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Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis , we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 is responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation and in which the connection between Wag31, PlrA, and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate the synthesis of the different layers of the mycobacterial cell wall in changing conditions., Importance: This study is performed in Mycobacterium smegmatis , which is used as a model to understand the basic physiology of pathogenic mycobacteria such as Mycobacterium tuberculosis . In this work, we examine the function and regulation of three proteins involved in regulating cell wall elongation in mycobacterial cells, which occurs at the cell tips or poles. We find that Wag31, a regulator of polar elongation, works partly through the regulation of MmpL3, a transporter of cell wall constituents and an important drug target. Our work suggests that, beyond its transport function, MmpL3 has another function in controlling cell wall synthesis broadly in response to stress., Competing Interests: The authors declare no conflict of interest.

Endolysins produced by bacteriophages hydrolyze host cell wall peptidoglycan to release newly assembled virions. D29 mycobacteriophage specifically infects mycobacteria including the pathogenic Mycobacterium tuberculosis. D29 encodes LysA endolysin, which hydrolyzes mycobacterial cell wall peptidoglycan. We previously showed that LysA harbors two catalytic domains (N-terminal domain [NTD] and lysozyme-like domain [LD]) and a C-terminal cell wall binding domain (CTD). While the importance of LD and CTD in mycobacteriophage biology has been examined in great detail, NTD has largely remained unexplored. Here, to address NTD's significance in D29 physiology, we generated NTD-deficient D29 (D29 ∆NTD ) by deleting the NTD-coding region from D29 genome using CRISPY-BRED. We show that D29 ∆NTD is viable, but has a longer latent period, and a remarkably reduced burst size and plaque size. A large number of phages were found to be trapped in the host during the D29 ∆NTD -mediated cell lysis event. Such poor release of progeny phages during host cell lysis strongly suggests that NTD-deficient LysA produced by D29 ∆NTD , despite having catalytically-active LD, is unable to efficiently lyse host bacteria. We thus conclude that LysA NTD is essential for optimal release of progeny virions, thereby playing an extremely vital role in phage physiology and phage propagation in the environment., (© 2024 John Wiley & Sons Ltd.)

Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase A1 (DesA1) is an essential Ca 2+ -binding protein that catalyses a key step in mycolic acid biosynthesis. To investigate the structural and functional significance of Ca 2+ binding, we introduced mutations at key residues in its Ca 2+ -binding βγ-crystallin motif to generate DesA1F303A, E304Q, and F303A-E304Q. Complementation of a conditional ΔdesA1 strain of Mycobacterium smegmatis, with the Ca 2+ non-binders F303A or F303A-E304Q, failed to rescue its growth phenotype; these complements also exhibited enhanced cell wall permeability. Our findings highlight the criticality of Ca 2+ in DesA1 function, and its implicit role in the maintenance of mycobacterial cellular integrity., (© 2024 Federation of European Biochemical Societies.)

Members of the order Mycobacteriales are distinguished by a characteristic diderm cell envelope, setting them apart from other Actinobacteria species. In addition to the conventional peptidoglycan cell wall, these organisms feature an extra polysaccharide polymer composed of arabinose and galactose, termed arabinogalactan. The nonreducing ends of arabinose are covalently linked to mycolic acids (MAs), forming the immobile inner leaflet of the highly hydrophobic MA membrane. The contiguous outer leaflet of the MA membrane comprises trehalose mycolates and various lipid species. Similar to all actinobacteria, Mycobacteriales exhibit apical growth, facilitated by a polar localized elongasome complex. A septal cell envelope synthesis machinery, the divisome, builds instead of the cell wall structures during cytokinesis. In recent years, a growing body of knowledge has emerged regarding the cell wall synthesizing complexes of Mycobacteriales., focusing particularly on three model species: Corynebacterium glutamicum, Mycobacterium smegmatis, and Mycobacterium tuberculosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 β-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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shaku, Ocius, Apostolos, Pires, VanNieuwenhze, Dhar and Kana.)

Mycobacterium tuberculosis has distinct cell wall composition that helps in intracellular survival of bacteria. Rv1900c, a two domain protein, has been grouped in lip gene family. The expression of rv1900c was upregulated under acidic, nutritive and iron stress conditions in M. tuberculosis H37Ra. To investigate the biological effect of Rv1900c in mycobacterium physiology, rv1900c gene was cloned in M. smegmatis, a surrogate host. Its counterpart MSMEG_4477 in M. smegmatis demonstrated 38% protein similarity with Rv1900c. MSMEG_4477 gene was knocked out in M. smegmatis by homologous recombination. rv1900c and MSMEG_4477 genes, cloned in pVV16, were expressed in the M. smegmatis knockout strain (M. smegmatis ΔMSMEG_4477). Gene knockout significantly altered colony morphology and growth kinetics of M. smegmatis. M. smegmatis ΔMSMEG_1900 (pVV16::rv1900c) colonies were less wrinkled and had smooth surface as compared to M. smegmatis ΔMSMEG_4477. The changes were reverted back to normal upon expression of MSMEG_4477 in knockout strain M. smegmatis ΔMSMEG_4477 (pVV16::MSMEG_4477). The expression of rv1900c enhanced the biofilm formation and survival of bacteria under various in vitro stresses like acidic, nutritive stress, including lysozyme, SDS and multiple antibiotics treatment in comparison to control. On the other hand the expression of rv1900c decreased the cell wall permeability. The resistance provided by M. smegmatis ΔMSMEG_4477 (pVV16::MSMEG_4477) was comparable to M. smegmatis having vector alone (MS_vec). The lipid content of M. smegmatis ΔMSMEG_1900 (pVV16::rv1900c) was observed to be different from M. smegmatis ΔMSMEG_4477 (pVV16::MSMEG_4477). M. smegmatis ΔMSMEG_1900 (pVV16::rv1900c) was more tolerant to stress conditions in comparison to M. smegmatis ΔMSMEG_4477 (pVV16::MSMEG_4477). Expression of rv1900c enhanced the intracellular survival of mycobacteria. Therefore, the present study suggested an association of Rv1900c to the stress tolerance by cell wall modification that might have resulted in enhanced intracellular survival of the mycobacteria., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in Mycobacterium smegmatis , we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. smegmatis , indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis., Competing Interests: AG, TK, CS, JP, SO, JJ, TG, YM, MS No competing interests declared

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure. IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer "myco" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens., (Copyright © 2021 Pohane et al.)

Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes cross-linked by cell wall transpeptidases (TP). While synthetic PG fragments containing l-lysine in the third position on the stem peptide are easier to access, those with meso -diaminopimelic acid ( m -DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on widely available building blocks to assemble meso -cystine ( m -CYT), which mimics key structural features of m -DAP. To demonstrate proper mimicry of m -DAP, cell wall probes were synthesized with m -CYT in place of m -DAP and evaluated for their metabolic processing in live bacterial cells. We found that m -CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m -DAP in their PG. Additionally, we have used hybrid quantum mechanical/molecular mechanical (QM/MM) and molecular dynamics (MD) simulations to explore the influence of m -DAP analogs on the PG cross-linking. The results showed that the cross-linking mechanism of transpeptidases occurred through a concerted process. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m -DAP containing organisms.

About one quarter of people worldwide are infected with tuberculosis, and multi-drug resistant tuberculosis (MDR-TB) remains a health threat. It is known that two-Component Signal Transduction Systems (TCSs) of Mycobacterium tuberculosis are closely related to tuberculosis resistance, but the mechanism by which orphan response protein Rv3143 regulates strain sensitivity to drug is still unclear. This study found that Rv3143 overexpression resulted in approximately two-fold increase in Mycobacterium smegmatis antibiotic sensitivity. Transcriptome sequencing indicated that 198 potential genes were regulated by Rv3143, affecting the sensitivity of the strain to rifampicin (RIF). MSMEG_4740 promoter binding with Rv3143, was screened out by surface plasmon resonance (SPR). Rv1524, the homologous gene of MSMEG_4740, belonging to the glycosyltransferase (Gtf) family, was related to cell wall modification. By measuring ethidium bromide (EB) accumulation, we found when Rv3143 or MSMEG_4740, or Rv1524 was overexpressed, the cell wall permeability of Mycobacterium smegmatis was increased. In addition, a combination of Rv3143 and RIF was observed. Our findings provide a new strategy for treating drug-resistant tuberculosis by increasing the expression of Rv3143 to enhance the strain sensitivity to antibiotics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Trehalose monomycolate (TMM) represents an essential element of the mycobacterial envelope. While synthesized in the cytoplasm, TMM is transported across the inner membrane by MmpL3 but, little is known regarding the MmpL3 partners involved in this process. Recently, the TMM transport factor A (TtfA) was found to form a complex with MmpL3 and to participate in TMM transport, although its biological role remains to be established. Herein, we report the crystal structure of the Mycobacterium smegmatis TtfA core domain. The phylogenetic distribution of TtfA homologues in non-mycolate containing bacteria suggests that TtfA may exert additional functions., (© 2019 Wiley Periodicals, Inc.)

Rv0518, a hypothetical protein of Mycobacterium tuberculosis, was designated as possible exported protein. In-silico analysis suggested that the protein belonged to the family of GDSL lipases. In this study, rv0518 gene was cloned and expressed in E. coli followed by purification and characterization. It possessed lipolytic activity, preferably hydrolyzed pNP-decanoate at pH 9.0 and 40 °C. The enzyme was stable till 50 °C and wider pH range (5.0-11.0). The predicted active site residues, Ser-46, Asp-205, His-208, Gly-87, Asn-120 were confirmed by site directed mutagenesis. rv0518 gene expression in M. tuberculosis H37Ra was up-regulated under nutrient starvation and the protein was detected in membrane fraction. The expression of rv0518 in M. smegmatis altered the colony morphology/growth kinetics, provided resistance to SDS, lysozyme, anti-TB drugs and enhanced in vitro survival of M. smegmatis under nutritive stress. The total lipid content and trehalose dimycolate was elevated in M. smegmatis expressing rv0518 gene. The presence of Rv0518 enhanced infection ability and intracellular survival capability of M. smegmatis. Hence, Rv0518 is a cell wall associated GDSL lipase might helped bacteria to utilize glycerol/lipids for its growth as well as provided resistance to various intracellular stresses by cell wall modulation resulting in its enhanced intracellular survival., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as an in vitro experimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenic Mycobacterium smegmatis in MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM 13-25KDa ) and LAM increased (LAM 37-100KDa ), when compared these molecules after bacteria culture in complete medium (LM 17-25KDa and LAM 37-50KDa ). These changes modified the cell surface hydrophobicity and susceptibility against H 2 O 2 . The infection of J774 macrophages with M. smegmatis, after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.

Cell wall hydrolases are enzymes that cleave bacterial cell walls by hydrolyzing specific bonds within peptidoglycan and other portions of the envelope. Two major sources of hydrolases in nature are from hosts and microbes. This study specifically investigated whether cell wall hydrolytic enzymes could be employed as exogenous reagents to augment the efficacy of antimicrobial agents against mycobacteria. Mycobacterium smegmatis cultures were treated with ten conventional antibiotics and six anti-tuberculosis drugs-alone or in combination with cell wall hydrolases. Culture turbidity, colony-forming units (CFUs), vital staining, and oxygen consumption were all monitored. The majority of antimicrobial agents tested alone only had minimal inhibitory effects on bacterial growth. However, the combination of cell wall hydrolases and most of the antimicrobial agents tested, revealed a synergistic effect that resulted in significant enhancement of bactericidal activity. Vital staining showed increased cellular damage when M. smegmatis and Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) were treated with both drug and lysozyme. Respiration analysis revealed stress responses when cells were treated with lysozyme and drugs individually, and an acute increase in oxygen consumption when treated with both drug and lysozyme. Similar trends were also observed for the other three enzymes (hydrolase-30, RipA-His 6 and RpfE-His 6 ) evaluated. These findings demonstrated that cell wall hydrolytic enzymes, as a group of biological agents, have the capability to improve the potency of many current antimicrobial drugs and render ineffective antibiotics effective in killing mycobacteria. This combinatorial approach may represent an important strategy to eliminate drug-resistant bacteria.

This study develops a flow cytometry analysis of the bacterial pathogens Escherichia coli and Staphylococcus aureus based on a ligand-bioreceptor interaction. We used fluorescently labeled plant lectins as natural receptors that could specifically interact with the cell wall carbohydrates of bacteria. An epifluorescence microscopy was used as an additional approach to confirm and visualize lectin-carbohydrate interactions. The binding specificity of plant lectins to E. coli and S. aureus cells was studied, and wheat germ agglutinin, which provided high-affinity interactions, was selected as a receptor. Using this method, bacterial pathogens can be detected in concentrations of up to 10 6  cells/mL within 5 min. Their accessibility and universality make lectin reagents a promising tool to control a wide range of bacterial pathogens.

Mycolyl-arabinogalactan-peptidoglycan (mAGP) is the major content of the mycobacterium cell wall structure and essential for mycobacterial survival. Peptidoglycan (PG) plays an important role in maintenance of cell division, cell wall integrity and pathogenesis. Mycobacterium smegmatis MSMEG_6281, a peptidoglycan amidase, is vital for mycobacterial cell division. However, the effects of MSMEG_6281on cell wall integrity and mycobacterial virulence remain unknown. In the current study, we demonstrate that MSMEG_6281gene knockout in M.smegmatis alters the microbiological characteristics. Our results revealed that MSMEG_6281gene knockout bacteria (M. sm-ΔM_6281) lost their acid-fastness, increased their sensitivity to lipophilic compounds and presented an abnormal morphology. Our results revealed that MSMEG_6281was related to maintaining the cell wall integrity. Furthermore, we investigated the effects of MSMEG_6281 inactivation on mycobacterial virulence using mice models infected by different M.smegmatis strains. MSMEG_6281 inactivation in the M sm-ΔM_6281 infected group caused less mycobacterial colonization, reduced pathological signs, decreased the anti-microbial enzymes production including iNOS and β-defensins in mouse lungs. Moreover, IL-1β and TLR2 expression were significantly down-regulated, while the production of IFN-γ and TNF-α was up-regulated. These findings indicated the diversity of host immune responses induced by different strains of M.smegmatis, suggesting that MSMEG_6281 inactivation impact mycobacterial virulence. In conclusion, the MSMEG_6281 protein plays important roles in maintaining cell wall integrity and mycobacterial virulence., (Copyright © 2019. Published by Elsevier Ltd.)

Rv1288, a conserved hypothetical protein of M. tuberculosis (M.tb) , was recently characterized as two-domain esterase enzyme by in silico study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from M.tb into E. coli for expression and purification. The purified rRv1288 and rEst proteins exhibited lipolytic activity with medium chain length esters as optimum substrates, while Lyt domain did not show enzymatic activity. However, presence of Lyt domain resulted in enhanced rate of protein aggregation at higher temperature. Both rRv1288 and rEst followed the similar patterns of substrate specificity, temperature and pH activity. Site directed mutagenesis confirmed the Ser-294, Asp-391 and His-425 as catalytic site residues. Rv1288 was found to be present in cell wall fraction of M.tb H37Ra. Peptidoglycan binding activity of Rv1288 and its domains demonstrated that the Lyt domain is essential for anchoring protein to the cell wall. Expression of rv1288 was up regulated in M.tb under nutrient starved condition. Over expression of rv1288 in surrogate host M. smegmatis led to change in colony morphology, enhanced pellicle and aggregate formation that might be linked with the changed lipid composition of bacterial cell wall. Cell wall of M. smegmatis expressing rv1288 had higher amount of lipids, with a significant increase in trehalose dimycolate content. Rv1288 also leads to increase in drug resistance of M. smegmatis . Rv1288 also enhanced the intracellular survival of M. smegmatis in Raw264.7 cell line. Overall, this study suggested that Rv1288, a cell wall localized carboxyl hydrolase with mycolyl-transferase activity, modulated the cell wall lipids to favor the survival of bacteria under stress condition.

Mycobacterial pathogens have evolved a unique secretory apparatus called the Type VII secretion system (T7SS) which comprises of five gene clusters designated as ESX1, ESX2, ESX3, ESX4, and ESX5. Of these the ESX3 T7SS plays an important role in the regulatory uptake of iron from the environment, thereby enabling the bacteria to establish successful infection in the host. However, ESX3 secretion system is conserved among all the mycobacterial species including the fast-growing nonpathogenic species M. smegmatis. Although the function of ESX3 T7SS is known to be absolutely critical for establishing infection by M. tuberculosis, its conserved nature in all the pathogenic and nonpathogenic mycobacterial species intrigues to explore the additional functional roles in Mycobacterium species through which potent targets for drugs can be identified and developed. In the present study, we investigated the possible role of EccD3, a transmembrane protein of the ESX3 T7SS in M. smegmatis by deleting the entire eccD3 gene by efficient allelic exchange method. The preliminary investigations through the creation of knockout mutant of the eccD3 gene indicate that this secretory apparatus has an important role in maintaining the cell wall integrity which was evident from the abnormal colony morphology, lack of biofilm formation and difference in cell wall permeability., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The distinctive cell walls of mycobacteria are characteristic features of these bacteria. Individual cell wall components influence diverse mycobacterial phenotypes, such as colony morphology, virulence and stress resistance. To investigate the role of the hypothetical protein Rv2387, we constructed a Mycobacterium smegmatis strain that heterologously expressed this ORF, and we observed that the M. smegmatis strain expressing Rv2387 exhibited altered colony morphology and cell wall lipid composition, leading to a marked decrease in the resistance against acidic conditions. This study demonstrates that due to its impact on cell wall remodeling, Rv2387 might play an important role in mycobacterial physiology., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Resuscitation-promoting factors (Rpfs) have previously been shown to act as growth-stimulatory molecules via their lysozyme-like activity on peptidoglycan in the bacterial cell wall. In this study, we investigated the ability of Mycobacterium smegmatis strains lacking rpf genes to form biofilms and tested their susceptibilities to cell wall-targeting agents. M. smegmatis contains four distinct rpf homologues, namely, MSMEG_5700 ( rpfA ), MSMEG_5439 ( rpfB ), MSMEG_4640 ( rpfE2 ), and MSMEG_4643 ( rpfE ). During axenic growth of the wild-type strain, all four mRNA transcripts were expressed to various degrees, but the expression of MSMEG_4643 was significantly greater during exponential growth. Similarly, all rpf mRNA transcripts could be detected in biofilms grown for 7, 14, and 28 days, with MSMEG_4643 expressed at the highest abundance after 7 days. In-frame unmarked deletion mutants (single and combinatorial) were generated and displayed altered colony morphologies and the inability to form typical biofilms. Moreover, any strain lacking rpfA and rpfB simultaneously exhibited increased susceptibility to rifampin, vancomycin, and SDS. Exogenous Rpf supplementation in the form of culture filtrate failed to restore biofilm formation. Liquid chromatography-mass spectrometry (LC-MS) analysis of peptidoglycan (PG) suggested a reduction in 4-3 cross-linked PG in the Δ rpfABEE2 mutant strain. In addition, the level of PG-repeat units terminating in 1,6-anhydroMurNAc appeared to be significantly reduced in the quadruple rpf mutant. Collectively, our data have shown that Rpfs play an important role in biofilm formation, possibly through alterations in PG cross-linking and the production of signaling molecules. IMPORTANCE The cell wall of pathogenic mycobacteria is composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer capsule. This inherent complexity renders it resistant to many antibiotics. Consequently, its biosynthesis and remodeling during growth directly impact viability. Resuscitation-promoting factors (Rpfs), enzymes with lytic transglycosylase activity, have been associated with the revival of dormant cells and subsequent resumption of vegetative growth. Mycobacterium smegmatis , a soil saprophyte and close relative of the human pathogen Mycobacterium tuberculosis , encodes four distinct Rpfs. Herein, we assessed the relationship between Rpfs and biofilm formation, which is used as a model to study drug tolerance and bacterial signaling in mycobacteria. We demonstrated that progressive deletion of rpf genes hampered the development of biofilms and reduced drug tolerance. These effects were accompanied by a reduction in muropeptide production and altered peptidoglycan cross-linking. Collectively, these observations point to an important role for Rpfs in mycobacterial communication and drug tolerance., (Copyright © 2018 American Society for Microbiology.)

Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using Mycobacterium smegmatis , we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in Escherichia coli We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31. IMPORTANCE The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli , but other proteins have critical regulatory functions that may be unique to the actinomycetes., (Copyright © 2018 American Society for Microbiology.)

M. tuberculosis is intrinsically tolerant to many antibiotics largely due to the imperviousness of its unusual mycolic acid-containing cell wall to most antimicrobials. The emergence and increasingly widespread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) revitalized keen interest in phage-inspired therapy. SWU1gp39 is a novel gene from mycobacteriophage SWU1 with unknown function. SWU1gp39 expressed in M. smegmatis conferred the host cell increased susceptibility to multiple antibiotics, including isoniazid, erythromycin, norfloxacin, ampicillin, ciprofloxacin, ofloxacin, rifampicin and vancomycin, and multiple environment stresses such as H2O2, heat shock, low pH and SDS. By using EtBr/Nile red uptake assays, WT-pAL-gp39 strain showed higher cell wall permeability than control strain WT-pAL. Moreover, the WT-pAL-gp39 strain produced more reactive oxygen species and reduced NAD(+)/NADH ratio. RNA-Seq transcriptomes of the WT-pAL-gp39 and WT-pAL revealed that the transcription of 867 genes was differentially regulated, including genes associated with lipid metabolism. Taken together, our results implicated that SWU1gp39, a novel gene from mycobacteriophage, disrupted the lipid metabolism of host and increased cell wall permeability, ultimately potentiated the efficacy of multiple antibiotics and stresses against mycobacteria.

The role of the unique proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins in the pathophysiology and virulence of Mycobacterium tuberculosis is not clearly understood. One of the PE family proteins, PE11 (LipX or Rv1169c), specific to pathogenic mycobacteria is found to be over-expressed during infection of macrophages and in active TB patients. In this study, we report that M. smegmatis expressing PE11 (Msmeg-PE11) exhibited altered colony morphology and cell wall lipid composition leading to a marked increase in resistance against various environmental stressors and antibiotics. The cell envelope of Msmeg-PE11 also had greater amount of glycolipids and polar lipids. Msmeg-PE11 was found to have better survival rate in infected macrophages. Mice infected with Msmeg-PE11 had higher bacterial load, showed exacerbated organ pathology and mortality. The liver and lung of Msmeg-PE11-infected mice also had higher levels of IL-10, IL-4 and TNF-α cytokines, indicating a potential role of this protein in mycobacterial virulence.

A series of arabinose glycosyl sulfamides with varying alkyl chain types and lengths were synthesised as mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Unprecedented conversion of the desired furanose to the thermodynamically more stable pyranose form occurred during final de-protection. Biological testing against Mycobacterium smegmatis revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which in the case of mono-substituted sulfamides was maximal for a C-10 chain., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension.

Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.

PE_PGRS proteins localize in the mycobacterial cell wall and the cell wall localization of PE_PGRS33 has been shown to be attributed to its PE domain. In this study, we expressed deletion mutants of PE_PGRS30 in Mycobacterium smegmatis to characterize the role of its domains in protein localization. It was revealed that, apart from the PE domain, the C-terminal domain present in few PE_PGRS proteins carries individual cell wall localization signals. Proteinase K sensitivity assay showed that PE_PGRS30 is exposed on the mycobacterial surface through its PGRS domain. PGRS domain was also shown to be responsible for polar localization of PE_PGRS30., (Copyright © 2014. Published by Elsevier B.V.)

We recently showed that two small membrane proteins of Mycobacterium tuberculosis, CwsA and CrgA, interact with each other, and that loss of CwsA in M. smegmatis is associated with defects in the cell division and cell wall synthesis processes. Here we show that CwsA overproduction also affected growth, cell division and cell shape of M. smegmatis and M. tuberculosis. CwsA overproduction in M. tuberculosis led to increased sensitivity to cefsulodin, a penicillin-binding protein (PBP) 1A/1B targeting beta (β) -lactam, but was unaffected by other β-lactams and vancomycin. A M. smegmatis cwsA overexpressing strain showed bulgy cells, increased fluorescent vancomycin staining and altered localization of Wag31-mCherry fusion protein. However, the levels of phosphorylated Wag31, important for optimal peptidoglycan synthesis and growth in mycobacteria, were not affected. Interestingly, CwsA overproduction in E. coli led to the formation of large rounded cells that eventually lysed whereas the overproduction of FtsZ along with CwsA reversed this phenotype. Together, our results emphasize that optimal levels of CwsA are required for regulated cell wall synthesis, hence maintenance of cell shape, and that CwsA likely interacts with and modulates the activities of other cell wall synthetic components including PBPs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The MtrAB histidine-aspartate signal transduction of mycobacteria includes the response regulator MtrA and sensor kinase MtrB. We recently showed that Mycobacterium smegmatis ΔmtrB is filamentous, defective for cell division, cell shape maintenance and shows compromised MtrA target gene expression. Interestingly, overproduction of phosphorylation competent M. tuberculosis MtrAY102C reverses the ΔmtrB mutant phenotype, although the genetic basis of phenotype reversal is unknown. Here we show that introduction of D56N mutation in MtrAY102C completely abolished its phosphorylation potential yet the double mutant protein retained a partial ability to reverse the mtrB mutant phenotype indicating that phosphorylation activity is not necessary for the function of MtrAY102C. The phosphorylation-defective MtrAD56N-Y102C protein bound its target promoters ripA and fbpB efficiently. Together, these results support a hypothesis that the gain-of-function phenotype of MtrAY102C is in part due to its ability to function as a constitutively active protein in the absence of phosphorylation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis., Results: We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain., Conclusions: The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG.

Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis. IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.

PPE represent a peculiar family of mycobacterial proteins characterized by a 180 aminoacids conserved N-terminal domain. Several PPE genes are co-transcribed with a gene encoding for a protein belonging to another family of mycobacterial specific proteins named PE. Only one PE-PPE couple has been extensively characterized so far (PE25-PPE41) and it was shown that these two proteins form a heterodimer and that this interaction is essential for PPE41 stability and translocation through the mycobacterial cell wall. In this study we characterize the PE11-PPE17 couple. In contrast with what was found for PE25-PPE41, we show that PPE17 is not secreted but surface exposed. Moreover, we demonstrate that the presence of PE11 is not necessary for PPE17 stability or for its localization on the mycobacterial surface. Finally, we show that the PPE domain of PPE17 targets the mycobacterial cell wall and that this domain can be used as a fusion partner to expose heterologous proteins on the mycobacterial surface.

Background: Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production., Results: Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility., Conclusions: Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane.

The ultrastructural functions of the electron-dense glycopeptidolipid-containing outermost layer (OL), the arabinogalactan-mycolic acid-containing electron-transparent layer (ETL), and the electron-dense peptidoglycan layer (PGL) of the mycobacterial cell wall in septal growth and constriction are not clear. Therefore, using transmission electron microscopy, we studied the participation of the three layers in septal growth and constriction in the fast-growing saprophytic species Mycobacterium smegmatis and the slow-growing pathogenic species Mycobacterium xenopi and Mycobacterium tuberculosis in order to document the processes in a comprehensive and comparative manner and to find out whether the processes are conserved across different mycobacterial species. A complete septal partition is formed first by the fresh synthesis of the septal PGL (S-PGL) and septal ETL (S-ETL) from the envelope PGL (E-PGL) in M. smegmatis and M. xenopi. The S-ETL is not continuous with the envelope ETL (E-ETL) due to the presence of the E-PGL between them. The E-PGL disappears, and the S-ETL becomes continuous with the E-ETL, when the OL begins to grow and invaginate into the S-ETL for constriction. However, in M. tuberculosis, the S-PGL and S-ETL grow from the E-PGL and E-ETL, respectively, without a separation between the E-ETL and S-ETL by the E-PGL, in contrast to the process in M. smegmatis and M. xenopi. Subsequent growth and invagination of the OL into the S-ETL of the septal partition initiates and completes septal constriction in M. tuberculosis. A model for the conserved sequential process of mycobacterial septation, in which the formation of a complete septal partition is followed by constriction, is presented. The probable physiological significance of the process is discussed. The ultrastructural features of septation and constriction in mycobacteria are unusually different from those in the well-studied organisms Escherichia coli and Bacillus subtilis.

Lipoarabinomannan (LAM) is a structurally heterogeneous amphipathic lipoglycan present in Mycobacterium spp. and other actinomycetes, which constitutes a major component of the cell wall and exhibits a wide spectrum of immunomodulatory effects. Analysis of Mycobacterium smegmatis subcellular fractions and spheroplasts showed that LAM and lipomannan (LM) were primarily found in a cell wall-enriched subcellular fraction and correlated with the presence (or absence) of the mycolic acids in spheroplast preparations, suggesting that LAM and LM are primarily associated with the putative outer membrane of mycobacteria. During the course of these studies significant changes in the LAM/LM content of the cell wall were noted relative to the age of the culture. The LAM content of the M. smegmatis cell wall was dramatically reduced as the bacilli approached stationary phase, whereas LM, mycolic acid, and arabinogalactan content appeared to be unchanged. In addition, cell morphology and acid-fast staining characteristics showed variations with growth phase of the bacteria. In the logarithmic phase, the bacteria were found to be classic rod-shaped acid-fast bacilli, while in the stationary phase M. smegmatis lost the characteristic rod shape and developed a punctate acid-fast staining pattern with carbolfuchsin. The number of viable bacteria was independent of LAM content and phenotype. Taken together, the results presented here suggest that LAM is primarily localized with the mycolic acids in the cell wall and that the cellular concentration of LAM in M. smegmatis is selectively modulated with the growth phase.

Arabinogalactan (AG) of mycobacterial cell wall consists of arabinan region, galactan region and disaccharide linker. The arabinan is composed of D-arabinofuranose residues, and decaprenyphosphoryl-D-arabinose (DPA) is the donor of the D-arabinofuranose residues. DPA is formed from phosphoribose diphosphate (PRPP) in a four-step process catalyzed by transferase, phosphatase and epimerase, respectively. Mycobacterium tuberculosis Rv3806c has been identified as PRPP: decaprenyl-phosphate 5-phosphoribosyltransferase, and heteromeric Rv3790/Rv3791 has epimerase activity. Rv3807c is putative phospholipid phosphatase. However, there is no direct biochemical evidence since expression of Rv3807c has been unsuccessful. Mycobacterium smegmatis MSMEG_6402 is ortholog of Rv3807c. To investigate the function of MSMEG_6402 on AG biosynthesis, a conditional MSMEG_6402 gene knock out (M. sm-ΔM_6402) strain was constructed through homologous recombination technique. The morphological and compositional changes of cell wall were examined in the M. sm-ΔM_6402 strain. The M. sm-ΔM_6402 strain grew at non-permissive temperature slower than that at permissive temperature, indicating that MSMEG_6402 is non-essential for growth of M. smegmatis. The change of cell shape and detectable bulging on the cell surface of M. sm-ΔM_6402 strain were observed by scanning electron microscopy, and curled as well as deformed cell wall of M. sm-ΔM_6402 strain was revealed by transmission electron microscopy. Analysis of sugar composition in the cell wall by HPLC indicated that the ratio of arabinofuran to galactofuran in M. sm-ΔM_6402 strain was changed to 1.7:1 comparing with 2:1 in the wild type. It demonstrates that the lacking MSMEG_6402 interferes the biosynthesis of arabinan. Analyzing 5' P-DPR and DPR from both M. sm-ΔM_6402 strain and wild type M. smegmatis is undergoing in this lab., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Active efflux systems and reduced cell-wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. In this study, we have compared the Mycobacterium smegmatis wild-type strain mc2155 with knockout mutants for porins MspA (the main porin of M. smegmatis) and MspC, the efflux pump LfrA (the main efflux pump system of M. smegmatis) and its repressor LfrR for their ability to transport ethidium bromide (EtBr) on a real-time basis. This information was then correlated with minimum inhibitory concentrations (MICs) of several antibiotics in the presence or absence of the efflux inhibitors chlorpromazine, thioridazine and verapamil., Results: In the absence of porins MspA and MspC, accumulation of ethidium bromide decreased and the cells became more resistant to several antibiotics, whereas the knockout mutant for the LfrA pump showed increased accumulation of EtBr and increased susceptibility to EtBr, rifampicin, ethambutol and ciprofloxacin. Moreover, the efflux inhibitors caused a reduction of the MICs of streptomycin, rifampicin, amikacin, ciprofloxacin, clarithromycin and erythromycin in most of the strains tested., Conclusions: The methodology used in this study demonstrated that porin MspA plays an important role in the influx of quaternary ammonium compounds and antibiotics and that efflux via the LfrA pump is involved in low-level resistance to several antimicrobial drugs in M. smegmatis. The results obtained with this non-pathogenic mycobacterium will be used in future studies as a model for the evaluation of the activity of the same efflux inhibitors on the susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to isoniazid and rifampicin.

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6'-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual beta-(1-->4) and beta-(1-->5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.

The PE family of Mycobacterium tuberculosis includes 98 proteins which share a highly homologous N-terminus sequence of about 110 amino acids (PE domain). Depending on the C-terminal domain, the PE family can be divided in three subfamilies, the largest of which is the PE_PGRS with 61 members. In this study, we determined the cellular localization of three PE proteins by cell fractionation and immunoelectron microscopy by expressing chimeric epitope-tagged recombinant proteins in Mycobacterium smegmatis. We demonstrate that the PE domain of PE_PGRS33 and PE11 (a protein constituted by the only PE domain) contains the information necessary for cell wall localization, and that they can be used as N-terminal fusion partners to deliver a sufficiently long C-terminus-linked protein domain on the mycobacterial cell surface. Indeed, we demonstrate that PE_PGRS33 and Rv3097c (a lipase belonging to the PE family) are surface exposed and localize in the mycobacterial cell wall. Moreover, we found that PE_PGRS33 is easily extractable by detergents suggesting its localization in the mycobacterial outer membrane. Beyond defining the cellular localization of these proteins, and a function for their PE domains, these data open the interesting possibility to construct recombinant mycobacteria expressing heterologous antigens on their surface for vaccine purposes.

Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.

To study the late events of cell wall assembly in Mycobacterium smegmatis, specific in vivo radiolabelling of exponentially growing liquid cultures over periods of less than one cell generation were carried out. N-Acetyl-[(14)C]glucosamine was used to label peptidoglycan and [(14)C]glucose to label arabinogalactan and arabinomannan. Over periods of several generations, radioactive cell wall material was turned over as soluble autolysis products into the culture fluid. However, turnover of newly synthesized and labelled cell wall was delayed for about one cell generation, implying inside-to-outside growth of the wall as observed in Bacillus. Little radioactive wall material was released into the culture fluid during the first generation of labelling in growing cultures, but the addition of amoxicillin plus the beta-lactamase inhibitor clavulanic acid, at the minimum inhibitory concentration of amoxicillin, led to the release of radioactive peptidoglycan that could be isolated by gel filtration chromatography and contained nearly 3 mol alanine per glutamic acid residue, indicating that it was linear, un-crosslinked peptidoglycan that had never been substantially cross-linked to the cell wall due to inhibition of transpeptidation by amoxicillin. This peptidoglycan had no covalently attached arabinogalactan. Radioactive arabinogalactan was synthesized and released from the amoxicillin-treated bacteria without attachment to peptidoglycan. The results indicate that during growth, incorporation of arabinogalactan into the cell wall requires its ligation to newly synthesized peptidoglycan and that the peptidoglycan must be undergoing concomitant cross-linking to the inner surface of the cell wall. Inhibition of peptidoglycan transpeptidation prevents ligation of arabinogalactan to peptidoglycan and its consequent incorporation into the wall.

d-Serine inhibited the growth of Mycobacterium smegmatis and induced the morphological alteration of the bacilli. The growth inhibitory action of d-serine was partially reduced by an equimolecular concentration of d-alanine. The combination of glycine with d-alanine reversed the growth inhibition produced by d-serine more than did d-alanine alone. In cells cultured in the presence of d-serine, the amounts of alanine, diaminopimelic acid, and glycine inserted into the cell wall mucopeptide were reduced, and serine was increased. The intracellular accumulation of a precursor of cell wall mucopeptide was increased by d-serine, and this accumulation was reduced by d-alanine. d-Serine competed with glycine for incorporation into the cell wall mucopeptide. The incorporation of l-aspartic acid into diaminopimelic acid residues in the cell wall mucopeptide was markedly inhibited by d-serine. Three mutants resistant to d-serine were isolated by nitrosoguanidine treatment. In these mutants the effects of d-serine on the sites of cell wall mucopeptide synthesis were all reduced. Thus, d-serine inhibition of the growth is due to replacement of glycine residues of the cell wall mucopeptide with d-serine and inhibition of the cell wall synthesis by blocking the formation of d-alanine and diaminopimelic acid.