty pGADT7linker was introduced into the yeast strains with the pGBKT7 derivatives in place of pPLDosR and the resulting yeast strains were used as negative controls. All yeast strains were spotted onto SD/-Leu/-Trp plates (+His) and histidine-deficient SD/-Leu/-Trp/-His plates containing 3AT (-His).

Fig. 4

Effect of PknB overexpression on expression of hspX, ald, and ahpC in M. smegmatis. For overexpression of PknB KD, PknH KD, and PknG in M. smegmatis, the pMH201 derived plasmids, pMHPknB, pMHPknH, and pMHPknG, were employed, respectively. (A) The promoter activity of hspX was measured by determining β-galactosidase activity in M. smegmatis strains harboring both an hspX::lacZ transcriptional fusion plasmid (pNChspX) and one of pMHPknB, pMHPknH, or pMHPknG. As a negative control, the M. smegmatis strain with both pNChspX and pMH201 was included in the experiment. The M. smegmatis strains were grown either aerobically to an OD₆₀₀ of 0.4 to 0.5 (Aer) or under hypoxic conditions for 20 h (Hyp) in the presence of 0.2% (w/v) acetamide. (B) The promoter activity of ald was determined using the M. smegmatis strains harboring an ald::lacZ transcriptional fusion plasmid (pALDLACZ) and either pMH201 or pMHPknB, which were grown aerobically to an OD₆₀₀ of 0.4 to 0.5 in the presence of both 0.2% (w/v) acetamide and 25 mM alanine. The promoter activity of ahpC was determined using an ahpC::lacZ transcriptional fusion plasmid (pNCM3) in M. smegmatis strains grown aerobically to an OD₆₀₀ of 0.4–0.5 in the presence of 0.2% (w/v) acetamide. All values provided are the averages of the results from three independent determinations. Error bars indicate standard deviations. Western blot analysis was conducted for detection of the expressed PknB KD, PknH KD, and PknG. Cell-free crude extracts (20 μg) were separated on SDS-PAGE, followed by Western blot analysis with a His-tag antibody.

Fig. 5

Effect of PknB overexpression on hypoxic growth of M. smegmatis and expression of hspX and devS at the transcriptional level. (A) The M. smegmatis strains harboring either pMHPknB or pMH201 were grown in 7H9 medium containing 0.2% (w/v) acetamide under hypoxic conditions and their growth was monitored spectrophotometrically at 600 nm. All values provided are the averages of the results from two independent determinations. Error bars indicate deviations from the means. (B) The M. smegmatis strains harboring pMHPknB or pMH201 were grown either aerobically to an OD₆₀₀ of 0.4 to 0.5 (Aer) or under hypoxic conditions for 11 and 20 h (Hyp) in the presence of 0.2% (w/v) acetamide. The expression levels of hspX and devS were determined by RT-PCR. RT-PCR of the 16S rRNA gene was conducted to ensure that the same amounts of total RNA were employed for RT-PCR.

Fig. 6

Effect of point mutations on phosphorylation of DevR by PknB KD. (A) 0.1 nmol of PknB KD was mixed with 0.1 nmol of WT or mutant forms of DevR in 20 μl of reaction buffer. Reactions were started by adding 100 μM cold ATP and 1,000 Ci/mole [γ-³²P] ATP, incubated at 30°C for 5 min, and terminated. Phosphorylated proteins were detected by autoradiography. The phosphorylation extent of the WT and mutant forms of DevR was estimated by using a densitometer program, ImageJ (v1.37). The phosphorylation extent of WT DevR is set at 100 and the relative values are expressed for the mutant forms of DevR. Before phosphorylation assay, the mixtures of 0.1 nmol of purified PknB KD and 0.1 nmol each of DevR protein were subjected to SDS-PAGE and the gel was stained by Coomassie brilliant blue (CBB). As a loading control, the stained gel is presented below the autoradiogram to compare the amounts of purified WT and mutant forms of DevR used in the experiment. (B) Phosphorylation reactions were performed using 0.05 nmol of PknB KD and 0.1 nmol of WT and T180A DevR in the time course. The reactions were terminated at the time points indicated. Phosphorylated proteins were detected by autoradiography.

Fig. 7

Effect of point mutations on the transcriptional activity of DevR in vivo. The transcriptional activities of the mutant forms of DevR were assessed by determining the expression level of hspX in M. smegmatis ΔdevR mutant strains complemented with the WT or mutant devR genes. To express devR, the genes encoding the WT and mutant forms of DevR (T180A, T198A, S205A, T180E, T198E, and S205E) were cloned into pMH201. The empty pMH201 vector and its derivatives were integrated into the chromosomal DNA of the M. smegmatis ΔdevR mutant harboring pNChspX. Complementation tests were performed by determining the β-galactosidase activity in strains grown under either 20 h hypoxic (A) or aerobic (B) conditions. All values provided are the averages of the results from three independent determinations. Error bars indicate standard deviations. Western blot analysis was performed for the detection of expressed DevR and DevR derivatives in M. smegmatis strains grown under 20 h hypoxic conditions. Crude extracts (50 μg) were separated on SDS-PAGE, followed by Western blot analysis with a His-tag antibody.

Fig. 8

EMSA showing the binding of purified WT and T180E mutant forms of DevR to the hspX control region. (A) The 204 bp DNA fragments (100 fmol corresponding to 12.6 ng) containing the DevR-binding site upstream of hspX were incubated with various concentrations of purified WT and T180E DevR. The amounts of WT DevR and T180E DevR are given above the lanes. The arrow indicates the bands of free DNA. (B) Quantitation of the band intensity of free DNA in panel A was performed using the ImageJ densitometry program (v1.37). The band intensity of the free DNA without DevR is set at 100 and the relative values are plotted as a function of the DevR concentration.