Tuning the Mycobacterium tuberculosis Alternative SigmaFactor SigF through the Multidomain Regulator Rv1364c andOsmosensory Kinase Protein Kinase D

Richa Misra,a Dilip Menon,b,f Gunjan Arora,a Richa Virmani,a Mohita Gaur,c Saba Naz,c,d Neetika Jaisinghani,b,f Asani Bhaduri,a

Ankur Bothra,b,f Abhijit Maji,a Anshika Singhal,a Preeti Karwal,a Christian Hentschker,e Dörte Becher,e Vivek Rao,b,f

Vinay K. Nandicoori,d Sheetal Gandotra,b,f Yogendra Singha,c

aAllergy and Infectious Disease Unit, CSIR-Institute of Genomics and Integrative Biology, Delhi, IndiabRespiratory Disease Biology, CSIR-Institute of Genomics and Integrative Biology, New Delhi, IndiacDepartment of Zoology, University of Delhi, Delhi, IndiadNational Institute of Immunology, Delhi, IndiaeInstitute of Microbiology, Ernst-Moritz-Arndt University Greifswald, Greifswald, GermanyfAcademy of Scientific and Innovative Research (AcSIR), New Delhi, India

ABSTRACT Bacterial alternative sigma factors are mostly regulated by a partner-switching mechanism. Regulation of the virulence-associated alternative sigma factorSigF of Mycobacterium tuberculosis has been an area of intrigue, with SigF havingmore predicted regulators than other sigma factors in this organism. Rv1364c isone such predicted regulator, the mechanism of which is confounded by thepresence of both anti-sigma factor and anti-sigma factor antagonist functions ina single polypeptide. Using protein binding and phosphorylation assays, wedemonstrate that the anti-sigma factor domain of Rv1364c mediates autophos-phorylation of its antagonist domain and binds efficiently to SigF. Furthermore,we identified a direct role for the osmosensor serine/threonine kinase PknD inregulating the SigF-Rv1364c interaction, adding to the current understandingabout the intersection of these discrete signaling networks. Phosphorylation ofSigF also showed functional implications in its DNA binding ability, which mayhelp in activation of the regulon. In M. tuberculosis, osmotic stress-dependent in-duction of espA, a SigF target involved in maintaining cell wall integrity, is cur-tailed upon overexpression of Rv1364c, showing its role as an anti-SigF factor.Overexpression of Rv1364c led to induction of another target, pks6, involved inlipid metabolism. This induction was, however, curtailed in the presence of os-motic stress conditions, suggesting modulation of SigF target gene expressionvia Rv1364c. These data provide evidence that Rv1364c acts an independent SigFregulator that is sensitive to the osmosensory signal, mediating the cross talk ofPknD with the SigF regulon.

IMPORTANCE Mycobacterium tuberculosis, capable of latently infecting the host andcausing aggressive tissue damage during active tuberculosis, is endowed with acomplex regulatory capacity built of several sigma factors, protein kinases, and phos-phatases. These proteins regulate expression of genes that allow the bacteria toadapt to various host-derived stresses, like nutrient starvation, acidic pH, and hyp-oxia. The cross talk between these systems is not well understood. SigF is one suchregulator of gene expression that helps M. tuberculosis to adapt to stresses and im-parts virulence. This work provides evidence for its inhibition by the multidomainregulator Rv1364c and activation by the kinase PknD. The coexistence of negativeand positive regulators of SigF in pathogenic bacteria reveals an underlying require-ment for tight control of virulence factor expression.

Citation Misra R, Menon D, Arora G, Virmani R,Gaur M, Naz S, Jaisinghani N, Bhaduri A, BothraA, Maji A, Singhal A, Karwal P, Hentschker C,Becher D, Rao V, Nandicoori VK, Gandotra S,Singh Y. 2019. Tuning the Mycobacteriumtuberculosis alternative sigma factor SigFthrough the multidomain regulator Rv1364cand osmosensory kinase protein kinase D.J Bacteriol 201:e00725-18. https://doi.org/10.1128/JB.00725-18.

Editor Tina M. Henkin, Ohio State University

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Sheetal Gandotra,sheetal.gandotra@igib.res.in, or YogendraSingh, ysingh@igib.res.in.

Received 26 November 2018Accepted 2 January 2019

Accepted manuscript posted online 14January 2019Published

RESEARCH ARTICLE

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KEYWORDS Mycobacterium tuberculosis, STPK, protein-protein interaction, serine/threonine protein kinase, sigma factor

Mycobacterium tuberculosis, responsible for an infection with the highest mortalitydue to any single bacterial species worldwide, is endowed with a genome

enriched in signal transduction and gene regulatory modules (1, 2). Alternative sigma(�) factors provide bacteria with the means to simultaneously regulate many genes inresponse to altered environmental or developmental signals encountered during theinfection process (3, 4). Sigma factor F (SigF), the general stress response � factor of M.tuberculosis, is implicated in persistence (5, 6). A sigF deletion mutant of M. tuberculosisis attenuated in the murine and guinea pig model of tuberculosis (7–9). Although theSigF regulon has been characterized in several studies (7, 10–14) and includes manygenes involved in cell surface modification and virulence factor secretion (7, 14), thereis a stark lack of uniformity in the phenotypic behavior of SigF mutant/overexpressionstrains in different studies, mostly attributed to the differences in the M. tuberculosisstrains used (CDC1551 versus H37Rv) (8, 10, 12). Furthermore, very little is understoodabout how signals perceived in the host enable switching to the alternative � factor.

The availability of most alternative � factors is governed by a complex partner-switching system controlled by phosphorylation-dependent regulation, best exempli-fied by the Bacillus subtilis general stress response � factor SigB (15). Under unstressedconditions, SigB is inactivated by the anti-� factor RsbW, which physically binds to itand thus prevents the association of SigB with RNA polymerase. The anti-� factorantagonist RsbV can bind and sequester RsbW in an unphosphorylated form, but thisis prevented by the kinase activity of RsbW. This system is, in turn, regulated by twophosphatases, RsbP and RsbU, which on sensing different stress signals dephosphor-ylate RsbV. Another set of anti-� factor and anti-� factor antagonist homologs controlsthe activity of RsbU. Upon dephosphorylation by either RsbP or RsbU, RsbV binds RsbW,thus enabling stress-dependent transcription by a SigB-containing holoenzyme (15).However, the regulation of the B. subtilis SigB homolog in M. tuberculosis, SigF, is notvery well understood. Cotranscribed with sigF, usfX encodes the cognate anti-�factor for SigF (16). Apart from this, other putative anti-� factor regulators (Rv0516c,Rv1364c, Rv1365c, Rv1904, Rv2638, Rv3687c) are present in the genome, and someof these have been characterized to be antagonists (16–23); however, ambiguityremains about their role vis-à-vis SigF. Although protein homology provides vitalclues, it is difficult to extrapolate the function of these regulators. A study byHatzios et al. revealed activation of the SigF regulon upon disruption of Rv0516c,questioning its role as an anti-� factor antagonist (24). The existence of multipleregulators for SigF suggests that the inhibition of the alternate � factor must alsobe crucial to its survival or pathogenesis. Rv1364c of M. tuberculosis is unique in itsdomain architecture, in that it mimics a tandem array of domains (sensor–phosphatase– kinase–anti-� factor antagonist) in a single polypeptide, where thekinase domain is predicted to work as the anti-� factor domain (17, 19, 20, 22, 23).Its function vis-à-vis an antagonist or agonist of SigF remains elusive, since bothregulatory domains are present in a single protein.

In the present work, we demonstrate that Rv1364c functions primarily as a bona fideanti-SigF factor. We show that the kinase activity of Rv1364c is essential for itsautophosphorylation of the anti-� antagonist domain and that Rv1364c is capable ofbinding to SigF in the autophosphorylated form. This may have significance in restrain-ing SigF activity under normal growth conditions. Through an independent mecha-nism, protein kinase D (PknD), a eukaryote-like serine/threonine protein kinase (STPK),induced the phosphorylation of both proteins and mobilized SigF release fromRv1364c. PknD overexpression has been shown in an earlier study to induce the SigFregulon indirectly (18); here we find evidence for a direct mechanistic link throughphosphorylation-dependent dissociation with its anti-� factor.

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RESULTSAutophosphorylation of recombinant Rv1364c and its effect on the interaction

with SigF. Previous studies on the characterization of Rv1364c reported the presenceof an active phosphatase domain in Rv1364c, with D211 and D328 being identified asthe active-site residues (19, 22). The kinase activity is questionable, as indicated by twoopposing reports, with both being uncertain about the activity of the full-lengthprotein (19, 20). The kinase domain of Rv1364c (RsbW) is reported to possess thecharacteristic Bergerat fold of the GHKL (gyrase, Hsp90, histidine kinase, MutL) ATPase/kinase superfamily (19, 20, 23). Our earlier attempts at characterizing this protein haddemonstrated an inability of the protein to execute [�-32P]ATP transfer to theC-terminal anti-� factor antagonist domain belonging to the sulfate transporter andanti-sigma factor antagonist (STAS) family (19). We reasoned that this could be due tothe dominant activity of the phosphatase in preventing retention of the transferred 32P.To further assess the activity of the anti-� factor domain, we probed the ATPase activityof the full-length protein and found that it can indeed hydrolyze ATP. The kinaseconserved active-site residues E444 and N448, based on sequence homology to B.subtilis proteins (19, 20, 23), were crucial for the ATPase activity (Fig. 1A). Surprisingly,the phosphomimetic mutant of the conserved phosphoacceptor residue (Rv1364cS600E)showed activity comparable to that of the full-length wild-type (WT) protein (Fig. 1A).So, we further probed if phosphorylation at the predicted S600 site was dependent onthe kinase activity of Rv1364c. Using the phosphoprotein stain Pro-Q Diamond, wemeasured the phosphorylation status of the purified full-length protein Rv1364c. Weobserved that Rv1364c was efficiently phosphorylated under standard purificationprocedures (Fig. 1B). To explore the mechanism of autophosphorylation and to rule outphosphorylation due to any Escherichia coli kinase, we also probed the phosphorylationstatus of Rv1364c variants. Mutations in the kinase domain (N448A and E444A) andconserved phosphoracceptor site S600 in the substrate domain abolished the phos-phorylation (Fig. 1B). On the other hand, mutations in conserved active-site residues ofthe phosphatase domain (D211A and D328A) had no effect on the phosphorylationstatus of the protein. Thus, our results indicate that Rv1364c is found in phosphorylatedform when expressed in E. coli and provide a plausible reason why previous attemptsat detecting the �-32P of ATP being transferred at this site failed (19). The autophos-phorylation at S600, however, does not confer any additional advantage to the inter-action of Rv1364c with SigF (Fig. 1C). In view of the results of our previous study (19)and the present results, we therefore establish that full-length Rv1364c possesses bothphosphatase and kinase activities and can efficiently bind to SigF independently of itsautophosphorylation. Since PknD is known to influence the SigF regulon (18), weexplored the cross talk of this STPK with the SigF-Rv1364c protein pair.

Rv1364c and SigF are reversibly phosphorylated by PknD in vitro. STPK-mediated phosphorylation fine-tunes a variety of cellular processes in Mycobacteriumspp. to help these organisms rapidly adapt to host-derived stresses (25, 26). Studieshave suggested the cross talk of STPK-mediated signaling and � factor regulatorysystems, indicating the importance of multimode regulation of transcription (18, 24,27). PknD helps M. tuberculosis to adapt to osmotic stress by regulating the SigFregulon; however, the exact mechanism of how SigF is activated (released from itscognate anti-� factors) is yet unknown (24). We performed an in vitro [�-32P]ATPtransfer assay with PknD and His6-tagged Rv1364c and found the efficient phosphor-ylation of Rv1364c by PknD (Fig. 2A). The kinase domains of PknD and Rv1364c areapproximately the same size; therefore, to authenticate phosphotransfer to Rv1364crather than the autophosphorylation of PknD in this assay, the reaction end productswere treated with tobacco etch virus (TEV) protease, which cleaves the affinity tag fromRv1364c. The retention of radioactivity on the digested product validates the PknD-mediated phosphorylation of Rv1364c (Fig. 2A). Moreover, the S600A mutant retainedthe ability to be phosphorylated by PknD, suggesting the presence of additionalphosphorylation sites on Rv1364c (Fig. 2A). Independently, Rv1364c cloned with abigger maltose binding protein (MBP) tag was also subjected to the kinase assay in the

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presence of WT PknD or WT PknD in the presence of its specific inhibitor, SP600125, orthe PknDD138N kinase-dead mutant, to confirm PknD-mediated phosphorylation ofRv1364c (Fig. 2B). The complete loss of phosphorylation of Rv1364c in the presence ofthe inhibitor and the PknD kinase-dead mutant confirmed the specificity of thereaction. The previously reported substrate of PknD, Rv0516c (18), was included as apositive control in these assays.

To test if extrinsic signals received by STPKs can also affect SigF directly, weperformed in vitro [�-32P]ATP transfer assays with purified full-length/kinase domains ofPknA, PknB, PknD, PknE, PknF, PknG, and PknH. The kinase domain of PknD performedthe most efficient phosphorylation of SigF (Fig. 2C), although other STPKs were alsocapable of phosphorylating it. The specificity of the PknD-mediated phosphorylation ofSigF was also confirmed with the PknD kinase-dead mutant (Fig. 2D). The PknD-

FIG 1 Conserved ATPase and autophosphorylation activity in Rv1364c and effect on interaction with SigF. (A) (Top) Thin-layerchromatography- and autoradiography-based ATPase activity of purified Rv1364c, its indicated variants, or the positive control, Rv1747possessing the ATPase domain, at 0 min and 60 min postincubation with [�-32P]ATP. Buffer alone acted as a negative control. SD, substratedomain of Rv1364c; PD, phosphatase domain of Rv1364c. (Bottom) Relative ADP formation, measured using densitometry and expressedas a percentage of the activity of the wild-type protein. The results from three independent experiments are presented here as themean � SEM. (B) (Top and middle) Pro-Q Diamond staining of recombinant Rv1364c and its variants. Pro-Q Diamond staining indicatesthe phosphorylation level, while SYPRO Ruby stains total protein. (Bottom) The ratio of the two densities normalized for each proteinvariant to the density of the wild-type protein. Data are means from three independent experiments, each performed in triplicate (mean �SEM). Analysis of variance and Dunnett’s posttest were used to compare all groups to wild-type Rv1364c (***, P � 0.001; **, P � 0.01; *,P � 0.05; ns, not significant). Lanes M, molecular mass markers. (C) His6-tagged Rv1364c and its phosphoablative variant, Rv1364c S600A,were immobilized (500 ng/well) on the surface of the microtiter plate and challenged with increasing concentrations (0 to 1,000 ng) ofGST-tagged fusion proteins, SigF, and GST alone (negative control) in solution. The error bars indicate the mean � SD for triplicatereadings.

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FIG 2 PknD phosphorylates both Rv1364c and SigF. (A) In vitro kinase assay of Rv1364c with STPK (PknD). Digestion of the reaction products with TEV proteaseresolves the phosphorylation of WT Rv1364c, Rv1364c S600A, and PknD. Phosphorylation of Rv1364c S600A by PknD reveals a site distinct from the conservedautophosphorylation site. (B) In vitro kinase assay of Rv1364c with WT PknD, WT PknD in the presence of the inhibitor SP600125, and kinase-dead mutant

(Continued on next page)

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mediated phosphorylation of Rv1364c and SigF was reversed by PstP (Fig. 2E and F), thecognate phospho-Ser/Thr phosphatase, denoting the reversible regulation of theireffector functions.

PknD phosphorylation attenuates the interaction of SigF with its anti-� factor,Rv1364c. The PknD-mediated phosphorylation of Rv1364c and SigF was validatedin vivo, using E. coli as a surrogate host (Fig. 3A). Rv1364c or SigF, coexpressed withPknD in vivo, were purified to assess phosphorylation. Pro-Q Diamond staining ofpurified Rv1364c (His tagged) and SigF (glutathione S-transferase [GST] tagged) showedphosphorylation (Fig. 3A). Two-dimensional gel electrophoresis and immunoblot anal-ysis of native Rv1364c and Rv1364c coexpressed with PknD in E. coli were alsoperformed and indicated that multiple acidic phosphoisoforms of Rv1364c were gen-erated by PknD, in contrast to the native Rv1364c, confirming the presence of addi-tional phosphorylation sites (Fig. 3B). Through mass spectrometry, we identified the

FIG 2 Legend (Continued)PknDD138N. The loss of phosphorylation by the PknD inhibitor and kinase-dead PknD shows that PknD specifically phosphorylates Rv1364c. Rv0516c wasincluded as a positive control. (C) In vitro kinase assay of M. tuberculosis STPKs (labeled on top) to check the phosphorylation of SigF in the presence of[�-32P]ATP. (D) An in vitro kinase assay of SigF with WT PknD and its kinase-dead mutant, PknDD138N, also shows the specificity of PknD-mediatedphosphorylation of SigF. (E and F) (Bottom) Autoradiographs of PknD-phosphorylated Rv1364c (E) and SigF (F) after incubation with purified M. tuberculosisphosphatase PstP for the indicated times at 25°C show the reversibility of the PknD-mediated phosphorylation. (Top) Equal amount of protein in all lanes basedon Coomassie brilliant blue (CBB) staining. (Bottom) Images of autoradiographs of the same dried protein gel shown at the top visualized by use of aPhosphorImager FLA 2000/GE Typhoon Trio imager. Lanes M, molecular mass markers.

FIG 3 PknD-mediated phosphorylation leads to decreased Rv1364c-SigF binding in vitro. Rv1364c (His6 tagged) and SigF (GST tagged) purifiedfrom cultures coexpressing MBP alone and MBP-PknD were evaluated for their phosphorylation by Pro-Q Diamond staining (A), two-dimensionalgel electrophoresis (B), and interaction by ELISA (C). (A) (Top) Pro-Q Diamond-stained gel; (bottom) Coomassie brilliant blue staining of the samegel shown at the top. pG-SigF and pRv1364c refer to the phosphorylated forms of GST-tagged SigF (G-SigF) and Rv1364c, respectively, obtainedfrom cultures coexpressing PknD. Lane M, molecular mass markers. (B) Two-dimensional gels of the Rv1364c protein purified from E. colioverexpressing pACYC-PknD-Rv1364c (top) or pACYC-Rv1364c (bottom). Approximately 500 pg of precipitated proteins was resolved on a 7-cmpH 4 to 7 linear gradient, followed by a second dimension on 10% SDS-polyacrylamide gels, and subjected to immunoblotting with anti-Rv1364cantibody. Rv1364c coexpressed with PknD in E. coli gets phosphorylated in vivo and shows additional acidic phosphoisoforms compared to thePknD-naive condition. (C) His6-tagged Rv1364c or pRv1364c was immobilized (500 ng/well) on the surface of the microtiter plate and challengedwith increasing concentrations (0 to 1,000 ng) of GST-tagged SigF (G-SigF) or pG-SigF in solution. GST alone acted as a negative control. The valueswere normalized to those for the negative control, GST. (Inset) The same experiment with only the phosphorylated forms of the interactingproteins pRv1364c and pG-SigF. The error bars indicate the mean � SD from three independent experiments, each with three technical replicates.Student’s t test was applied for comparing means across groups (*, P � 0.05; **, P � 0.01).

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PknD-mediated phosphorylation of Rv1364c on multiple threonine and serine residues(Thr54, Thr81, Thr299, Thr390, Thr520, Thr568, and Ser506) (see Fig. S1 in the supple-mental material). The results suggest that PknD directly phosphorylates the alternative� factor SigF and its regulator, Rv1364c.

To examine the effect of PknD-mediated phosphorylation on the SigF-Rv1364cinteraction in vitro, SigF and Rv1364c were coexpressed with and without PknD in thepACYC three-way protein expression system to obtain the phosphorylated and un-phosphorylated forms of these proteins (Fig. 3A). The protein-protein interaction wasanalyzed by a sandwich enzyme-linked immunosorbent assay (ELISA). Although thephosphorylation of only one of the interacting proteins (SigF or Rv1364) did not leadto a significant loss of the interaction (Fig. 3C), as shown in the inset in Fig. 3C, theinteraction between SigF and Rv1364c was reduced significantly upon the in vivophosphorylation of both proteins by PknD.

PknD-phosphorylated SigF mediates tighter complex formation with RNApolymerase. We questioned whether PknD-phosphorylated SigF retains its ability torecruit RNA polymerase to its cognate promoter. Purified SigF bound to the sigFpromoter in an RNA core polymerase-dependent manner (Fig. 4A). Using the sigF

FIG 4 Phosphorylated SigF binds tightly to the target gene promoter. (A) Binding of SigF to the sigF promoter in the presence of E. colicore RNA polymerase, measured using EMSA. (B and C) E. coli core RNA polymerase recruitment by SigF or pSigF to the sigF promoter,measured using EMSA. The percentage of DNA in complex was calculated using the following formula: 100 · (amount of bound probe/totalamount of probe). (C) The bar graph represents the mean � SD from three independent experiments. (D and E) Strength and specificityof DNA binding by SigF-RNA polymerase versus pSigF-RNA polymerase complexes measured by EMSA (D). The percentage of releasedDNA was calculated using the following formula: 100 · (1 � ΔFPc/ΔFP), where ΔFP � amount of free probe in the absence of SigF or pSigF� amount of free probe in the presence of SigF or pSigF; the subscript c indicates the presence of the competing cold probe. The graph(E) represents the mean � SD from four independent experiments. The t test was used for comparing the means between SigF and pSigFfor each DNA concentration (*, P � 0.05).

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promoter as the target sequence, we checked the ability of SigF and PknD-phosphorylated SigF (pSigF) to recruit E. coli core RNA polymerase to this DNA byelectrophoretic mobility shift assay (EMSA). We found that purified SigF and pSigFenabled DNA-protein complex formation with RNA polymerase in a dose-dependentmanner (Fig. 4B). The phosphorylated protein reached saturation of binding at a lowerprotein concentration (Fig. 4B and C). Additional protein-DNA complexes of lower andhigher mobility were found with PknD-phosphorylated SigF than with SigF (Fig. 4B,asterisks). Competition with a nonradioactive probe confirmed the specificity of theinteraction in both cases. However, the release of only 50% of the radioactive probefrom the pSigF-RNA polymerase-DNA complex compared to 80% of the probe from theunphosphorylated SigF counterpart at even 40 times the competing probe concentra-tion suggested tighter complex formation by pSigF than by SigF (Fig. 4D and E). In viewof the previous reports of studies in which PknD overexpression led to activation of theSigF regulon (18), our results show direct evidence of the effect of the PknD-mediatedphosphorylation of SigF on its DNA binding ability and, thereby, its effect on theregulon. Thus, phosphorylation of SigF by PknD not only is responsible for the disso-ciation with its anti-� factor but also causes tight binding of pSigF-RNA polymeraseto DNA.

Rv1364c overexpression quenches SigF target gene espA induction underosmotic stress. To understand how Rv1364c regulates SigF under stress, we looked atits role in the osmosensory signaling pathway. Hatzios et al. illustrated the PknD-mediated osmosensory activation of the SigF regulon gene espA, which enables M.tuberculosis to adapt to osmotic stress by cell wall remodeling and virulence factorproduction (24). We studied whether the induction of this gene is affected by the actionof Rv1364c as either an anti-SigF or an anti-SigF antagonist. Approximately 25-foldoverexpression of Rv1364c was achieved by the plasmid pTC0X1-Rv1364c in M. tuber-culosis H37Rv (Fig. 5A). As shown earlier (24), osmotic stress led to a modest butstatistically significant increase in the expression of espA (Fig. 5B). The ability to increaseespA expression in response to osmotic stress was curtailed in cultures harboring theoverexpression plasmid pTC0X1-Rv1364c (Fig. 5B). This suggested that Rv1364c func-tions as an anti-SigF factor. We studied another probable SigF target gene, theexpression of which is not regulated by osmotic stress in wild-type cells, pks6 (7, 24).Expression of pks6 was indeed not induced by osmotic stress in the empty vector-containing strain (Fig. 5C). Cells overexpressing Rv1364c were found to express 50%higher levels of pks6 than cells of the control strain, probably due to an anti-SigFantagonist function in the basal state. Exposure to osmotic stress under this scenarioled to an approximately 50% reduction in the expression of pks6, proposing activationof the anti-SigF property of Rv1364c by osmotic stress. Together, these data verify the

FIG 5 Rv1364c regulates the virulence factor espA under osmotic stress. The relative expression of Rv1364c (A), espA (B), and pks6 (C)in M. tuberculosis cultures harboring pTC0X1-Rv1364c versus pTC-mcs in the presence or absence of osmotic stress was measured byqRT-PCR. Cultures were exposed to 140 mM NaCl for 1 h to induce osmotic stress. Gene expression was normalized to 16S rRNAexpression. Values are the mean � SEM from six independent cultures. The t test was used for comparing means between theindicated groups (ns, not significant; *, P � 0.05; **, P � 0.01).

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role of osmotic stress-mediated control of SigF target genes via Rv1364c in M. tuber-culosis.

Distribution of Rv1364c-, SigF-, and PknD-like sequences in actinomycetes.Earlier reports of divergence in SigF orthologs in M. tuberculosis and M. smegmatis and,in general, in pathogenic versus nonpathogenic mycobacteria suggest that divergentregulatory circuits of SigF activation may play an important role in the virulence-associated features of this alternative � factor (28, 29). The phylogenetic analysis ofRv1364c orthologs revealed that members from pathogenic mycobacteria form adistinct clade and appear to be phylogenetically closer to some nonmycobacterialactinobacterial members (Fig. S2A). We observed that the multidomain architecture isconserved in some actinobacterial species, such as Actinoplanes friuliensis, Nakamurellamultipartita, Rhodococcus ruber, Modestobacter marinus, Amycolatopsis vancoresmycina,and Actinomadura madurae, apart from the pathogenic Mycobacterium. While A. ma-durae is an opportunistic human pathogen (30), R. ruber is a species closely related toa known opportunistic human pathogen, Rhodococcus equi. A. friuliensis and A. van-coresmycina are producers of friulimicin and vancoresmycin, respectively (31, 32). Sincethese environmental organisms are relatively less studied, we suggest that such aunique multidomain fusion event may endow these organisms with the means to adaptto environmental and host-derived stresses. The nonpathogenic mycobacterial cladeconspicuously lacked the phosphatase-kinase-STAS occurrence. SigF orthologs formedthree distinct clades, consisting of pathogenic Mycobacterium spp., nonpathogenicMycobacterium spp., and nonmycobacterial actinomycetes (Fig. S2B). Interestingly, M.tuberculosis PknD sequence-specific features (viz., the kinase domain plus NHL repeats)are mostly conserved in the pathogenic Mycobacterium spp. only (Fig. S2C). Thissuggests the presence of a coregulatory mechanism for SigF-Rv1364c-PknD in the faceof the specific stresses faced by these pathogenic members.

DISCUSSION

M. tuberculosis SigF draws parallels with B. subtilis SigB, by virtue of sequencehomology. Compared to the paradigm of stressosome-dependent regulation of SigB inB. subtilis (33–35), a stressosome-independent control mechanism exists in M. tubercu-losis for its homolog, SigF (Fig. 6). Since Bacillus possesses only stand-alone proteinspossessing regulatory domains, M. tuberculosis probably evolved an alternate strategyto influence this interaction. While phosphorylation and dephosphorylation mediatedby the GHKL family of kinases and PP2C phosphatases, respectively, are known togovern the opposing activities of regulatory proteins of SigB in B. subtilis (15), theregulation of M. tuberculosis SigF is not yet completely clear. Interestingly, bioinformat-ics analyses revealed a similar coevolution pattern for SigF and its regulator, Rv1364c,among members of the Mycobacterium genus (see Fig. S2 in the supplementalmaterial). The domain architecture of Rv1364c possesses a unique arrangement ofsensor-PP2C phosphatase-GHKL kinase-STAS domains conserved only among thepathogenic mycobacterial members (19, 20, 23), suggesting additional ways to controlvirulence-associated SigF in the face of stresses (Fig. 6 and S2). While Rv1364c possessesboth phosphatase and kinase activities, it primarily acts as a SigF anti-� factor, withdominant autophosphorylation occurring at the conserved serine residue of the STASdomain (Fig. 1). The signal required for the switch to phosphatase activity is not yetknown. The eukaryote-like STPK PknD has been implicated in activation of the SigFregulon under osmotic stress, albeit indirectly (18, 24). Here we describe a direct crosstalk between PknD and SigF and its regulator, Rv1364c (Fig. 2 and 3). The PknD-mediated phosphorylation sites in Rv1364c identified in this work map to the differentdomains of the protein. These findings provide new directions toward understandingthe regulation of SigF function.

While STPKs have been described to target other mycobacterial � factor regulators(27, 36), the role of phosphorylation of a � factor has never been described in bacteria.In the plant Arabidopsis thaliana, SIG1 phosphorylation leads to inhibition of RNApolymerase recruitment to the photosystem I (PS-I) promoter, thereby helping in the

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switch to PS-II in response to redox stress (37). Phosphorylation-dependent relief fromantagonists and the simultaneous activation of RNA polymerase recruitment wouldform a positive feed-forward loop for activation of the target � factor regulon. Our workprovides evidence for such a positive feed-forward activation mechanism of SigF by anextracellular sensor kinase, PknD, by not only destabilization of its interaction with itsanti-� factor, Rv1364c, but also simultaneous activation of its RNA polymerase recruit-ment function (Fig. 3 and 4). One of the consequences may be to initiate more frequentpulses of transcription initiation events, a phenomenon associated with amplification ofthe output in response to stress (38). Phosphorylated SigF may variably occupy certaintargets of the SigF regulon, depending on the affinity of the promoter, leading todifferential gene expression at various time points under stress conditions. It remains tobe deciphered what signals modulate activation of the Ser/Thr phosphatase PstP thatdrives bacterial transcriptional regulation in the opposite direction.

A high density of SigF targets is involved in lipid metabolism and cell surfacechanges (11, 14). PknD activation by osmotic stress was shown to regulate espA via

FIG 6 Schematic representation of the regulation of SigF in M. tuberculosis. (A) Domain architecture of Rv1364c indicatingeach of its domains. (B) Model of SigF regulation based on findings from previous studies (16, 18, 19, 24) and the resultsfrom the current study. Two anti-� factors, the cognate UsfX and the multidomain regulator Rv1364c, bind to andnegatively regulate SigF, the M. tuberculosis homolog of B. subtilis SigB. Osmotic stress-stimulated activation of PknD maylead to the phosphorylation of Rv1364c and SigF, thereby affecting their ability to interact with each other and releasingSigF. Both the native unphosphorylated and phosphorylated SigF can recruit RNA polymerase and may control differentgenes of its regulon in response to osmotic stress. Overexpression of Rv1364c may either increase the expression of somegenes, such as pks6, or negatively influence the expression of others, such as espA, suggesting the influence of other SigFregulators that can directly interact with Rv1364c and influence the SigF regulon.

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Rv0516c in the CDC1551 strain of M. tuberculosis (24). espA, an essential component ofthe ESX-1 secretion system, is involved in maintaining the integrity of the cell wall (24,39). The lack of evidence for a direct interaction between Rv0516c and SigF left thepathway unlinked between phosphorylation of Rv0516c by PknD and induction of theSigF target, espA. Our data reveal the osmotic induction of espA in M. tuberculosis H37Rvand inhibition of this induction upon overexpression of Rv1364c, indicating its role asan anti-SigF factor under osmotic stress conditions (Fig. 5 and 6). Rv1364c-mediatedrepression of pks6, a gene involved in cell wall lipid synthesis (40), by osmotic stress alsopoints toward its role as an anti-SigF factor under osmotic stress. Interestingly, theinduction of pks6 upon overexpression of Rv1364c points toward an anti-SigF antag-onist function which could be mediated through interaction with other SigF regulators.These results put forward another example of the intersection of signaling pathwaysmediated by eukaryote-like STPKs and alternative sigma factors, highlighting the tightregulation of the signal transduction mechanism in M. tuberculosis.

MATERIALS AND METHODSBacterial strains and growth conditions. E. coli DH5� (Novagen) was used as a host strain for

cloning purposes, and E. coli BL21(DE3) (Stratagene) was used as a host strain for the expression ofrecombinant proteins. E. coli cells were grown at 37°C in Luria-Bertani (LB) broth or LB agar (Difco) platessupplemented with 100 �g/ml of ampicillin and/or 25 �g/ml of kanamycin, when needed. M. tuberculosisH37Rv was grown in Middlebrook 7H9 broth (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80,and albumin-dextrose-catalase (Difco) at 37°C with shaking (150 rpm).

Constructs and gene manipulation. pTC-mcs and pTC0X1L were kind gifts from Dirk Schnappinger,Weill Cornell Medical College (catalog numbers 20317 and 20315, respectively; Addgene) (41). The clonesfor GST- or His6-tagged STPKs and PstPcatalytic domain from previous studies were used (42–44). The plasmidcoding for M. tuberculosis SigF, pLCD1, was kindly provided by W. R. Bishai (Johns Hopkins School ofMedicine, Baltimore, MD). For the coexpression studies, the dual-expression vectors pETDuet-1 (Nova-gen) and pACYCDuet-1 (Novagen) were used (45, 46). The pETDuet-PknD construct from a previous studywas used (45). SigF was subsequently cloned into the multiple-cloning site 1 (MCS1) region of the vectorat HindIII-NotI sites. The PknD kinase domain amplicon was digested with NdeI and XhoI and cloned intothe corresponding sites in MCS2 of pACYCDuet-1. Rv1364c was cloned into MCS1 of the pACYCDuet-1and pACYC-PknD constructs at BamHI and HindIII sites. Rv1364c was also cloned into the pMAL vectorto obtain MBP-Rv1364c. Rv0516c was cloned into the pGEX-5X-3 vector. All the DNA manipulations werecarried out according to the standard protocols. The gene segment encoding Rv1364c cloned inpProExHTc and Rv1364cphosphatase domain, Rv1364ckinase domain, Rv1364csubstrate domain, Rv3287c (usfx) clonedin the pGEX-5X-3 vector from a previous study were used (19). Mutagenesis of the active-site residues ofRv1364c (D211A, D328A, E444A, N448A, S600A, S600E) and the PknD kinase active-site residue (D138N)was carried out using the HTc-Rv1364c construct and pGEX-PknD1–378 as templates, respectively, and aQuikChange XL site-directed mutagenesis kit (Stratagene) as described previously (19). The sequences ofall clones were confirmed by DNA sequencing. The primers and constructs used in this study aredescribed in Tables 1 and 2, respectively.

Purification of recombinant proteins. All the recombinant constructs were expressed and purifiedwith Ni-nitrilotriacetic acid, glutathione-Sepharose, or amylose resin affinity columns (Qiagen/NEB) asHis6-, GST-, or MBP-tagged fusion proteins from E. coli per the manufacturer’s instructions and asdescribed previously (42, 45). The purified proteins were assessed by SDS-PAGE, and all the pure fractionswere pooled and dialyzed with buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10% glycerol). The proteinconcentrations were estimated by use of a Bio-Rad protein assay kit, and fractions were aliquotedand stored at �80°C until further use. To get phosphorylated and unphosphorylated forms of SigF,pETDuet-1 constructs coexpressing His6-SigF, MBP-PknD/His6-SigF, or MBP alone were transformed intoE. coli BL21(DE3) Codon Plus cells (Stratagene). Cultures were induced with IPTG (isopropyl-�-D-thiogalactopyranoside) and further grown for 12 to 16 h at 18°C. Cells were harvested, and His6-taggedSigF and His6-tagged phospho-SigF were purified using the procedure mentioned above. The three-waycoexpression of SigF and Rv1364c with PknD was obtained by cotransforming the pGEX-SigF plasmidwith the pACYC-MBP-PknD-1364c and pACYC-MBPalone-1364c plasmids in E. coli to get phosphorylatedand unphosphorylated forms of the proteins. The His6-tagged Rv1364c and GST-tagged SigF proteinswere obtained from the overexpressed cultures by the purification procedure mentioned above.

ATPase activity assay. The ATPase activity assay was performed essentially as described previously(47), with slight modifications. ATPase activity was determined in a reaction mixture containing 50 mMTris-HCl, pH 7.4, 1 mM MgCl2, 1 mM dithiothreitol, 1.0 �Ci of [�-32P]ATP (20 �Ci/mmol; Board of Radiationand Isotope Technology [BRIT], Hyderabad, India) and 3 �g purified protein of Rv1364c, Rv1364cD211A,Rv1364cD328A, Rv1364cN444A, Rv1364cN448A, Rv1364cS600A, Rv1364cS600E, or Rv1747 (positive control [48]).After 0 and 60 min of incubation at 25°C, the products were separated by thin-layer chromatography onpolyethyleneimine cellulose sheets (Merck) using 0.75 M potassium phosphate buffer, pH 3.75, as thesolvent. The radioactive signal on the dried sheets was visualized with a PhosphorImager FLA 2000imager (Fujifilm), and ATPase activity was assayed by measuring the relative intensity of the product,ADP, by the use of ImageQuant software.

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Phosphorylation state detection by fluorescent staining. For detection of the phosphorylationstate of Rv1364c, Pro-Q Diamond phosphoprotein staining (Invitrogen) was performed per the manu-facturer’s instructions. Briefly, equal amounts of the wild-type and mutant proteins were electrophoresedby SDS-PAGE, and the gels were fixed twice in a solution of 50% (vol/vol) methanol and 10% (vol/vol)acetic acid and subsequently washed four times with Milli-Q water. The gels were stained with Pro-QDiamond stain for 1.5 h. To remove the nonspecific background, the gels were destained three times with20% acetonitrile, 50 mM sodium acetate (pH 4), followed by two additional washing steps. The gels werescanned using a Typhoon Trio� scanner (GE Biosciences) (excitation source, 532-nm laser; long-passemission filter, 560 nm). The same gels were then stained with SYPRO Ruby for total protein detection.To differentiate between phosphorylated and unphosphorylated proteins, the ratio of Pro-Q Diamonddye to SYPRO Ruby dye signal intensities for each band was determined by the use of imaging software.The molecular weight marker run in parallel with the proteins also served as a control.

In vitro kinase assay. The in vitro kinase assays were performed by incubating 3 �g of Rv1364c orSigF and 0.5 to 3 �g of STPKs to obtain for each specific kinase the optimal autophosphorylation activityin a 25-�l reaction mixture containing 20 mM PIPES [piperazine-N,N=-bis(2-ethanesulfonic acid); pH 7.2],5 mM MnCl2, 5 mM MgCl2, and 1 �Ci [�-32P]ATP (BRIT, Hyderabad, India) for 30 min at 25°C. The PknDinhibitor SP600125 was dissolved in dimethyl sulfoxide and diluted in water, and the solution was addedat a concentration of 20 �M to the reaction mixture (whenever required), as described previously (18).The reactions were terminated by adding Laemmli sample loading buffer, followed by boiling at 100°Cfor 5 min. The proteins were separated by 10% SDS-PAGE, stained with Coomassie blue, dried, andvisualized by use of a PhosphorImager FLA 2000 (Fujifilm)/GE Typhoon Trio imager. For visualization ofthe phosphorylation signal on cleaved proteins, removal of recombinant tags was achieved by additionof proteases (thrombin [Novagen] for recombinant SigF and TEV for recombinant Rv1364c or its mutants)after the kinase reaction according to the manufacturer’s instructions. The reactions were stopped byusing SDS buffer and resolved on 8 to 10% SDS-PAGE gels. Likewise, for the dephosphorylation ofphosphorylated SigF and Rv1364c, the reaction mixtures were incubated with PstPcat (1 �g) for anadditional 0, 5, 30, and 60 min at 25°C and resolved on a 10% SDS-PAGE gel. The signals were visualizedby autoradiography.

Analysis of protein isoforms by two-dimensional PAGE and immunoblotting. To assess thephosphorylation status of native Rv1364c and PknD-phosphorylated Rv1364c, equal amounts of proteinswere subjected to two-dimensional PAGE, followed by immunoblotting, as described earlier (27, 49).Briefly, each sample was rehydrated into 7-cm-long immobilized pH gradient (IPG) strips with a pH range

TABLE 1 Primers used in this study

Primer namea Primer sequence (5= ¡ 3=)Rv1364c N448A F.P TCCGAATTCGTCGAGGCCGCGGTCGAACACGGATACRv1364c N448A R.P GTATCCGTGTTCGACCGCGGCCTCGACGAATTCGGARv1364c E444A F.P CGTGCACGCGATCTCCGCATTCGTCGAGAACGCGRv1364c E444A R.P CGCGTTCTCGACGAATGCGGAGATCGCGTGCACGRv1364c S600A F.P GTCACCCACCTTGGTGCGGCCGGCGTCGGCGCCRv1364c S600A R.P GGCGCCGACGCCGGCCGCACCAAGGTGGGTGACRv1364c S600E F.P GTCACCCACCTTGGTGAGGCCGGCGTCGGCGCCRv1364c S600E R.P GGCGCCGACGCCGGCCTCACCAAGGTGGGTGACPknD D138N F.P GGCGTAACGCACCGCAACGTAAAACCGGPknD D138N R.P CCGGTTTTACGTTGCGGTGCGTTACGCCpETDuet_SigF_F.P CGACGGGCGGCATCAAGCTTGTGACGGCGCGCGCpETDuet_SigF_R.P CTCGCCGAGATCAAGTAGGCGGCCGCCTACTCCAACTGATCCCGpGEX_SigF_F.P GCCCGACGGGCGGGATCCAGCAGGTGACGGpGEX_SigF_R.P CTCGCCGAGATCAAGTAAGGCGGCCGCCTACTCCAACTGATCCCGpGEX_Rv0516c_F.P CGACGGAGAACGAGGATCCTGATGACTACCACGATCCCpGEX_Rv0516c_R.P CACAACGACGACCCGCGGCCGCTTTAGGCTGACCpMAL_1364_F.P GGTCCGTAGGAGGGACGGATCCATGGCGGCCGAAATGGpMAL_1364_R.P CGTGCAGGCTCGTTGAAGCTTCTACTCCTGGGCGAAGATGpACDuet_PknD F.P GACCTAGTGAAGGGAATTCGCATATGAGCGATGCCGTTCCGpACDuet_PknD R.P GCCGACGACGGCCTCGAGCTTCCGTTTGTTGCCGGCpACDuet_Rv1364c F.P GTCCGTAGGAGGGATCCCCAAATGGCGGCCGAAATGGpACDuet_Rv1364c R.P CCGTGCAGGCTCGTTGAAGCTTCTACTCCTGGGCGAAGPromoter_SigF F.P GCGGCTGGAAATCCCGGCATCGCGGGPromoter_SigF R.P GGTCGGACCTGCTGGTAGTGGGGATCTAACGCRv1364c_RTF TCGGTGCGGCCGAGGATGTACGRv1364c_RTR TAGACCTCCCGAGCGGGCTGTC16S rRNA_RTF TACGTTCCCGGGCCTTGT16S rRNA_RTR AATCGCCGATCCCACCTTespA_RTF TCCGGGTGATGGCTGGTTAGespA_RTR GGTCGTGGATCAGGCTGATGpks6_RTF CGTAGTGCTGACTCGTTAAGpks6_RTR TCGGTCAGAAAGTCCCATAGaF.P and F, forward primer; R.P and R, reverse primer.

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TAB

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of 4 to 7 or 3.9 to 5.1, as appropriate (Bio-Rad). Isoelectric focusing was performed for 15,000 V-h in aProtean isoelectric focusing cell (Bio-Rad). After equilibration, strips were loaded and resolved in thesecond dimension through a 10% SDS-PAGE gel. The proteins were electrotransferred, and immunoblotanalysis was performed using anti-Rv1364c polyclonal serum (customized antibody from BangaloreGenei India Pvt. Ltd.). The blots were developed using an Immobilon Western chemiluminescenthorseradish peroxidase substrate kit (Millipore) according to the manufacturer’s instructions.

ELISA. Enzyme-linked immunosorbent assay (ELISA) was done using the method described by Luet al. (50) with some modifications. Briefly, the His6-tagged proteins were dissolved in coating buffer(0.1 M NaHCO3, pH 9.2) at a concentration of 5 �g/ml and adsorbed (100 �l/well) onto the surface of a96-well ELISA plate (MaxiSorp; Nunc) overnight at 4°C. After rinsing the wells five times with wash buffer(phosphate-buffered saline, pH 7.4, 0.05% Tween 20), the reactive sites were blocked with blocking buffer(2% bovine serum albumin in wash buffer) for 2 h at room temperature. After five washes, the adsorbedproteins were challenged with various concentrations of GST-tagged interacting proteins (100 �l/well)dissolved in binding buffer (50 mM Tris, pH 7.4, 200 mM NaCl, 0.02% NP-40, 10% glycerol) for 1 h at roomtemperature. Following five washes, the wells were treated with horseradish peroxidase-conjugated GSTantibody (catalog number ab3416; Abcam) at a 1:10,000-fold dilution for 1 h at room temperature. Afterfive washes with wash buffer and an additional wash with phosphate-buffered saline, pH 7.4, thechromogenic substrate o-phenylenediamine dihydrochloride (0.4 mg/ml in 0.1 M phosphate-citrate buf-fer, pH 5) and H2O2 were added to visualize the interaction. After addition of stop solution (2.5 M H2SO4),the absorbance was read at 490 nm.

EMSA. DNA-� factor binding was carried out by an electrophoretic mobility shift assay (EMSA) asdescribed previously (51). A DNA fragment derived from the upstream region of usfX containing thepredicted SigF-specific promoter sequence (11, 16) was amplified from the M. tuberculosis H37Rv genomeusing specific primers (Table 1). After purification, the 118-bp PCR product (1 �g) was labeled with T4polynucleotide kinase (Roche Applied Science) and [�-32P]ATP (BRIT, Hyderabad, India) per the manu-facturer’s instructions. The radiolabeled PCR fragment was purified free of [�-32P]ATP and polynucleotidekinase using a nucleotide removal kit (Qiagen). EMSAs were performed by incubation of 0.2 U of E. colicore polymerase enzyme (Epicentre Technologies) (11, 12) with various concentrations of purified SigFand pSigF in binding buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 0.1 mMdithiothreitol, 5% [vol/vol] glycerol) at 37°C for 10 min, followed by addition of 0.03 pmol labeled DNAprobe. After incubation for an additional 30 min, 6� DNA loading buffer (Fermentas) was added, thereaction mixtures were electrophoresed at 4°C on 5% nondenaturing polyacrylamide gels in 0.5� TBE(Tris-borate-EDTA) buffer for 2 h at 200 V, and the products were visualized by autoradiography (personalmolecular imager system; Bio-Rad). To quantify the amount of DNA bound, ImageQuant data analysissoftware was used.

Gene expression analysis. M. tuberculosis strains containing pTC-mcs, pTC0X1-Rv1364c, andpTC0X1-Rv1364c-S600A were grown in Sauton’s medium. Osmotic stress treatment was performed asdescribed previously (24). Cultures were subcultured to an optical density (OD) of 0.05 in Sauton’smedium and grown to an OD of 0.6, and then NaCl (final concentration, 140 mM) or an equal volume ofwater was added, followed by incubation in a shaker incubator for 1 h at 37°C. The cultures wereharvested by addition of equal volumes of 4 M guanidine isothiocyanate (GITC), followed by centrifu-gation. The culture pellet was resuspended in the TRIzol LS reagent, and RNA was extracted as reportedpreviously (52). cDNA synthesis was performed using random hexamers, and quantitative reversetranscription-PCR (qRT-PCR) was performed using the gene-specific primers listed in Table 1.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/JB

.00725-18.SUPPLEMENTAL FILE 1, PDF file, 1.4 MB.

ACKNOWLEDGMENTSWe thank Rakesh Sharma, Bhupesh Taneja, and Rajesh S. Gokhale for useful discus-

sions during the course of this work and Ulf Gerth for helping with the mass spec-trometry analysis.

This work was funded by CSIR Task Force projects BSC0403 and BSC0123, a DSTPurse grant, and a JC Bose fellowship (SERB) to Y.S. Saba Naz was funded through aCSIR-Senior research fellowship.

We declare no conflict of interest with the contents of this article.S. Gandotra and Y. Singh guided the study. R. Misra and S. Gandotra wrote the

manuscript. R. Misra performed all experiments in E. coli and with recombinant proteinswith contributions from G. Arora, R. Virmani, M. Gaur, S. Naz, A. Bothra, A. Maji, and A.Singhal; D. Menon and N. Jaisinghani performed the experiments with M. tuberculosis;C. Hentschker and D. Becher performed the mass spectrometry analysis; R. Misra and A.Bhaduri performed the phylogenetic analysis; V. Rao and V. K. Nandicoori providedstrains and reagents; and V. Rao, P. Karwal, and V. K. Nandicoori provided inputs indiscussion.

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