Results and discussion Spontaneous mutants of M. Both are the long-sought beta-hydroxyacyl-ACP dehydratases in the fatty acid elongation cycle. In previous studies, we have shown in Corynebacterium glutamicum that MSH is engaged in redox regulation and thiol-protection of proteins under hypochlorite stress by the formation of mixed disulfides, termed as S-mycothiolations 9. Palmitoyl-CoA allosterically inactivates it. The redox-sensitive low molecular weight protein-tyrosine-phosphatase PtpA showed Zur is active as transcriptional repressor in the Zn-bound form, while Zn-deficiency leads to Zur inactivation and derepression of its regulon consisting of Zn-transporters, Zn-containing ribosomal proteins and the immunodominant ESAT-6 proteins
Zn, iron or FeS-clusters. A The treemap legend shows the classification of the M. Using RNA-seq transcriptomics, the regulatory impact of thiol-oxidation of NaOCl-sensitive transcription factors on the changes in gene expression was analyzed.
S-mycothiolated proteins include also antioxidant enzymes, such as peroxiredoxins Tpx, Mpx and methionine sulfoxide reductases MsrA. DNA sequence analysis of targeted genes in these seven strains revealed the absence of mutations in the genes known to mediate co-resistance to INH and ETH, namely inhA the gene or its promoter region and ndh. The minimal inhibitory concentration MIC of platensimycin against M. Platensimycin selectively inhibits cellular lipid biosynthesis. The WHO estimates that 4. Using RNA-seq transcriptomics, the regulatory impact of thiol-oxidation of NaOCl-sensitive transcription factors on the changes in gene expression was analyzed.
Brown, , unpublished data. One potential complementing cosmid was isolated, sequenced and shown to contain the mshA gene, a gene characterized as mediating the first step in the biosynthesis of mycothiol Newton et al. Thus, S-cysteinlyation cannot compensate for the loss of MSH in protein protection and redox-regulation. AhpC has been shown to function as peroxidase and peroxinitrite reductase together with AhpD, the dihydrolipoamide succinyl transferase SucD and the NADH-dependent dihydrolipoamide dehydrogenase Lpd , thus linking the antioxidant response to regeneration of important enzymes of the intermediary metabolism Both are the long-sought beta-hydroxyacyl-ACP dehydratases in the fatty acid elongation cycle.
In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Zn-containing ribosomal proteins are suggested to serve as reservoir for Zn-storage The redox-sensitive low molecular weight protein-tyrosine-phosphatase PtpA showed A phenylalanine residue blocks this channel in ecFabH, and constrains specificity to acetyl-CoA while in mtFabH there is a threonine which permits longer acyl chains to bind Scarsdale et al. Rv is a target for dehydratase inhibitors Bhowruth et al. Compared with the wild-type MabA, the TD mutant dramatically decreased ketoacyl reductase activity and significantly inhibited the de novo biosynthesis of mycolic acids Veyron-Churlet et al.
Brown, , unpublished data. Compared with the wild-type MabA, the TD mutant dramatically decreased ketoacyl reductase activity and significantly inhibited the de novo biosynthesis of mycolic acids Veyron-Churlet et al. Thus, humans can make oleic acid, but cannot synthesis linoleic acid or linolenic acid. Biochemical studies suggested that the mechanism of ethionamide resistance in mshA mutants was likely due to a defect in ethionamide activation.
Thus, M. In bacteria, these activities are found on separate enzymes and are not part of a complex. A minor part of Cys residues UmaA is S-mycothiolated at the conserved Cys76 that is required for S-adenosylmethionine binding. Non-modified proteins are colored in grey and S-mycothiolated proteins that were not identified using the OxICAT approach are shown in pink.
Desaturation of Fatty Acids Fatty acids are synthesized in the saturated form and desaturation occurs later.
In contrast to the hydroxylated intermediate of beta oxidation, the beta intermediate here is in the D-configuration. Desaturation of Fatty Acids Fatty acids are synthesized in the saturated form and desaturation occurs later. Figure 3 Schematics of the glycerol catabolism, glycolysis, TCA cycle, glyoxylate cycle and gluconeogenesis in M. However, most Gram-positive bacteria do not produce GSH 1. Both GuaB homologs are S-mycothiolated at their conserved active sites, Cys and Cys, respectively, that form the thioimidate intermediate Fig.
Light and heavy ICAT-labeled peptide pairs show a mass difference of 9 Da after separation using mass spectrometry. This analysis provided the evidence that these strains possessed mutations that conferred INH and ETH resistance and had not been previously identified in M. Precise null deletion mutations of the mshA gene were generated by specialized transduction in three different strains of M.
In the wild-type control, Cys residues B The total spectral counts determine the cell size of each protein identified in the proteome dataset and classified according to TIGRfam. Thus, the central carbon metabolism and the fatty acid biosynthesis pathways include many NaOCl-sensitive proteins that are important for cellular survival in mycobacteria.
Results and discussion Spontaneous mutants of M. In previous studies, we have identified S-cysteinylated proteins in the absence of bacillithiol and mycothiol 9 , Contributors Synthesis of fatty acids occurs in the cytoplasm and endoplasmic reticulum of the cell and is chemically similar to the beta-oxidation process , but with a couple of key differences. Figure 1 M. Samples of three independent M.