Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) - the variability of gene expression in response to environmental changes - remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also find that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Nanopore Identification of N-Acetylation by Hydroxylamine Deacetylation (NINAHD).

N-Acetyl modification, a chemical modification commonly found on biomacromolecules, plays a crucial role in the regulation of cell activities and is related to a variety of diseases. However, due to the instability of N-acetyl modification, accurate and rapid identification of N-acetyl modification with a low measurement cost is still technically challenging. Here, based on hydroxylamine deacetylation and nanopore single molecule chemistry, a universal sensing strategy for N-acetyl modification has been developed. Acetohydroxamic acid (AHA), which is produced by the hydroxylamine deacetylation reaction and serves as a reporter for N-acetylation identification, is specifically sensed by a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA). With this strategy, N-acetyl modifications on RNA, DNA, proteins, and glycans were identified, demonstrating its generality. Specifically, histones can be treated with hydroxylamine deacetylation, from which the generated AHA can represent the amount of N-acetyl modification detected by a nanopore sensor. The unique event features of AHA also demonstrate the robustness of sensing against other interfering analytes in the environment.

The C-terminus is essential for the stability of the mycobacterial channel protein MspA.

Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.

An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics.

D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential therapeutic. However, the regulatory mechanism of LysA during the phage lytic cycle remains ill defined. Here, we show that during D29 lytic cycle, structural and functional regulation of LysA not only orchestrates host cell lysis but also is critical for maintaining phage-host population dynamics by governing various phases of lytic cycle. We report that LysA exists in two conformations, of which only one is active, and the protein undergoes a host peptidoglycan-dependent conformational switch to become active for carrying out endogenous host cell lysis. D29 maintains a pool of inactive LysA, allowing complete assembly of phage progeny, thus helping avoid premature host lysis. In addition, we show that the switch reverses after lysis, thus preventing exogenous targeting of bystanders, which otherwise negatively affects phage propagation in the environment.

Deciphering the molecular mechanism and regulation of formaldehyde detoxification in Mycobacterium smegmatis.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

Mycobacterial FtsEX-RipC interaction is required for normal growth and cell morphology in rifampicin and low ionic strength conditions.

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), remains a major global health problem ranking as the second leading cause of death from a single infectious agent. One of the major factors contributing toward Mtb's success as a pathogen is its unique cell wall and its ability to counteract various arms of the host's immune response. A recent genome-scale study profiled a list of candidate genes that are predicted to be essential for Mtb survival of host-mediated responses. One candidate was FtsEX, a protein complex composed of an ATP-binding domain, FtsE, and a transmembrane domain, FtsX. FtsEX functions through interaction with a periplasmic hydrolase, RipC. Homologs of FtsEX exist in other bacteria and have been linked with playing a key role in regulating peptidoglycan hydrolysis during cell elongation and division. Here, we report on Mycobacterium smegmatis, FtsE, FtsX, and RipC and their protective roles in stressful conditions. We demonstrate that the individual genes of FtsEX complex and RipC are not essential for survival in normal growth conditions but conditionally essential in low-salt media and antibiotic-treated media. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. Our results suggest that FtsE, FtsX, and RipC are required for both normal cell elongation and division and ultimately for survival in stressful conditions. IMPORTANCE: Mycobacterial cell growth and division are coordinated with regulated peptidoglycan hydrolysis. Understanding cell wall gene complexes that govern normal cell division and elongation will aid in the development of tools to disarm the ability of mycobacteria to survive immune-like and antibiotic stresses. We combined genetic analyses and scanning electron microscopy to analyze morphological changes of mycobacterial FtsEX and RipC mutants in stressful conditions. We demonstrate that FtsE, FtsX, FtsEX, and RipC are conditionally required for the survival of Mycobacterium smegmatis during rifampicin treatment and in low-salt conditions. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. We also show that the FtsEX-RipC interaction is essential for the survival of M. smegmatis in rifampicin. Our results suggest that FtsE, FtsX, and RipC are required for normal cell wall regulation and ultimately for survival in stressful conditions.

The accumulation of methylated porphyrins in dormant cells of Mycolicibacterium smegmatis is accompanied by a decrease in membrane fluidity and an impede of the functioning of the respiratory chain.

Transition of Mycolicibacterium smegmatis (Msm) and Mycobacterium tuberculosis to dormancy in vitro is accompanied by an accumulation of free methylated forms of porphyrins (tetramethyl coproporphyrin - TMC) localized in the cell wall of dormant bacteria. A study of the fluorescence anisotropy of BODIPY based fluorescent probes on individual cell level using confocal microscope revealed significant changes in this parameter for BODIPY FL C16 from 0.05 to 0.22 for vegetative and dormant Msm cells correspondingly. Similarly, the increase of TMC concentration in vegetative Msm cells grown in the presence of 5-aminolevulinic acid (a known inducer of porphyrin synthesis) resulted in an increase of BODIPY FL C16 anisotropy. These changes in TMC concentration and membrane fluidity were accompanied by an inhibition of the activity of the respiratory chain measured by oxygen consumption and a reduction of the DCPIP redox acceptor. During the first 8 h of the reactivation of the dormant Msm cells, the porphyrin content and probe fluorescent anisotropy returned to the level for vegetative bacteria. We suggested that upon transition to dormancy, an accumulation of TMC in membranes leads to a decrease in membrane fluidity, resulting in an inhibition of the respiratory chain activity. However, direct interactions of TMC with membrane bound enzymes cannot also be excluded. This, in turn, may result in the down regulation of many metabolic energy-dependent reactions as a part of mechanisms accompanying the transition to a hypometabolic state of mycobacteria.

Understanding the structural basis of the binding specificity of c-di-AMP to M. smegmatis RecA using computational biology approach.

Mycobacterium tuberculosis RecA (MtRecA), a protein involved in DNA repair, homologous recombination and SOS pathway, contributes to the development of multidrug resistance. ATP binding-site in RecA has been a drug target to disable RecA dependent DNA repair. For the first time, experiments have shown the existence and binding of c-di-AMP to a novel allosteric site in the C-terminal-Domain (CTD) of Mycobacterium smegmatis RecA (MsRecA), a close homolog of MtRecA. In addition, it was observed that the c-di-AMP was not binding to Escherichia coli RecA (EcRecA). This article analyses the possible interactions of the three RecA homologs with the various c-di-AMP conformations to gain insights into the structural basis of the natural preference of c-di-AMP to MsRecA and not to EcRecA, using the structural biology tools. The comparative analysis, based on amino acid composition, homology, motifs, residue types, docking, molecular dynamics simulations and binding free energy calculations, indeed, conclusively indicates strong binding of c-di-AMP to MsRecA. Having very similar results as MsRecA, it is highly plausible for c-di-AMP to strongly bind MtRecA as well. These insights from the in-silico studies adds a new therapeutic approach against TB through design and development of novel allosteric inhibitors for the first time against MtRecA.Communicated by Ramaswamy H. Sarma.

Mycobacterial PE12 protein promotes bacterial survival through inhibiting cell apoptosis.

Mycobacterial PE_PGRS family proteins play key roles in pathogen-host interaction. However, the function of most PE_PGRS proteins remains unknown. In this study, we characterized the role of PE12 of Mycobacterium bovis (M. bovis) on bacterial growth, bacterial survival, and host cell apoptosis. Transcriptome sequencing of infected THP-1 cells was also performed. Compared to Ms_Vec, we found that M. bovis PE12 did not alter the colony morphology of M. smegmatis. The survival of Ms_PE12 was obviously higher than that of Ms_Vec. Furthermore, PE12 significantly suppressed the apoptosis of THP-1 induced by M. smegmatis infection. Transcriptome analysis results showed that there were 70 downregulated genes in the Ms_PE12 infection group in comparison with the Ms_Vec infection group, and these differentially expressed genes were enriched in 240 downregulated GO terms and 6 KEGG pathways. The downregulated expression genes are involved in cell adhesion, phagocytosis, apoptosis, inflammatory response, glycolysis and transmembrane transporter activity. Taken together, our study reveals that PE12 can suppress apoptosis and inhibit proinflammatory cytokine response. We propose that PE12 is related to macrophage phagocytosis and apoptosis, providing useful information to the pathogenic mechanisms of M. bovis.

RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis.

Drug resistance is one of the most difficult challenges facing tuberculosis (TB) control. Drug efflux is among the mechanisms leading to drug resistance. In our previous studies, we partially characterized the ABC-type MSMEG-3762/63 efflux pump in Mycobacterium smegmatis, which shares high percentage of identity with the Mycobacterium tuberculosis Rv1687/86c pump. MSMEG-3762/63 was shown to have extrusion activity for rifampicin and ciprofloxacin, used in first and second-line anti-TB treatments. Moreover, we described the functional role of the TetR-like MSMEG-3765 protein as a repressor of the MSMEG_3762/63/65 operon and orthologous Rv1687/86/85c in M. tuberculosis. Here we show that the operon is upregulated in the macrophage environment, supporting a previous observation of induction triggered by acid-nitrosative stress. Expression of the efflux pump was also induced by sub-inhibitory concentrations of rifampicin or ciprofloxacin. Both these drugs also prevented the binding of the MSMEG-3765 TetR repressor protein to its operator in the MSMEG_3762/63/65 operon. The hypothesis that these two drugs might be responsible for the induction of the efflux pump operon was assessed by bioinformatics analyses. Docking studies using a structural model of the regulator MSMEG-3765 showed that both antibiotics abolished the ability of this transcriptional repressor to recognize the efflux pump operon by interacting with the homodimer at different binding sites within the same binding pocket. Reduced binding of the repressor leads to induction of the efflux pump in M. smegmatis, and reduced efficacy of these two anti-mycobacterial drugs.

The PPE2 protein of Mycobacterium tuberculosis is secreted during infection and facilitates mycobacterial survival inside the host.

Mycobacterium tuberculosis secrets various effector proteins to evade host immune responses for facilitating its intracellular survival. The bacterial genome encodes several unique PE/PPE family proteins, which have been implicated to play important role in mycobacterial pathogenesis. A member of this family, PPE2 have been shown to contain a monopartite nuclear localization signal (NLS) and a DNA binding domain. In this study, we demonstrate that PPE2 protein is present in the sera of mice infected with either M. smegmatis expressing PPE2 or a clinical strain of M. tuberculosis (CDC1551). It was found that exogenously added PPE2 can permeate through the macrophage cell membrane and eventually translocate into the nucleus which requires the presence of NLS which showed considerable homology to HIV-tat like cell permeable peptides. Exogenously added PPE2 could inhibit NO production and decreased mycobacterial survival in macrophages. PPE2-null mutant of M. tuberculosis failed to inhibit NO production and had poor survival in macrophages which could be rescued by complementation with full-length PPE2. PPE2-null mutants also had poor survival in the lungs of infected mice indicating that PPE2 even when present in the bloodstream can confer a survival advantage to mycobacteria.

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

Overexpression of a membrane transport system MSMEG_1381 and MSMEG_1382 confers multidrug resistance in Mycobacterium smegmatis.

Mycobacterium tuberculosis is a leading cause of human mortality worldwide, and the emergence of drug-resistant strains demands the discovery of new classes of antimycobacterial that can be employed in the therapeutic pipeline. Previously, a secondary metabolite, chrysomycin A, isolated from Streptomyces sp. OA161 displayed potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis and different species of mycobacteria. The antibiotic inhibits mycobacterial topoisomerase I and DNA gyrase, leading to bacterial death, but the mechanisms that could cause resistance to this antibiotic are currently unknown. To further understand the resistance mechanism, using M. smegmatis as a model, spontaneous resistance mutants were isolated and subjected to whole-genome sequencing. Mutation in a TetR family transcriptional regulator MSMEG_1380 was identified in the resistant isolates wherein the gene was adjacent to an operon encoding membrane proteins MSMEG_1381 and MSMEG_1382. Sequence analysis and modeling studies indicated that MSMEG_1381 and MSMEG_1382 are components of the Mmp family of efflux pumps and over-expression of either the operon or individual genes conferred resistance to chrysomycin A, isoniazid, and ethambutol. Our study highlights the role of membrane transporter proteins in conferring multiple drug resistance and the utility of recombinant strains overexpressing membrane transporters in the drug screening pipeline.

The Cytotoxic Mycobacteriophage Protein Phaedrus gp82 Interacts with and Modulates the Activity of the Host ATPase, MoxR.

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity.

A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Introduction: Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Methods: Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. Results and discussion: In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and ß-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

Development of a novel target-based cell assay, reporter of the activity of Mycobacterium tuberculosis protein-O-mannosyltransferase.

The Protein-O-mannosyltransferase is crucial for the virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This enzyme, called MtPMT (Rv1002c), is responsible for the post-translational O-mannosylation of mycobacterial proteins. It catalyzes the transfer of a single mannose residue from a polyprenol phospho-mannosyl lipidic donor to the hydroxyl groups of selected Ser/Thr residues in acceptor proteins during their translocation across the membrane. Previously, we provided evidence that the loss of MtPMT activity causes the absence of mannoproteins in Mycobacterium tuberculosis, severely impacting its intracellular growth, as well as a strong attenuation of its pathogenicity in immunocompromised mice. Therefore, it is of interest to develop specific inhibitors of this enzyme to better understand mycobacterial infectious diseases. Here we report the development of a "target-based" phenotypic assay for this enzyme, assessing its O-mannosyltransferase activity in bacteria, in the non-pathogenic Mycobacterium smegmatis strain. Robustness of the quantitative contribution of this assay was evaluated by intact protein mass spectrometry, using a panel of control strains, overexpressing the MtPMT gene, carrying different key point-mutations. Then, screening of a limited library of 30 compounds rationally chosen allowed us to identify 2 compounds containing pyrrole analogous rings, as significant inhibitors of MtPMT activity, affecting neither the growth of the mycobacterium nor its secretion of mannoproteins. These molecular cores could therefore serve as scaffold for the design of new pharmaceutical agents that could improve treatment of mycobacterial diseases. We report here the implementation of a miniaturized phenotypic activity assay for a glycosyltransferase of the C superfamily.

Septum site placement in Mycobacteria - identification and characterisation of mycobacterial homologues of Escherichia coli MinD.

A major virulence trait of Mycobacterium tuberculosis (M. tb) is its ability to enter a dormant state within its human host. Since cell division is intimately linked to metabolic shut down, understanding the mechanism of septum formation and its integration with other events in the division pathway is likely to offer clues to the molecular basis of dormancy. The M. tb genome lacks obvious homologues of several conserved cell division proteins, and this study was aimed at identifying and functionally characterising mycobacterial homologues of the E. coli septum site specification protein MinD (Ec MinD). Sequence homology based analyses suggested that the genomes of both M. tb and the saprophyte Mycobacterium smegmatis (M. smegmatis) encode two putative Ec MinD homologues - Rv1708/MSMEG_3743 and Rv3660c/ MSMEG_6171. Of these, Rv1708/MSMEG_3743 were found to be the true homologues, through complementation of the E. coli ∆minDE mutant HL1, overexpression studies, and structural comparisons. Rv1708 and MSMEG_3743 fully complemented the mini-cell phenotype of HL1, and over-expression of MSMEG_3743 in M. smegmatis led to cell elongation and a drastic decrease in c.f.u. counts, indicating its essentiality in cell-division. MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif. Interaction of Rv1708 with the chromosome associated proteins ScpA and ParB, implied a link between its septum formation role, and chromosome segregation. Comparative structural analyses showed Rv1708 to be closer in similarity to Ec MinD than Rv3660c. In summary we identify Rv1708 and MSMEG_3743 to be homologues of Ec MinD, adding a critical missing piece to the mycobacterial cell division puzzle.

Sub-operon promoter arrangement of disA facilitates c-di-AMP homeostasis and selective stress responses in Mycobacterium smegmatis.

Bacterial second messenger signaling often plays an important role in cellular physiology. In this study, we have attempted to understand how c-di-AMP synthesis and degradation are transcriptionally regulated in Mycobacterium smegmatis. We found that although a c-di-AMP synthesis gene, disA, exists in a multi-gene operon, a sub-operon promoter arrangement facilitates disA gene expression under normal conditions to maintain intracellular c-di-AMP concentration and is induced further during certain stress adaptations. Individual gene-specific promoters also play a key role under various genotoxic stress conditions to shut down c-di-AMP synthesis, which could otherwise be detrimental for cells. Further, we learned that a high c-di-AMP concentration plays a role in the autoregulation of the disA promoter to limit intracellular c-di-AMP concentration. This study was helpful to understand how c-di-AMP synthesis is regulated under normal and stress conditions linked to its physiological relevance in M. smegmatis.