scholarly journals Genome-wide identification of novel genes involved in Corynebacteriales cell envelope biogenesis using Corynebacterium glutamicum as a model

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0240497
Author(s):  
Célia de Sousa-d’Auria ◽  
Florence Constantinesco-Becker ◽  
Patricia Constant ◽  
Maryelle Tropis ◽  
Christine Houssin
Keyword(s):  
Cell Wall ◽  
Corynebacterium Glutamicum ◽  
Acid Metabolism ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Bioinformatics Analyses ◽  
Mycolic Acids ◽  
Genome Wide ◽  
Altered Cell ◽  
Covalent Complex

Corynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-cell wall antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.

scholarly journals Genome-wide identification of novel genes involved in Corynebacteriales cell envelope biogenesis using Corynebacterium glutamicum as a model

2020 ◽  
Author(s):  
Célia de Sousa-d’Auria ◽  
Florence Constantinesco-Becker ◽  
Patricia Constant ◽  
Maryelle Tropis ◽  
Christine Houssin
Keyword(s):  
Cell Wall ◽  
Corynebacterium Glutamicum ◽  
Acid Metabolism ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Bioinformatics Analyses ◽  
Mycolic Acids ◽  
Genome Wide ◽  
Altered Cell ◽  
Covalent Complex

AbstractCorynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-arabinogalactan antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.

scholarly journals Two Accessory Proteins Govern MmpL3 Mycolic Acid Transport in Mycobacteria

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Allison Fay ◽  
Nadine Czudnochowski ◽  
Jeremy M. Rock ◽  
Jeffrey R. Johnson ◽  
Nevan J. Krogan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Complex Structure ◽  
Mycolic Acid ◽  
Accessory Proteins ◽  
Acid Transport ◽  
Content Type ◽  
Mycolic Acids ◽  
Antibiotic Target

ABSTRACT Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named “TMM transport factor A”, TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis. IMPORTANCE The cell envelope of Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.

scholarly journals Arylamine N-Acetyltransferase Is Required for Synthesis of Mycolic Acids and Complex Lipids in Mycobacterium bovis BCG and Represents a Novel Drug Target

2004 ◽  
Vol 199 (9) ◽  
pp. 1191-1199 ◽  
Author(s):  
Sanjib Bhakta ◽  
Gurdyal S. Besra ◽  
Anna M. Upton ◽  
Tanya Parish ◽  
Carolyn Sholto-Douglas-Vernon ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Bovis ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Phenotypic Traits ◽  
Mycobacterium Bovis Bcg ◽  
Mycolic Acids ◽  
Altered Cell ◽  
Novel Target ◽  
Unique Cell

Mycolic acids represent a major component of the unique cell wall of mycobacteria. Mycolic acid biosynthesis is inhibited by isoniazid, a key frontline antitubercular drug that is inactivated by mycobacterial and human arylamine N-acetyltransferase (NAT). We show that an in-frame deletion of Mycobacterium bovis BCG nat results in delayed entry into log phase, altered morphology, altered cell wall lipid composition, and increased intracellular killing by macrophages. In particular, deletion of nat perturbs biosynthesis of mycolic acids and their derivatives and increases susceptibility of M. bovis BCG to antibiotics that permeate the cell wall. Phenotypic traits are fully complemented by introduction of Mycobacterium tuberculosis nat. We infer from our findings that NAT is critical to normal mycolic acid synthesis and hence other derivative cell wall components and represents a novel target for antituberculosis therapy. In addition, this is the first report of an endogenous role for NAT in mycobacteria.

scholarly journals The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan

2020 ◽  
Vol 295 (15) ◽  
pp. 5110-5123 ◽  
Author(s):  
Lin Shen ◽  
Albertus Viljoen ◽  
Sydney Villaume ◽  
Maju Joe ◽  
Iman Halloum ◽  
...  
Keyword(s):  
Cell Wall ◽  
Structural Changes ◽  
Cell Envelope ◽  
Wall Structure ◽  
Tubercle Bacillus ◽  
Bioinformatics Analyses ◽  
The Past ◽  
Biochemical Assays ◽  
Mycobacterial Protein

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

scholarly journals Redundant Function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis Cyclopropanation of Oxygenated Mycolates

2010 ◽  
Vol 192 (14) ◽  
pp. 3661-3668 ◽  
Author(s):  
Daniel Barkan ◽  
Vivek Rao ◽  
George D. Sukenick ◽  
Michael S. Glickman
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Genetic Analysis ◽  
Biosynthetic Pathway ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Acid Modification ◽  
Mycolic Acids ◽  
Powerful Approach ◽  
Long Chain

ABSTRACT The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2, mmaA2 and cmaA1, or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.

scholarly journals MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

2019 ◽  
Vol 116 (23) ◽  
pp. 11241-11246 ◽  
Author(s):  
Chih-Chia Su ◽  
Philip A. Klenotic ◽  
Jani Reddy Bolla ◽  
Georgiana E. Purdy ◽  
Carol V. Robinson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Native Mass Spectrometry ◽  
Mycolic Acids ◽  
Mycobacterial Cell ◽  
Exact Function ◽  
Species Specific ◽  
Mycobacterial Cell Wall ◽  
Trehalose Monomycolate ◽  
Lipid Transporter

The cell envelope ofMycobacterium tuberculosisis notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure ofMycobacterium smegmatisMmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

scholarly journals Carbon Source-Induced Modifications in the Mycolic Acid Content and Cell Wall Permeability of Rhodococcus erythropolis E1

2003 ◽  
Vol 69 (12) ◽  
pp. 7019-7027 ◽  
Author(s):  
Ivana Sokolovská ◽  
Raoul Rozenberg ◽  
Christophe Riez ◽  
Paul G. Rouxhet ◽  
Spiros N. Agathos ◽  
...  
Keyword(s):  
Cell Wall ◽  
Carbon Source ◽  
Acid Content ◽  
Rhodococcus Erythropolis ◽  
Mycolic Acid ◽  
Mycolic Acids ◽  
Carbon Chains ◽  
Linear Alkanes ◽  
Wall Permeability ◽  
Cell Wall Permeability

ABSTRACT The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.

scholarly journals The Assessment of Viability of M. Tuberculosis after Exposure to CPC Using Different Methods

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Gomathi Sekar ◽  
R. Lakshmi ◽  
N. Selvakumar
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Metabolic Activity ◽  
Acid Content ◽  
Reference Strain ◽  
Vital Staining ◽  
Mycolic Acid ◽  
Viable Count ◽  
Mycolic Acids ◽  
Minimal Reduction

Settings. National Institute for Research in Tuberculosis, Chennai. Objective. To assess the proportion of metabolically active cells of Mycobacterium tuberculosis after exposed to CPC using FDA-EB vital staining and viable counts on LJ medium. Mycolic acid content in M. tuberculosis after exposure to CPC was estimated using HPLC. Methods. Clinical isolates of M. tuberculosis and standard reference strain M. tuberculosis H37Rv were used for FDA-EB, viable count, and HPLC. Results. FDA/EB consistently stained 70–90% of log phase cells as green and the remaining cells as red-orange. After CPC treatment, 65–70% of the cells stained red-orange. The viability counts were comparable to 0-day controls. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms. Conclusion. The cells are metabolically inactive during storage with CPC but these cells grew well on LJ medium after removal of CPC. The viability of M. tuberculosis was maintained in CPC with minimal reduction. Mycolic acid content was reduced if the cells of M. tuberculosis were treated with CPC for 7 days. All the above findings provide yet another evidence for the damage of cell wall of M. tuberculosis.

scholarly journals Genome sequencing of the bacteriophage CL31 and interaction with the host strain Corynebacterium glutamicum ATCC 13032

2021 ◽  
Author(s):  
Max Hünnefeld ◽  
Ulrike Viets ◽  
Vikas Sharma ◽  
Astrid Wirtz ◽  
Aël Hardy ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Genome Sequencing ◽  
Cell Envelope ◽  
Gc Content ◽  
Model Organism ◽  
Proteome Analysis ◽  
Mycolic Acid ◽  
Host Strain ◽  
Modification System

AbstractIn this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4 %). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage Φ16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the Δresmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

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