ABSTRACT: (Coryno)mycolate is a α-branched, β-hydroxylated long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. It forms an outer membrane and functions as a permeability barrier conferring pathogenic mycobacteria to resistance to antibiotics. Whereas mycolate biosynthetic pathway has been intensively studied, the studies on the transcriptional regulation of genes involved in the pathway are limited. Here, we report that the previously uncharacterized extracytoplasmic function σ factor, σD, is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD-binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprises acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase, and mycolyltransferases, all of which are involved in mycolate synthesis. Actually, deletion or overexpression of sigD encoding σD modified the amount of extractable mycolate. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD-dependent promoter. Another σD regulon member, L,D-transpeptidase, conferred lysozyme resistance. Thus, σD modifies cell wall composition and enhances mycolate synthesis to provide resistance to environmental stress.
ORGANISM(S): Corynebacterium glutamicum Corynebacterium glutamicum R
Project description:(Coryno)mycolate is a α-branched, β-hydroxylated long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. It forms an outer membrane and functions as a permeability barrier conferring pathogenic mycobacteria to resistance to antibiotics. Whereas mycolate biosynthetic pathway has been intensively studied, the studies on the transcriptional regulation of genes involved in the pathway are limited. Here, we report that the previously uncharacterized extracytoplasmic function σ factor, σD, is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD-binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprises acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase, and mycolyltransferases, all of which are involved in mycolate synthesis. Actually, deletion or overexpression of sigD encoding σD modified the amount of extractable mycolate. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD-dependent promoter. Another σD regulon member, L,D-transpeptidase, conferred lysozyme resistance. Thus, σD modifies cell wall composition and enhances mycolate synthesis to provide resistance to environmental stress.
Project description:(Coryno)mycolate is a α-branched, β-hydroxylated long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. It forms an outer membrane and functions as a permeability barrier conferring pathogenic mycobacteria to resistance to antibiotics. Whereas mycolate biosynthetic pathway has been intensively studied, the studies on the transcriptional regulation of genes involved in the pathway are limited. Here, we report that the previously uncharacterized extracytoplasmic function σ factor, σD, is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD-binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprises acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase, and mycolyltransferases, all of which are involved in mycolate synthesis. Actually, deletion or overexpression of sigD encoding σD modified the amount of extractable mycolate. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD-dependent promoter. Another σD regulon member, L,D-transpeptidase, conferred lysozyme resistance. Thus, σD modifies cell wall composition and enhances mycolate synthesis to provide resistance to environmental stress.
Project description:(Coryno)mycolate is a α-branched, β-hydroxylated long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. It forms an outer membrane and functions as a permeability barrier conferring pathogenic mycobacteria to resistance to antibiotics. Whereas mycolate biosynthetic pathway has been intensively studied, the studies on the transcriptional regulation of genes involved in the pathway are limited. Here, we report that the previously uncharacterized extracytoplasmic function σ factor, σD, is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD-binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprises acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase, and mycolyltransferases, all of which are involved in mycolate synthesis. Actually, deletion or overexpression of sigD encoding σD modified the amount of extractable mycolate. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD-dependent promoter. Another σD regulon member, L,D-transpeptidase, conferred lysozyme resistance. Thus, σD modifies cell wall composition and enhances mycolate synthesis to provide resistance to environmental stress.
Project description:(Coryno)mycolate is a α-branched, β-hydroxylated long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. It forms an outer membrane and functions as a permeability barrier conferring pathogenic mycobacteria to resistance to antibiotics. Whereas mycolate biosynthetic pathway has been intensively studied, the studies on the transcriptional regulation of genes involved in the pathway are limited. Here, we report that the previously uncharacterized extracytoplasmic function σ factor, σD, is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD-binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprises acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase, and mycolyltransferases, all of which are involved in mycolate synthesis. Actually, deletion or overexpression of sigD encoding σD modified the amount of extractable mycolate. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD-dependent promoter. Another σD regulon member, L,D-transpeptidase, conferred lysozyme resistance. Thus, σD modifies cell wall composition and enhances mycolate synthesis to provide resistance to environmental stress.
Project description:(Coryno)mycolate is a α-branched, β-hydroxylated long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. It forms an outer membrane and functions as a permeability barrier conferring pathogenic mycobacteria to resistance to antibiotics. Whereas mycolate biosynthetic pathway has been intensively studied, the studies on the transcriptional regulation of genes involved in the pathway are limited. Here, we report that the previously uncharacterized extracytoplasmic function σ factor, σD, is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD-binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprises acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase, and mycolyltransferases, all of which are involved in mycolate synthesis. Actually, deletion or overexpression of sigD encoding σD modified the amount of extractable mycolate. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD-dependent promoter. Another σD regulon member, L,D-transpeptidase, conferred lysozyme resistance. Thus, σD modifies cell wall composition and enhances mycolate synthesis to provide resistance to environmental stress.
Project description:Acclimation of cyanobacterium Synechocystis sp. PCC6803 to suboptimal conditions is largely dependent on adjustments of gene expression, which is highly controlled by the σ factor subunits of RNA polymerase (RNAP). The SigB and SigD σ factors are close homologues. Here we show that sigB and sigD genes are both induced in bright light and high temperature stresses. Comparison of transcriptomes of the control strain (CS), ΔsigB, ΔsigD, ΔsigBCE (SigD is an only functional group 2 σ factor), and ΔsigCDE (SigD is an only functional group 2 σ factor) strains in standard, bright light and high temperature conditions revealed that the SigB and SigD factors regulate different set of genes, and that SigB and SigD regulons are highly dependent on stress conditions. The SigB regulon is bigger than the SigD regulon at high temperature, whereas in bright light the SigD regulon is bigger the SigB regulon. Furthermore, our results show that favoring the SigB or SigD factor by deleting other group 2 σ factors do not lead to superior acclimation to bright light or high temperature conditions, indicating that all group 2 σ factors play roles in acclimation processes.
Project description:In this study, disruption and overexpression of sigD were performed in Corynebacterium glutamicum and analyzed by transcriptome sequencing (RNA-seq) to understand the SigD regulon in C. glutamicum. For the effect of sigD overexpression, the relative abundance of mRNA was compared in WT(pVWEx1-sigD) without IPTG or with 50 M of IPTG. For the effect of sigD disruption, the abundance was compared between the sigD disrupted mutant and the wild type strain.
Project description:Bacteria generally possess multiple σ factors that, based on structural and functional similarity, divide into two families: σD and σN. Among the seven σ factors in Escherichia coli, six belongs to the σD family. Each σ factor recognizes a group of promoters, providing effective control of differential gene expression. Many studies have shown that σ factors of the σD family compete with each other for function. In contrast, the competition between σN and σD families has yet to be fully explored. Here we report a global antagonistic effect on gene expression between two alternative σ factors, σN (RpoN) and σS (RpoS), a σD family protein. Mutations in rpoS and rpoN inversely affected a number of cellular traits, such as expression of flagellar genes, σN-controlled growth on poor nitrogen sources, and σS-directed expression of acid phosphatase AppA. Transcriptome analysis reveals that 40% of genes in the RpoN regulon were under reciprocal RpoS control. Furthermore, loss of RpoN led to increased levels of RpoS, while RpoN levels were unaffected by rpoS mutations. Expression of the flagellar σF factor (FliA), another σD family protein, was controlled positively by RpoN but negatively by RpoS. These findings unveil a complex regulatory interaction among σN, σS and σF, and underscore the need to employ systems biology approaches to assess the effect of such interaction of σ factors on cellular functions, including motility, nutrient utilization, and stress response.
Project description:Bacteria generally possess multiple σ factors that, based on structural and functional similarity, divide into two families: σD and σN. Among the seven σ factors in Escherichia coli, six belongs to the σD family. Each σ factor recognizes a group of promoters, providing effective control of differential gene expression. Many studies have shown that σ factors of the σD family compete with each other for function. In contrast, the competition between σN and σD families has yet to be fully explored. Here we report a global antagonistic effect on gene expression between two alternative σ factors, σN (RpoN) and σS (RpoS), a σD family protein. Mutations in rpoS and rpoN inversely affected a number of cellular traits, such as expression of flagellar genes, σN-controlled growth on poor nitrogen sources, and σS-directed expression of acid phosphatase AppA. Transcriptome analysis reveals that 40% of genes in the RpoN regulon were under reciprocal RpoS control. Furthermore, loss of RpoN led to increased levels of RpoS, while RpoN levels were unaffected by rpoS mutations. Expression of the flagellar σF factor (FliA), another σD family protein, was controlled positively by RpoN but negatively by RpoS. These findings unveil a complex regulatory interaction among σN, σS and σF, and underscore the need to employ systems biology approaches to assess the effect of such interaction of σ factors on cellular functions, including motility, nutrient utilization, and stress response. Precise deletion mutants of rpoS or rpoN of MG1655 were constructed and employed in this study. Cultures were inoculated in triplicate in M9 minimal media (0.2% glucose) at a starting OD of 0.0001 and grown aerobically at 37C. Cultures were harvested at OD600 = 0.3 in exponential phase. For RNA extraction, cultures were mixed directly with a boiling lysis buffer containing SDS and EDTA followed by acidic hot phenol to minimize RNA degradation. RNA samples were hybridized to Affymetrix E. coli Genome 2.0 Array according to Affymetrix's standard protocols.
Project description:Mycobacterium tuberculosis has a complex cell envelope that is remodelled throughout infection to respond and survive the hostile and variable intracellular conditions within the host. Despite the importance of cell wall homeostasis in pathogenicity, little is known about the environmental signals and regulatory networks controlling cell wall biogenesis in mycobacteria. The mycolic acid desaturase regulator (MadR) is a transcriptional repressor responsible for regulation of the essential aerobic desaturases desA1 and desA2 that are differentially regulated throughout infection along with mycolate modification genes and thus, likely involved in mycolic acid remodelling. Here we generated a madR null mutant in M. smegmatis that exhibited traits of an impaired cell wall with increased permeability, susceptibility to rifampicin and cell surface disruption as a consequence of desA1/desA2 dysregulation. Analysis of mycolic acids revealed the presence of a highly desaturated mycolate in the null mutant that exists in relative trace amounts in the wildtype, but increases in abundance upon cell surface disruption as a result of relieved repression on the desA1/desA2 promoters. Transcriptomic profiling confirmed MadR as a cell surface disruption responsive regulator of desA1/desA2 and further implicating it in the control of bespoke β-oxidation pathways and transport evolutionarily diversified subnetworks associated with virulence. In vitro characterisation of MadR using electromobility shift assays and analysis of binding affinities is suggestive of a unique acyl-CoA pool sensing mechanism, whereby MadR is able to bind a range of acyl-CoA but MadR repression of desA1/desA2 promoters is only relieved upon binding of saturated acyl-CoA of chain length C16-C24. We propose this acyl effector ligand mechanism as distinct to other regulators of mycolic acid biosynthesis or fatty acid desaturases and places MadR as the key regulatory checkpoint that coordinates mycolic acid remodelling in response to host derived cell surface perturbation