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Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H37Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr(65) and Thr(74) in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg(55) is also identified as an important residue for N-HupB-DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H37Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

During protein synthesis, translation elongation factor Tu (Ef-Tu) is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. The activity of Ef-Tu is dependent on its interaction with GTP. Posttranslational modifications, such as phosphorylation, are known to regulate the activity of Ef-Tu in several prokaryotes. Although a study of the Mycobacterium tuberculosis phosphoproteome showed Ef-Tu to be phosphorylated, the role of phosphorylation in the regulation of Ef-Tu has not been studied. In this report, we show that phosphorylation of M. tuberculosis Ef-Tu (MtbEf-Tu) by PknB reduced its interaction with GTP, suggesting a concomitant reduction in the level of protein synthesis. Overexpression of PknB in Mycobacterium smegmatis indeed reduced the level of protein synthesis. MtbEf-Tu was found to be phosphorylated by PknB on multiple sites, including Thr118, which is required for optimal activity of the protein. We found that kirromycin, an Ef-Tu-specific antibiotic, had a significant effect on the nucleotide binding of unphosphorylated MtbEf-Tu but not on the phosphorylated protein. Our results show that the modulation of the MtbEf-Tu-GTP interaction by phosphorylation can have an impact on cellular protein synthesis and growth. These results also suggest that phosphorylation can change the sensitivity of the protein to the specific inhibitors. Thus, the efficacy of an inhibitor can also depend on the posttranslational modification(s) of the target and should be considered during the development of the molecule.

Background: The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs., Principal Findings: PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+)-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr(137), Thr(141), Thr(174) and Thr(290) being the target residues of PknB while Thr(137) and Thr(174) are phosphorylated by PknA. The Mn(2+)-ion binding residues Asp(38) and Asp(229) are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c) (D38G) are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+)/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent., Conclusions and Significance: This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

Reversible protein phosphorylation is a prevalent signaling mechanism which modulates cellular metabolism in response to changing environmental conditions. In this study, we focus on previously uncharacterized Mycobacterium tuberculosis Ser/Thr protein kinase (STPK) PknJ, a putative transmembrane protein. PknJ is shown to possess autophosphorylation activity and is also found to be capable of carrying out phosphorylation on the artificial substrate myelin basic protein (MyBP). Previous studies have shown that the autophosphorylation activity of M. tuberculosis STPKs is dependent on the conserved residues in the activation loop. However, our results show that apart from the conventional conserved residues, additional residues in the activation loop may also play a crucial role in kinase activation. Further characterization of PknJ reveals that the kinase utilizes unusual ions (Ni(2+), Co(2+)) as cofactors, thus hinting at a novel mechanism for PknJ activation. Additionally, as shown for other STPKs, we observe that PknJ possesses the capability to dimerize. In order to elucidate the signal transduction cascade emanating from PknJ, the M. tuberculosis membrane-associated protein fraction is treated with the active kinase and glycolytic enzyme Pyruvate kinase A (mtPykA) is identified as one of the potential substrates of PknJ. The phospholabel is found to be localized on serine and threonine residue(s), with Ser(37) identified as one of the sites of phosphorylation. Since Pyk is known to catalyze the last step of glycolysis, our study shows that the fundamental pathways such as glycolysis can also be governed by STPK-mediated signaling.

Mycobacterium tuberculosis profoundly exploits protein phosphorylation events carried out by serine/threonine protein kinases (STPKs) for its survival and pathogenicity. Forkhead-associated domains (FHA), the phosphorylation-responsive modules, have emerged as prominent players in STPK mediated signaling. In this study, we demonstrate the association of the previously uncharacterized FHA domain-containing protein Rv0019c with cognate STPK PknB. The consequent phosphorylation of Rv0019c is shown to be dependent on the conserved residues in the Rv0019c FHA domain and activation loop of PknB. Furthermore, by creating deletion mutants we identify Thr(36) as the primary phosphorylation site in Rv0019c. During purification of Rv0019c from Escherichia coli, the E. coli protein chloramphenicol acetyltransferase (CAT) specifically and reproducibly copurifies with Rv0019c in a FHA domain-dependent manner. On the basis of structural similarity of E. coli CAT with M. tuberculosis PapA5, a protein involved in phthiocerol dimycocerosate biosynthesis, PapA5 is identified as an interaction partner of Rv0019c. The interaction studies on PapA5, purified as an unphosphorylated protein from E. coli, with Rv0019c deletion mutants reveal that the residues N-terminal to the functional FHA domain of Rv0019c are critical for formation of the Rv0019c-PapA5 complex and thus constitute a previously unidentified phosphoindependent binding motif. Finally, PapA5 is shown to be phosphorylated on threonine residue(s) by PknB, whereas serine/threonine phosphatase Mstp completely reverses the phosphorylation. Thus, our data provides initial clues for a possible regulation of PapA5 and hence the phthiocerol dimycocerosate biosynthesis by PknB, either by direct phosphorylation of PapA5 or indirectly through Rv0019c.

Mycobacterium tuberculosis encodes for 11 eukaryotic-like serine/threonine protein kinases. Genetic and biochemical studies show that these kinases regulate various cellular processes including cell shape and morphology, glucose and glutamine transport, phagosome-lysosome fusion and the expression, and/or activity of transcription factors. PknK is the largest predicted serine/threonine protein kinase in M. tuberculosis. Here, we have cloned, overexpressed, and purified protein kinase K (PknK) to near homogeneity and show that its ability to phosphorylate proteins is dependent on the invariant lysine (Lys(55)), and on two conserved threonine residues present in its activation loop. Despite being devoid of any apparent transmembrane domain, PknK is localized to the cell wall fraction, suggesting probable anchoring of the kinase to the cell membrane region. The pknK gene is located in the vicinity of the virS gene, which is known to regulate the expression of the mycobacterial monooxygenase (mymA) operon. We report here for the first time that VirS is in fact a substrate of PknK. In addition, four of the proteins encoded by mymA operon are also found to be substrates of PknK. Results show that VirS is a bona fide substrate of PknK in vivo, and PknK-mediated phosphorylation of VirS increases its affinity for mym promoter DNA. Reporter assays reveal that PknK modulates VirS-mediated stimulation of transcription from the mym promoter. These findings suggest that the expression of mymA operon genes is regulated through PknK-mediated phosphorylation of VirS.

Phosphorylation of the mycobacterial transcriptional activator, EmbR, is essential for transcriptional regulation of the embCAB operon encoding cell wall arabinosyltransferases. This signaling pathway eventually affects the resistance to ethambutol (a frontline antimycobacterial drug) and the cell wall Lipoarabinomannan/Lipomannan ratio (an important determinant for averting the host immune response). In this study, further biochemical characterization revealed that EmbR, as a transcriptional regulator, interacts with RNA polymerase and possesses a phosphorylation-dependent ATPase activity that might play a role in forming an open complex between EmbR and RNA polymerase. EmbR was recently shown to be phosphorylated by the cognate mycobacterial serine/threonine (Ser/Thr) kinase, PknH. Using bioinformatic analysis and in vitro assays, we identified additional novel regulators of the signaling pathway leading to EmbR phosphorylation, namely the Ser/Thr protein kinases PknA and PknB. A previously unresolved question raised by this signaling scheme is the fate of phosphorylated kinases and EmbR at the end of the signaling cycle. Here we show that Mstp, a mycobacterial Ser/Thr phosphatase, antagonizes Ser/Thr protein kinase-EmbR signaling by dephosphorylating Ser/Thr protein kinases, as well as EmbR, in vitro. Additionally, dephosphorylation of EmbR reduced its ATPase activity, interaction with Ser/Thr protein kinases and DNA-binding activity, emphasizing the antagonistic role of Mstp in the EmbR-Ser/Thr protein kinase signaling system.

EmbR, a putative transcriptional regulator from Mycobacterium tuberculosis, is homologous to the OmpR class of transcriptional regulators that possess winged helix-turn-helix DNA binding motifs. In contrast to other OmpR-like response regulators that are usually phosphorylated and controlled by histidine kinases, EmbR was recently shown to be phosphorylated by the cognate mycobacterial serine/threonine kinase PknH. Despite the in vitro evidence of phosphorylation and interaction between the kinase and regulator, the physiological function of the PknH-EmbR pair is still unknown. We identify the embCAB operon encoding arabinosyltransferases in M. tuberculosis as the cellular target of EmbR. Phosphorylation of EmbR enhances its DNA binding activity towards promoter regions of embCAB genes. In vivo studies involving expression of PknH in Mycobacterium smegmatis established its positive regulatory effect on transcription of the embCAB operon via phosphorylation of EmbR. Interestingly, increased transcription of embC, catalyzing arabinosylation of lipomannan (LM) to lipoarabinomannan (LAM), results in a high LAM/LM ratio, which in turn is a crucial factor in mycobacterial virulence. The PknH-mediated increase in the transcription of embAB genes significantly alters resistance to ethambutol, a frontline antituberculosis drug known to target embAB genes. These findings and in vivo upregulation of PknH inside the host macrophages suggest a functionally relevant signaling mechanism involving the PknH-EmbR-embCAB system.

Background: Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ. Methodology/Principal Findings: In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA. Conclusions/Significance: Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes. Author Summary: Mycobacterium ulcerans is a slow growing pathogen, which is prevalent in many tropical and sub-tropical countries. M. ulcerans possesses unique signaling pathways with only 13 STPK containing genes. This is strikingly different from its closest homolog Mycobacterium marinum and surprisingly closer to the human pathogen, Mycobacterium tuberculosis. PknQ, MupFHA and MupDivIVA are regulatory proteins encoded by the virulence determining plasmid pMUM001 of M. ulcerans. In addition to characterizing the STPK, we focused on deciphering the basis of interaction between the three partner proteins leading to the identification of critical residues. Present study describes the newly identified phosphoserine-based interactions, which is unique amongst the FHA-domain containing proteins. We confirmed our results using structural analysis via specific mutants and their interaction profiles. Importantly, these data highlight the significance of FHA domains and their role in understanding cellular signaling. This work will encourage further studies to elucidate role of M. ulcerans signaling systems. It will also raise questions like how less studied tropical bacterial pathogens acquire eukaryotic-like Ser/Thr protein kinase and exhibit unusual mechanisms to interact with its partner domains. [ABSTRACT FROM AUTHOR]