Dual control of RegX3 transcriptional activity by SenX3 and PknB

The mycobacterial SenX3–RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator. This system is a phosphorelay-based regulatory system involved in sensing environmental Pi levels and induction of genes required for Pi acquisition under Pi-limiting conditions. Here we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KDMtb) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3Mtb and RegX3Ms, respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3Mtb through phosphorylation events at Thr-100, Thr-191, and Thr-217. Electrophoretic mobility shift assays disclosed that phosphorylation of Thr-191 and Thr-217 abolishes the DNA-binding ability of RegX3Mtb and that Thr-100 phosphorylation likely prevents RegX3Mtb from being activated through conformational changes induced by SenX3-mediated phosphorylation. We propose that the convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate environmental Pi signals with the cellular replication state to adjust gene expression in response to Pi availability.

The adaptation and survival of Mycobacterium tuberculosis (Mtb) 2 under hostile host environments require exquisite regulation of gene expression in response to changing environments (1,2). Phosphorelay through proteins is a major mechanism by which environmental signals are transmitted to elicit appropriate adaptive responses (3,4). Two-component systems (TCSs), which constitute the primary regulatory systems utiliz-ing phosphorelay in prokaryotes, consist of sensory histidine kinases (HKs) and their cognate response regulators (RRs). In response to a specific ligand or environmental signal, an HK is autophosphorylated on a conserved histidine residue. The phosphoryl group is subsequently transferred from the HK to an aspartate residue conserved in the N-terminal receiver domain of the partner RR to activate the RR effector domain. Because most RRs contain the helix-turn-helix DNA-binding motif as an effector domain, the activated RRs normally serve as active transcription factors to regulate gene expression (5)(6)(7)(8)(9)(10). Mtb possesses 11 paired TCSs and five orphan RRs (11)(12)(13). Of 11 paired TCSs, the SenX3-RegX3 TCS has been suggested to play an important role mainly in the adaptation of mycobacteria to P i -limiting conditions (14,15). Other roles have also been suggested with regard to virulence (16 -19), persister formation (20), sensing of diatomic gases (O 2 , NO, and CO) (21), and membrane vesicle biogenesis (22). When the level of P i falls below a certain threshold value in the environment, the SenX3 HK is activated to phosphorylate the RegX3 RR. The activated RegX3 RR positively regulates expression of many genes, including those implicated in the acquisition of P i under P i -limiting conditions, such as the pstSCAB operon encoding a highaffinity phosphate-specific ABC transporter (Pst) and the phoA gene encoding alkaline phosphatase (14,23,24). Although the mechanism by which the SenX3 HK senses P i availability has not yet been fully elucidated, SenX3 appears not to sense P i levels by itself. The kinase/phosphatase activity of SenX3 has been suggested to be regulated by the functional state of the Pst uptake system with the assistance of other auxiliary proteins (PhoU in Mycobacterium smegmatis and PhoY in Mtb). This suggestion was made on the basis of the finding that inactivation of either Pst transporter or PhoU (PhoY) by mutation brings about constitutive activation of the SenX3-RegX3 TCS and constitutive expression of the RegX3 regulon independent of P i availability (20,24,25). According to the suggested model, the Pst transporter generates an inhibitory signal under P i -replete conditions that is transmitted by PhoU (PhoY) to the SenX3 HK. The inhibitory signal shifts the equilibrium of SenX3 activity from the kinase mode to the phosphatase mode, resulting in repression of the RegX3 regulon. The PhoU and PhoY proteins have been suggested to function as adaptor proteins between the Pst transporter and SenX3 through their interactions between the Per-ARNT-Sim (PAS) domain of SenX3 and the PstB ATPase subunit of the Pst system (26 -28).
Under P i -limiting conditions, the inhibitory signal is diminished or removed, which renders SenX3 active to phosphorylate the RegX3 RR. Intriguingly, a recent study demonstrated that the SenX3 HK is a hemoprotein with a b-type heme accommodated in its PAS domain and that oxidation of the heme from a ferrous to a ferric state enhances SenX3 autokinase activity, whereas binding of NO or CO to the heme leads to inhibition of autokinase activity (21). This finding suggests the possibility that SenX3 can also serve as a sensor kinase for diatomic gases such as O 2 , NO, and CO.
Because a eukaryotic-like Ser/Thr protein kinase (STPK) was first characterized in Myxococcus xanthus (29), many STPKs have been identified and characterized in bacteria, and increasing attention has been paid to the importance of STPKs in prokaryotic signaling pathways related to stress responses, development, virulence, regulation of central metabolism, as well as cell division and morphology (30 -33). In contrast to HKs that have a strict substrate specificity, STPKs can normally phosphorylate multiple substrates, resulting in pleiotropic responses from a single signal in the signal transduction pathway (31). The genomes of Mtb and M. smegmatis contain 11 and 13 STPK genes, respectively (11,34). The membranebound PknB is one of 11 STPKs in Mtb and is conserved in all mycobacteria (34). It has been shown to be essential for both Mtb and M. smegmatis (35)(36)(37) and to be involved in cell elongation, division, peptidoglycan biosynthesis, and regulation of oxygen-dependent cell replication (38 -40).

Protein-protein interactions between RegX3 and PknB
We demonstrated previously that purified RegX3 Mtb was strongly phosphorylated by purified PknB-KD Mtb in vitro (50). Because phosphorylation of RegX3 Mtb by PknB Mtb seems to require protein-protein interactions between them, we determined protein interactions between RegX3 Mtb and PknB Mtb by yeast two-hybrid (Y2H) assay. PknE (one of 11 STPKs in Mtb) and SenX3 of Mtb (SenX3 Mtb ) were included in the experiment as negative and positive controls, respectively (Fig. 1). For the Y2H assay, the regX3 Mtb gene was cloned into the prey vector pGADT7linker, whereas the gene portions encoding the KDs of PknB Mtb , PknE Mtb , and SenX3 Mtb were cloned into the bait vector pGBKT7. Consistent with a previous report (51), the yeast strain coexpressing RegX3 Mtb and SenX3 Mtb grew well in the absence of histidine (ϪHis), indicating protein-protein interactions between RegX3 Mtb and its cognate SenX3 Mtb HK. Coexpression of PknB Mtb with RegX3 Mtb led to growth of yeast in the absence of histidine whereas coexpression of PknE Mtb with RegX3 Mtb did not. As expected, the yeast strains expressing either SenX3 Mtb or PknB Mtb alone without expression of RegX3 Mtb did not grow on ϪHis medium. Taken together, the Y2H results suggest a possible interaction of RegX3 Mtb with PknB Mtb .

Overexpression of PknB-KD Mtb inhibits RegX3 transcriptional activity in vivo
To assess the effect of RegX3 phosphorylation by PknB on the transcriptional activity of RegX3, we overexpressed PknB-KD Mtb in M. smegmatis and examined the transcriptional activity of RegX3 by determining the expression level of the phoA gene, which encodes alkaline phosphatase and is regulated by the SenX3-RegX3 TCS. Because RegX3 Ms (MSMEG_0937) shares 93% identity with RegX3 Mtb at the amino acid level (Fig.  S1), we employed M. smegmatis overexpressing PknB-KD Mtb in place of Mtb to examine the overexpression effect of PknB-KD Mtb on RegX3 transcriptional activity. The gene portion encoding PknB-KD Mtb was overexpressed from an acetamideinducible promoter on pMHPknB that is a derivative of the pMH201 integration vector. Before determining the effect of PknB-KD Mtb overexpression on the transcriptional activity of RegX3, we examined the overexpression effects of PknB-KD Mtb on aerobic growth of M. smegmatis under P i -replete and P i -limiting conditions (Fig. S2). The M. smegmatis control strain with pMH201 grew under P i -replete conditions approximately three times faster than the same strain under P i -limiting conditions. Although growth of the M. smegmatis strain was severely inhibited by PknB-KD Mtb expressed from pMHPknB under both P ireplete and P i -limiting conditions, the optical density at 600 nm (A 600 ) and colony-forming unit values of the cultures of the M. smegmatis strain with pMHPknB were increased over time, indicating that overexpression of PknB-KD Mtb is not lethal to M. smegmatis but significantly inhibits its growth under conditions tested in this study. The expression level of phoA in M. smegmatis was determined using a phoA::lacZ transcriptional fusion plasmid, pNCphoA. As shown in Fig. 2A, phoA were cloned into pGBKT7 (encoding the GAL4 DNA-binding domain). Yeast strains cotransformed with both pPLRegX3 and pGBKT7 derivatives were used for the Y2H assay. To distinguish false positive interactions, the empty pGADT7linker vector was introduced into the yeast strains containing the pGBKT7 derivatives in place of pPLRegX3, and the resulting yeast strains were used as negative controls. All yeast strains were spotted onto SD/ϪLeu/ϪTrp plates (ϩHis) and histidine-deficient SD/ϪLeu/ϪTrp/ϪHis plates (ϪHis).

Inhibition of RegX3 transcriptional activity by PknB
expression was strongly induced in the M. smegmatis strain with both pNCphoA and pMH201 grown under P i -limiting conditions compared with the same strain grown under P ireplete conditions. However, the M. smegmatis strain harboring both pNCphoA and pMHPknB showed a significantly decreased level of phoA expression under P i -limiting conditions relative to the control strain with pNCphoA and pMH201 grown under P i -limiting conditions. As expected, virtually no ␤-gal activity was detected in the M. smegmatis strains with the pNC empty vector. The overexpression effect of PknB-KD Mtb on expression of phoA was also examined at the transcript level by RT-PCR analysis ( Fig. 2A, inset). We additionally determined the expression of pstS and ahpC in the RT-PCR analysis as controls. The pstSCAB operon is known to be under control of the SenX3-RegX3 TCS-like phoA (14), whereas the ahpC gene for alkyl hydroperoxide reductase is known to be regulated by FurA and Crp but not by RegX3 (52,53). RT-PCR analysis revealed that expression of phoA and pstS was significantly inhibited by PknB-KD Mtb overexpression. In contrast, overexpression of PknB-KD Mtb resulted in a slight increase in ahpC expression, which is consistent with our previous observation (50). Overexpression of PknB-KD Mtb in the M. smegmatis strain harboring pMHPknB was verified by Western blot analysis using a His tag antibody. Altogether, the results indicate that overexpression of PknB-KD Mtb inhibits the transcriptional activity of RegX3 Ms . To determine whether phoA expression correlates with the expression level of PknB-KD Mtb , the expression level of phoA was determined in the M. smegmatis strain with both pMHPknB and pNCphoA after it had been grown under P i -limiting conditions with treatment of increasing concentrations of acetamide. As shown in Fig. 2B, the expression level of phoA was gradually reduced with increasing concentrations of acetamide used in cultures. Western blot analysis showed that the amount of expressed PknB-KD Mtb was proportional to the concentration of treated acetamide. Taken together, the results indicate that the transcriptional activity of RegX3 Ms is inversely related to the expression extent of PknB-KD Mtb . It has been suggested that PknB and PknH might serve as the top-tier master STPKs in the hierarchical STPK-signaling cascade of Mtb (54). We examined whether overexpression of PknH-KD Mtb (amino acids 1-310) also inhibits phoA expression using an M. smegmatis strain overexpressing PknH-KD Mtb (Fig. S3). It has been demonstrated previously by Western blot analysis that PknH-KD Mtb is overexpressed in the M. smegmatis strain with pMHPknH grown in the presence of acetamide (50). When M. smegmatis strains were grown under P i -limiting conditions in the presence of acetamide, the expression level of phoA was only marginally decreased in the M. smegmatis strain carrying pMHPknH relative to that in the M. smegmatis strain with pMH201. This result suggests that overexpression of PknH-KD Mtb does not affect RegX3 transcriptional activity and that the observed inhibitory effect of phoA expression by PknB-KD Mtb overexpression is at least to some extent specific to PknB.
We next examined whether overexpression of PknB-KD Mtb also inhibits the transcriptional activity of RegX3 Mtb in

Inhibition of RegX3 transcriptional activity by PknB
vivo using a ⌬regX3 conditional mutant strain of M. smegmatis. The ⌬regX3 conditional mutant of M. smegmatis with deletion of its own regX3 Ms gene carries the acetamide-inducible regX3 Mtb gene on the chromosomal DNA. Therefore, the mutant was expected to exhibit mutant phenotypes in the absence of acetamide, whereas RegX3 Mtb was expected to be overexpressed in the mutant in the presence of acetamide. The transcriptional activity of RegX3 Mtb was examined by determining the expression level of phoA by RT-PCR and realtime qPCR (Fig. 3). Because the pAZI9018b-derived pAZIP-knB, which was used for overexpression of PknB-KD Mtb , carries the gene encoding PknB-KD Mtb that is under the control of an IPTG-inducible promoter, addition of IPTG to the growth medium led to overexpression of PknB-KD Mtb . When grown under P i -limiting conditions in the presence of both IPTG and acetamide, the ⌬regX3 conditional mutant strain with pAZIP-knB showed a 43% decrease in phoA expression relative to the mutant strain with the empty pAZI9018b vector grown under the same conditions. When grown under P i -limiting conditions without acetamide, phoA expression was almost abolished in both the mutant strains because of the lack of RegX3 Mtb expression. In contrast to the strong inhibition (ϳ90%) of RegX3 Ms transcriptional activity by overexpression of PknB-KD Mtb in the WT strain of M. smegmatis ( Fig. 2A), overexpression of PknB-KD Mtb in the ⌬regX3 conditional mutant of M. smegmatis reduced the transcriptional activity of RegX3 Mtb to a lesser extent, which might be attributable to the overexpression effect of RegX3 Mtb in the ⌬regX3 conditional mutant strain grown in the presence of acetamide. Considering the results shown in Figs. 2 and 3, we suggest that the increased kinase activity of PknB Mtb inhibits the transcriptional activity of both RegX3 Mtb and RegX3 Ms .

Inhibition of RegX3 transcriptional activity by PknB-KD Mtb overexpression results from the kinase activity of PknB-KD Mtb
It is conceivable that the inhibition of RegX3 Ms transcriptional activity in M. smegmatis by PknB-KD Mtb overexpression is a consequence of the sequestration of RegX3 Ms by the overexpressed PknB-KD Mtb through their protein-protein interactions rather than by increased PknB kinase activity. To examine this possibility, we determined the overexpression effect of inactive PknB-KD Mtb with the K40M mutation (37, 38) on phoA expression in M. smegmatis (Fig. 4). The mutant form of PknB-KD Mtb was overexpressed using pMHPknBK40M, which has the same construct as pMHPknB except for the K40M mutation in pknB. The transcriptional activity of RegX3 Ms was determined by measuring the promoter activity of phoA in M. smegmatis strains harboring pNCphoA. When grown under P i -limiting conditions, the M. smegmatis strain with both pMH201 and pNCphoA exhibited a high expression level of

Inhibition of RegX3 transcriptional activity by PknB
phoA, whereas overexpression of PknB-KD Mtb almost abolished phoA expression in the M. smegmatis strain with pMHP-knB and pNCphoA, which is consistent with the result shown in Fig. 2A. However, overexpression of the K40M mutant form of PknB-KD Mtb in the M. smegmatis strain grown under P i -limiting conditions led to merely a slight decrease in phoA expression compared with the control strain with pMH201 grown under the same conditions. Western blot analysis revealed that the similar amounts of the WT and K40M mutant forms of PknB-KD Mtb were synthesized in the M. smegmatis strains carrying pMHPknB and pMHPknBK40M, respectively. Taken together, these results unequivocally suggest that the inhibitory effect of PknB-KD Mtb overexpression on phoA expression was exerted by the increased PknB kinase activity in M. smegmatis.

Phosphorylation of Thr-100, Thr-191, and Thr-217 leads to inactivation of RegX3 transcriptional activity
To identify the amino acid residues of RegX3 Mtb that are phosphorylated by PknB-KD Mtb , we determined the sites (amino acids) of phosphorylation by LC-electrospray ionization MS/MS using purified RegX3 Mtb phosphorylated by purified PknB-KD Mtb (Fig. S4). The phosphorylation reaction was conducted with purified RegX3 Mtb and PknB-KD Mtb at a 1:1 stoichiometry for 1 h at 30°C. The RegX3 Mtb protein phosphorylated by PknB-KD Mtb was subjected to MS/MS analysis after in-gel digestion by trypsin, and phosphoresidues were identified from the MS/MS spectra. It was revealed that six Thr res-idues of RegX3 Mtb , corresponding to Thr-29, Thr-100, Thr-151, Thr-191, Thr-193, and Thr-217, were phosphorylated by PknB-KD Mtb . To confirm the MS result, we introduced point mutations (T29E, T100E, T151E, T191E, T193E, and T217E) into RegX3 Mtb by site-directed mutagenesis and then performed an in vitro phosphorylation assay using the WT and mutant forms of RegX3 Mtb and purified PknB-KD Mtb . The phosphorylation reactions were performed with the RegX3 Mtb proteins and PknB-KD Mtb at a 2:1 stoichiometry to reduce nonspecific phosphorylation of RegX3 Mtb . As shown in Fig. 5, the phosphorylation assay revealed that phosphorylation of T100E and T191E RegX3 Mtb by PknB-KD Mtb was significantly reduced compared with of WT RegX3 Mtb . Phosphorylation of T217E RegX3 Mtb by PknB-KD Mtb was also slightly diminished compared with WT RegX3 Mtb . In contrast, the T29E, T151E, and T193E mutations did not affect RegX3 Mtb phosphorylation by PknB-KD Mtb . A cumulative effect of T100E and T191E mutations on RegX3 Mtb phosphorylation was observed for RegX3 Mtb with double mutations (T100E/T191E) (Fig. S5). The T100E/ T191E mutant form of RegX3 Mtb was only marginally phosphorylated by PknB-KD Mtb , which could be explained by slight phosphorylation of Thr-217. Taken together, these results suggest that Thr-100 and Thr-191 in RegX3 Mtb are the major sites of phosphorylation by PknB Mtb . The detection of Thr-29, Thr-151, and Thr-193 phosphorylation in the MS analysis likely resulted from nonspecific phosphorylation of the residues in  (Fig. 6). The ⌬regX3 conditional mutant of M. smegmatis was complemented by introducing the pNBV1 derivatives that carry the WT or mutant regX3 Mtb genes (pNBV1RegX3WT, pNBV1RegX3T29E, pNBV1RegX3T100E, pNBV1RegX3T151E, pNBV1RegX3T191E, pNBV1RegX3T193E, and pNBV1RegX3T217E), and the expression level of phoA in the strains grown under P i -limiting conditions was measured by RT-PCR. As expected, the phoA gene was strongly expressed in the ⌬regX3 conditional mutant with the pNBV1 empty vector, which was grown in the presence of acetamide, whereas phoA expression occurred only marginally in the same strain grown in the absence of acetamide. In good agreement with this result, Western blot analysis revealed that His 6 -tagged RegX3 Mtb was detected in the ⌬regX3 conditional mutant grown in the presence of acetamide but not in the mutant strain grown in the absence of acetamide, confirming conditional expression of regX3 Mtb in the mutant depending on the presence or absence of acetamide. Expression of the WT, T29E, or T151E RegX3 Mtb complemented the ⌬regX3 conditional mutant grown in the absence of acetamide, as judged by phoA expression, whereas that of T100E, T191E, T193E, and T217E RegX3 Mtb did not. Western blot analysis showed that all mutant forms of RegX3 Mtb were expressed from the pNBV1 derivatives. The result shown in Fig. 6 implies that phosphorylation of Thr-100, Thr-191, Thr-193, and Thr-217 leads to inactivation of RegX3 transcriptional activity.
To determine whether the DNA-binding affinity of RegX3 Mtb is affected by phosphorylation of Thr-100, Thr-191, and Thr-217, we examined the binding of the corresponding phosphomimetic mutant forms (T100E, T191E, and T217E) of RegX3 Mtb to DNA fragments containing the upstream regulatory region of phoA by EMSA. As shown in Fig. 7A, more DNA fragments were shifted with increasing concentrations of WT and T100E RegX3 Mtb . In contrast, almost no band shift was observed for T191E RegX3 Mtb , and only a marginal band shift occurred for T217E RegX3 Mtb at high concentrations of the protein. To directly ascertain whether phosphorylation of RegX3 Mtb influences its DNA binding affinity, we performed EMSA using the RegX3 Mtb protein subjected to the phosphorylation reactions with the WT or K40M mutant form of PknB-KD Mtb . When the phosphorylation reaction of RegX3 Mtb was done without PknB-KD Mtb , the DNA fragments with the phoA upstream region were shifted in the EMSA (Fig. 7B). Phosphorylation of RegX3 Mtb by the active PknB-KD Mtb abolished binding of RegX3 Mtb to the DNA fragments, whereas RegX3 Mtb subjected to the phosphorylation reaction with the inactive K40M PknB-KD Mtb retained its DNA-binding ability. The WT or mutant form of PknB-KD Mtb alone without RegX3 Mtb did not bind to the DNA fragments. Taken together, the results in Fig. 7 suggest that phosphorylation of RegX3 Mtb on Thr-191 and Thr-217 by PknB Mtb significantly reduces the binding affinity of RegX3 Mtb for its target DNA sequence whereas phosphorylation of RegX3 Mtb on Thr-100 by PknB Mtb does not.

Discussion
The pknB gene forms an operon with pknA, pbpA, rodA, pstP (encoding a metal-dependent Ser/Thr protein phosphatase), and two genes coding for forkhead-associated domaincontaining proteins. This genetic locus was found to be conserved near the replication origin in the chromosomes of Acti- The ⌬regX3 conditional mutant strains of M. smegmatis with pNBV1RegX3WT, pNBV1RegX3T29E, pNBV1RegX3T100E, pNBV1RegX3T151E, pNBV1RegX3T191E, pNBV1RegX3T193E, or pNBV1RegX3T217E were used for complementation analysis. The complementation test was performed by determining the expression level of phoA in M. smegmatis strains grown under P i -limiting conditions in the absence of acetamide by RT-PCR. As a control, the ⌬regX3 conditional mutant with the empty pNBV1 vector, which was grown under P i -limiting conditions in the absence (ϪAce) or presence of (ϩAce) of acetamide, was included in the experiment. RT-PCR for 16S rRNA was conducted to ensure that the same amounts of total RNA were employed for RT-PCR. Protein levels of the WT and mutant forms of RegX3 Mtb expressed in the strains were determined by Western blot analysis with a His tag antibody, and the result is shown below the RT-PCR result.

Inhibition of RegX3 transcriptional activity by PknB
nobacteria, including mycobacteria (34,55,56). PknA and PknB have been suggested to be essential STPKs for mycobacteria and to be implicated in signal transduction regulating cell wall synthesis and cell shape (38,39,(57)(58)(59)(60). PknB is composed of the N-terminal KD and the C-terminal extracytoplasmic domain consisting of four penicillin-binding protein and Ser/ Thr kinase-associated (PASTA) repeats (61). These kinds of PASTA repeats are also found in PknB-like STPKs of Grampositive bacteria, including Actinobacteria (62,63). Several lines of evidence for phosphorylation of RRs by STPKs in mycobacteria have been reported. In 2010, phosphorylation of the DosR (DevR) RR by PknH was first reported in Mtb (43). PknH has been demonstrated to phosphorylate DosR (DevR) on Thr-198 and Thr-205 to enhance DosR (DevR) transcriptional activity. Recently, we revealed that purified PknB-KD Mtb strongly phosphorylated six RRs (RegX3, NarL, KdpE, TrcR, DosR/ DevR, and MtrA) of Mtb in vitro. Based on this finding, we began to study signal convergence between PknB and TCSs in mycobacteria and demonstrated that overexpression of PknB-KD Mtb in M. smegmatis resulted in a significant inhibition of DosR (DevR) transcriptional activity by phosphorylating Thr-180 of DosR (DevR) (50). We chose the SenX3-RegX3 TCS to further study the cross-talk between PknB and TCSs in mycobacteria because well-known reporter genes such as phoA and pstS, which are under strict control of the SenX3-RegX3 TCS, were available to determine the transcriptional activity of RegX3, and the activation condition of the SenX3-RegX3 TCS (P i -limiting condition) was established (14).
We observed that the transcriptional activity of RegX3 Ms and RegX3 Mtb was reduced with increased expression and activity of PknB-KD Mtb (Figs. 2-4). Furthermore, MS/MS analysis following the in vitro phosphorylation reaction with purified RegX3 Mtb and PknB-KD Mtb led to detection of six phosphorylation sites (Thr-29, Thr-100, Thr-151, Thr-191, Thr-193, and Thr-217) on RegX3 Mtb (Fig. S4). Because our MS/MS results did not show the phosphorylation extent of the identified Thr residues, we performed an in vitro phosphorylation assay with nonphosphorylatable Thr-to-Glu mutant forms of RegX3 Mtb and purified PknB-KD Mtb (Fig. 5), which revealed that Thr-100 and Thr-191 of RegX3 Mtb are the major sites of phosphorylation, whereas Thr-217 is the minor phosphorylation site. In addition, the phosphomimetic T100E, T191E, and Thr217E mutant forms of RegX3 Mtb were shown to lose transcriptional activity, as judged by the complementation result (Fig. 6). Altogether, these results indicate that PknB kinase activity inhibits the transcriptional activity of RegX3 Mtb mainly through phosphorylation of Thr-100 and Thr-191 (Thr-98 and Thr-189 in the case of RegX3 Ms ), which are located in the receiver domain and the DNA-binding helix-turn-helix motif of RegX3, respectively (Fig. S6). From the location of Thr-191 on RegX3 Mtb , it was assumed that phosphorylation of Thr-191 might inhibit the transcriptional activity of RegX3 Mtb by perturbing DNA binding of RegX3 Mtb . Indeed, our EMSA revealed that the phosphomimetic T191E mutation in RegX3 Mtb led to abolishment of the DNA-binding ability of RegX3 Mtb (Fig. 7). The location of Thr-217 near the helix-turn-helix motif accounts for abolishment of the transcriptional activity and DNA-binding ability of T217E RegX3 Mtb . Despite the lack of its transcriptional activity, the T100E mutant form of RegX3 Mtb has been shown to retain the same degree of DNA-binding ability as the WT form of RegX3 Mtb (Fig. 7), indicating that the lack of transcriptional activity of T100E RegX3 Mtb is not the result of its inability to bind DNA. The RR of the TCS contains six wellconserved residues (three acidic amino acids, one Thr/Ser, one Tyr/Phe, and one Lys) that are functionally important for phosphorylation of the RR and activation of the effector domain through phosphorylation-induced conformational changes (64 -66). In the case of RegX3 Mtb , the six residues correspond to Glu-8, Asp-9, Asp-52, Thr-79, Tyr-98, and Lys-101. The location of Thr-100 between Tyr-98 and Lys-101 implies that the

Inhibition of RegX3 transcriptional activity by PknB
T100E mutation (and, therefore, phosphorylation of Thr-100 by PknB) is likely to make it unlikely that RegX3 Mtb is activated through phosphorylation-induced conformational changes.
As mentioned above, six RRs of the 11 paired TCSs found in Mtb were shown to be robustly phosphorylated by PknB-KD Mtb . Four (RegX3, KdpE, TrcR, and MtrA) of the six RRs are structurally related to the OmpR family of RRs. The Thr residues corresponding to Thr-191 and Thr-217 of RegX3 Mtb are conserved in the four RRs (RegX3, KdpE, TrcR, and MtrA), whereas the Thr-100 -corresponding residues are conserved in RegX3, KdpE, and TrcR but not in MtrA (Fig. S6). Given the strong phosphorylation of KdpE, TrcR, and MtrA by PknB-KD Mtb like RegX3 and the conservation of at least two of three Thr residues corresponding to Thr-100, Thr-191, and Thr-217 of RegX3 Mtb , as well as the good conservation of amino acid sequences around the Thr residues corresponding to Thr-100 of RegX3 Mtb , it is possible that PknB might inhibit the transcriptional activity of KdpE, TrcR, and MtrA as well.
The signal convergence between TCSs and STPKs was also observed in several Gram-positive bacteria besides mycobacteria. In most cases, an STPK phosphorylates an RR to change the transcriptional activity of the RR. The WalR RR of the WalKR TCS in Bacillus subtilis has been reported to be phosphorylated by PrkC (a PknB homolog) both in vitro and in vivo (49). WalR shares a relatively high sequence similarity (48% identity and 81% similarity) with RegX3 Mtb over the whole protein sequence (Fig. S7). Phosphorylation of WalR on Thr-101, which corresponds to Thr-100 of RegX3 Mtb , has been shown to cause changes in WalR transcriptional activity. It is noteworthy that the amino acid residues around the Thr residues are very well conserved in WalR and RegX3 (Fig. S7), which reinforces phosphorylation of RegX3 Mtb on Thr-100 by PknB. In group A and group B streptococci, phosphorylation of the CovR RR on Thr-65 by a PknB-like STPK (Stk or Stk1) has been shown to inhibit acetyl phosphate-dependent phosphorylation of CovR (41,48). The RR06 and RitR RRs of Streptococcus pneumoniae have been shown to be phosphorylated by a PknB-like STPK, StkP (42,45). The GraR RR of the GraSR TCS of Staphylococcus aureus has been demonstrated to be phosphorylated on Thr-128, Thr-130, and Thr-149 by Stk1, which resulted in an increase in the DNA-binding affinity of GraR (46). Phosphorylation of the VraR RR of the S. aureus VraSR TCS on Thr-106 and Thr-119 in the receiver domain and on Thr-175 and Thr-178 within the DNA-binding domain by Stk1 has been shown to reduce its DNA binding affinity (47). The observation that several RRs are phosphorylated by multiple kinases, including their cognate HKs and STPKs, suggests that signaling through TCSs in bacteria could be more complex than anticipated.
The PASTA repeats of PknB have been demonstrated to serve as binding modules for muropeptides of peptidoglycan, and binding of muropeptides to the PASTA repeats has been suggested to activate PknB kinase activity (67). PknB has been demonstrated to be localized to the septa and poles of the dividing bacterial cell, where higher local concentrations of muropeptides are available because of high rates of peptidoglycan turnover and synthesis (54,68). These facts, in conjunction with the observation that the cellular abundance of PknB is significantly reduced in the nonreplicating Mtb cells relative to that in actively dividing cells (69), suggest that the kinase activity of PknB might be much higher in actively dividing mycobacteria than in nonreplicating mycobacteria, which raises the possibility that PknB might play a role in the regulation of gene expression and cellular processes as a sensor kinase that coordinates the replication state with the regulation of intracellular metabolism and gene expression in mycobacteria.
From this viewpoint, we propose a model for the dual control of RegX3 transcriptional activity by SenX3 and PknB. The major sensory kinase regulating the transcriptional activity of RegX3 in response to P i availability is the SenX3 HK. Under favorable growth conditions with sufficient P i supplementation, the RegX3 RR is not activated by the SenX3 HK, and the high activity of PknB might further inhibit the residual transcriptional activity of RegX3 by functioning as an "auxiliary switch" or "safety lock" to minimize leaky expression of the RegX3 regulon. Under P i -limiting conditions, where RegX3 is activated by SenX3, reduced PknB activity because of inhibited replication of mycobacterial cells might mitigate the inhibitory effect of PknB on RegX3, enabling full activation of RegX3 transcriptional activity.
In conclusion, we found that overexpression of PknB-KD Mtb inhibits the transcriptional activity of RegX3 Mtb by phosphorylating Thr-100, Thr-191, and Thr-217 (Thr-98, Thr-189, and Thr-215 for RegX3 Ms ). Convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate two different signals, the environmental P i level and the replication state, to regulate gene expression in response to changing P i availability.

Bacterial strains, plasmids, and culture conditions
The bacterial strains and plasmids used in this study are listed in Table S1. Escherichia coli strains were grown in Luria-Bertani medium at 37°C. M. smegmatis strains were grown in Middlebrook 7H9 medium (Difco, Detroit, MI) supplemented with 0.2% (w/v) glucose as a carbon source and 0.02% (v/v) Tween 80 as an anticlumping agent at 37°C. For P i -limiting and replete growth conditions, MOPS minimal medium (25 mM MOPS (pH 7.2), 25 mM KCl, 10 mM Na 2 SO 4 , 20 mM NH 4 Cl, 10 M FeCl 3 , 2 mM MgSO 4 , and 0.1 mM CaCl 2 ) supplemented with 50 M and 10 mM K 2 HPO 4 , respectively, were used. M. smegmatis strains were grown aerobically on a gyratory shaker (200 rpm) to an A 600 of 0.4 -0.5. Ampicillin (100 g/ml for E. coli), kanamycin (50 g/ml for E. coli and 15 or 30 g/ml for M. smegmatis), and hygromycin (200 g/ml for E. coli and 50 g/ml for M. smegmatis) were added to the growth medium when required. Overexpression of the genes encoding PknB-KD Mtb and RegX3 Mtb from pMH201-derived plasmids was induced by addition of acetamide to the growth medium to a final concentration of 0.2% (w/v) unless specific concentrations of acetamide are stated. Construction of the plasmids and a ⌬regX3 conditional mutant of M. smegmatis is described in the supporting information.

Determination of colony-forming units
Samples (1 ml) were collected from M. smegmatis cultures at the indicated time points. The collected samples were homoge-

Inhibition of RegX3 transcriptional activity by PknB
neously resuspended by passing them ten times through a 25-gauge needle to break up cell clumps. 200 l of the samples appropriately diluted with MOPS minimal medium were plated on glucose-MOPS agar plates supplemented with either 10 mM or 50 M K 2 HPO 4 and 0.2% (w/v) acetamide. The bacterial colonies growing on agar plates were counted after 72-h incubation at 37°C.

DNA manipulation and electroporation
Recombinant DNA manipulations were conducted in accordance with standard protocols and the manufacturer's instructions (70). Transformation of M. smegmatis with plasmids was carried out by electroporation as described previously (71).

Site-directed mutagenesis
To introduce point mutations into the genes encoding RegX3 Mtb and PknB-KD Mtb , PCR-based mutagenesis was conducted using the QuikChange site-directed mutagenesis procedure (Stratagene, La Jolla, CA). Synthetic oligonucleotides 33 bases long and containing a mutated codon in the middle of their sequences were used to mutagenize the original codons (Table S2). Mutations were verified by DNA sequencing.

␤-Galactosidase activity assay and determination of protein concentration
␤-Galactosidase activity was measured spectrophotometrically as described elsewhere (72). A Bio-Rad protein assay kit was used to determine the protein concentration.

RT-PCR and real-time qPCR
RNA isolation from M. smegmatis strains, preparation of complementary DNA, RT-PCR, and real-time qPCR were conducted as described previously (73). The primers used for complementary DNA synthesis, RT-PCR, and real-time qPCR are listed in Table S2.

Protein purification
E. coli strains overexpressing RegX3 Mtb or PknB-KD Mtb were grown aerobically at 37°C in Luria-Bertani medium containing 100 g/ml ampicillin (for E. coli strains containing the pT7-7 derivatives) or 50 g/ml kanamycin (for the E. coli strain carrying pETpknBhis) to an A 600 of 0.4 -0.6. Expression of the regX3 Mtb and pknB Mtb genes was induced by addition of IPTG to a final concentration of 0.5 mM, and then cells were grown further for 4 h at 30°C. Harvested cells from 200 ml of culture were resuspended in 5 ml of buffer A (20 mM Tris-Cl (pH 8.0) and 100 mM NaCl) in the presence of DNase I (10 units/ml) and 10 mM MgCl 2 and disrupted by two passages through a French pressure cell. Cell-free crude extracts were obtained by centrifugation twice at 23,708 ϫ g for 15 min. 500 l of the 50% (v/v) slurry (bed volume, 250 l) of Ni-Sepharose high-performance resin (GE Healthcare) was packed into a column. After equilibration of the resin with 10 bed volumes of buffer A, cell-free crude extracts were loaded into the column. The resin was washed with 60 bed volumes of buffer A containing 5 mM imidazole and 60 bed volumes of buffer A containing 10 mM imidazole, and then His 6 -tagged RegX3 Mtb and His 6 -tagged PknB-KD Mtb were finally eluted with 10 bed volumes of buffer A containing 250 mM imidazole. The eluted proteins were desalted using a PD-10 desalting column (GE Healthcare) equilibrated with the appropriate buffer.

Western blot analysis
To determine the amount of expressed His 6 -tagged RegX3 Mtb and PknB-KD Mtb in cells, Western blot analysis was performed as described previously (74). To detect His 6 -tagged proteins, mouse monoclonal IgG against His-3 (Santa Cruz Biotechnology, Santa Cruz, CA; sc8036) was used at a 1:2,000 dilution. Alkaline phosphatase-conjugated anti-mouse IgG produced in rabbits (Sigma, A4312) was used at a 1:10,000 dilution for detection of the primary antibody.

Analysis of in vivo protein-protein interactions
Saccharomyces cerevisiae AH109 strains cotransformed with both the pGADT7linker and pGBKT7 derivatives were grown in synthetic defined dropout (SD) medium (Clontech, Palo Alto, CA) lacking leucine and tryptophan (SD/ϪLeu/ϪTrp). The overnight cultures were diluted with distilled water to an A 600 of 0.6 and spotted onto both solid SD/ϪLeu/ϪTrp plates and histidine-deficient SD/ϪLeu/ϪTrp/ϪHis plates for a spotting assay. These plates were incubated at 30°C for 3-5 days.

LC-MS/MS analysis for phosphorylated RegX3 Mtb
Phosphorylation reactions of 0.4 nmol of WT RegX3 Mtb were conducted in the presence of 0.4 nmol of PknB-KD Mtb in 84 l of reaction buffer (20 mM Tris-Cl (pH 7.5), 50 mM NaCl, 10 mM MgCl 2 , 10 mM MnCl 2 , and 100 M ATP) for 1 h at 30°C. The reactions were terminated by adding 30 l of gel-loading buffer containing 100 mM EDTA. The proteins were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB), and the RegX3 Mtb bands were excised. The excised protein bands from SDS-PAGE gels were cut into small pieces, washed three times with 200 l of HPLC-grade water, and destained with 200 l of 1:1 (v:v) mixture of acetonitrile and ammonium bicarbonate (100 mM (pH 8.0)). The gel pieces were dehydrated for 5 min with 500 l of 100% acetonitrile and incubated in a solution of 10 mM DTT in 100 mM ammonium bicarbonate (50 l) for 30 min at 56°C, followed by 55 mM iodoacetamide in 100 mM ammonium bicarbonate (50 l) for 20 min at room temperature in the dark. Thereafter, the gel pieces were dehydrated again with 100% acetonitrile and rehydrated in a solution of 13 ng/l of sequencing-grade modified trypsin (Promega, Madi-Inhibition of RegX3 transcriptional activity by PknB son, WI) in 10 mM ammonium bicarbonate. The digestion was completed overnight at 37°C. Peptides were extracted by incubating the gel pieces in a 1:2 (v:v) mixture of 5% formic acid and acetonitrile, and the solution was dried by vacuum centrifugation.
Peptide samples were reconstituted in 7 l of 0.1% formic acid and injected from an auto sampler into a reverse-phase C 18 column (20 cm ϫ 75 m inner diameter, 3 m, 300 Å, packed in-house; Dr. Maisch GmbH) on an Eksigent multidimensional liquid chromatography system at a flow rate of 300 nl/min. Before use, the column was equilibrated with 95% mobile phase A (0.1% formic acid in H 2 O) and 5% mobile phase B (0.1% formic acid in acetonitrile). The peptides were eluted with a linear gradient from 10%-35% B over 100 min, followed by washing with 70% B and re-equilibration with 5% B at a flow rate of 300 nl/min with a total run time of 130 min. The HPLC system was connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA) operated in data-dependent acquisition mode. Survey full-scan MS spectra (m/z 400 -2,000) were acquired in the Orbitrap with a resolution of 60,000. Source ionization parameters were as follows: spray voltage, 1.9 kV; capillary temperature, 275°C. The MS/MS spectra of the 10 most intense ions from the MS1 scan with a charge state of 1 or more were acquired in the ion trap with the following options: isolation width, 2.0 m/z; normalized collision energy, 45%; dynamic exclusion, 60 s.
The acquired MS/MS spectra were subjected to a search against the in-house database (containing RegX3 Mtb and PknB-KD Mtb sequences with a common contaminant database) using the SEQUEST HT software in Proteome Discoverer 2.2 (Thermo Fisher Scientific). Two missed trypsin cleavages were allowed, and the peptide mass tolerances for MS/MS and MS were set to Ϯ0.6 Da and Ϯ10 ppm, respectively. Other parameters used for the SEQUEST HT searches included fixed modification of carbamidomethylation at cysteine (ϩ57.02 Da), variable modification of oxidation at methionines (ϩ15.99 Da), and phosphorylation at serine, threonine, or tyrosine (ϩ79.97 Da). ptmRS was run in PhosphoRS mode to localize the phosphorylation site. A probability of 75% or higher was considered to confidently indicate a phosphorylation site.

EMSA
123-bp DNA fragments including a RegX3-binding site upstream of the phoA gene were used in the assay (specific DNA). The DNA fragments were generated by PCR using the primer set F_phoA_EMSA_123 (5Ј-AGTCAAGCTTGCTCT-CGACGCCGTCGTG-3Ј) and R_phoA_EMSA_123 (5Ј-AGT-CGAATTCTGATCGCGAGTCACATAAGC-3Ј) and pNC-phoA as a template. 60-bp control DNA fragments without the RegX3-binding site were amplified by PCR using pUC19 as a template (control DNA) and the primers pUC19_EMSA_F (5Ј-CCTCTAGAGTCGACCTGC-3Ј) and pUC19_EMSA_R (5Ј-AGGAAACAGCTATGACCATG-3Ј). Purified RegX3 Mtb proteins were incubated with 100 fmol each of specific and control DNA in buffer (20 mM MOPS (pH 8.0) containing 150 mM KCl) in a final volume of 10 l for 20 min at 25°C. To examine the effect of RegX3 Mtb phosphorylation by PknB-KD Mtb on DNA binding, phosphorylation reactions were performed in 10 l of kinase buffer (20 mM Tris-Cl (pH 7.5), 25 mM NaCl, 10 mM MgCl 2 , and 20 mM MnCl 2 ) and 100 M ATP containing 60 pmol of RegX3 Mtb and 30 pmol of the WT or mutant form (K40M) of PknB-KD Mtb for 30 min at 30°C, and then 100 fmol each of the specific and control DNA were added to the phosphorylation reaction mixtures. The binding reaction mixtures were incubated for 20 min at 25°C. After addition of 2 l of 6ϫ loading buffer (0.25% (w/v) bromphenol blue, 0.25% (w/v) xylene cyanol, and 40% (w/v) sucrose), the mixtures were subjected to nondenaturing PAGE (6% (w/v) acrylamide) using 0.5ϫ TBE buffer (41.5 mM Tris borate and 0.5 mM EDTA (pH 8.3)) at 50 V/cm for 1 h 50 min at 4°C. The gels were stained with SYBR Green staining solution (Invitrogen) for 30 min. Bands were visualized using an UV illuminator.