Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate, a component of the mycolic acid layer of the cell envelope

2003 ◽  
Vol 180 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Sven Brand ◽  
Karsten Niehaus ◽  
Alfred P�hler ◽  
J�rn Kalinowski
Keyword(s):  
Functional Analysis ◽  
Corynebacterium Glutamicum ◽  
Cell Envelope ◽  
Mycolic Acid

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.

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 Genome Sequence of the Bacteriophage CL31 and Interaction with the Host Strain Corynebacterium glutamicum ATCC 13032

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 495
Author(s):  
Max Hünnefeld ◽  
Ulrike Viets ◽  
Vikas Sharma ◽  
Astrid Wirtz ◽  
Aël Hardy ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Cell Envelope ◽  
Gc Content ◽  
Model Organism ◽  
Proteome Analysis ◽  
Distance Matrix ◽  
Mycolic Acid ◽  
Host Strain ◽  
Modification System

In 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 ∆resmod 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.

scholarly journals Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria

Open Biology ◽  
2017 ◽  
Vol 7 (7) ◽  
pp. 170087 ◽  
Author(s):  
Yi Ting Tsai ◽  
Valentina Salzman ◽  
Matías Cabruja ◽  
Gabriela Gago ◽  
Hugo Gramajo
Keyword(s):  
Cell Envelope ◽  
Phospholipid Composition ◽  
Mycolic Acid ◽  
Transcriptional Level ◽  
Direct Role ◽  
Conditional Mutant ◽  
In The Wild ◽  
Fas Ii

One of the dominant features of the biology of Mycobacterium tuberculosis , and other mycobacteria, is the mycobacterial cell envelope with its exceptional complex composition. Mycolic acids are major and very specific components of the cell envelope and play a key role in its architecture and impermeability. Biosynthesis of mycolic acid (MA) precursors requires two types of fatty acid synthases, FAS I and FAS II, which should work in concert in order to keep lipid homeostasis tightly regulated. Both FAS systems are regulated at their transcriptional level by specific regulatory proteins. FasR regulates components of the FAS I system, whereas MabR and FadR regulate components of the FAS II system. In this article, by constructing a tight mabR conditional mutant in Mycobacterium smegmatis mc 2 155, we demonstrated that sub-physiological levels of MabR lead to a downregulation of the fasII genes, inferring that this protein is a transcriptional activator of the FAS II system. In vivo labelling experiments and lipidomic studies carried out in the wild-type and the mabR conditional mutant demonstrated that under conditions of reduced levels of MabR, there is a clear inhibition of biosynthesis of MAs, with a concomitant change in their relative composition, and of other MA-containing molecules. These studies also demonstrated a change in the phospholipid composition of the membrane of the mutant strain, with a significant increase of phosphatidylinositol. Gel shift assays carried out with MabR and P fasII as a probe in the presence of different chain-length acyl-CoAs strongly suggest that molecules longer than C 18 can be sensed by MabR to modulate its affinity for the operator sequences that it recognizes, and in that way switch on or off the MabR-dependent promoter. Finally, we demonstrated the direct role of MabR in the upregulation of the fasII operon genes after isoniazid treatment.

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 Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

2015 ◽  
Vol 197 (24) ◽  
pp. 3797-3811 ◽  
Author(s):  
Stevie Jamet ◽  
Yves Quentin ◽  
Coralie Coudray ◽  
Pauline Texier ◽  
Françoise Laval ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Stringent Response ◽  
Mycolic Acid ◽  
Content Type ◽  
New Targets ◽  
Key Enzyme ◽  
Unique Cell ◽  
Translational Apparatus

ABSTRACTMycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that thehadA,hadB, andhadCgenes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of thehadABCgenes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of thehadABCoperon with genes of the translational apparatus and with genes required for the modification of the mycolates.IMPORTANCEMycobacterium tuberculosisinfects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response.M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight againstM. tuberculosis.

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