Mycofactocin is associated with ethanol metabolism in Mycobacteria
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PROVIDER: PRJNA497261 | ENA |
REPOSITORIES: ENA
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Project description:Mycobacterium tuberculosis (Mtb) assimilates cholesterol during chronic infection, and its in vitro growth in presence of cholesterol requires Mycofactocin (MFT) biosynthesis genes, although the basis of this requirement is unclear. MFT belongs to the class of ribosomally synthesized and post-translationally modified peptides conserved in many Actinobacteria, and its biological function and structure requires further confirmation. To identify the function of MFT, we characterized mft gene deletion mutants constructed in M. smegmatis, M. marinum and Mtb. We found that the growth deficit of mft deletion mutants in cholesterol – a phenotypic basis for gene essentiality prediction – is ethanol-dependent. Furthermore, functionality of MFT was strictly required for mycobacterial growth in ethanol, implicating its role in ethanol metabolism. Although ethanol is a poor growth substrate, it perturbed the cellular metabolism profoundly. Transcriptional analysis revealed that the disruption of MFT caused respiration dysfunction and associated redox imbalance, which is proposed as an underlying mechanism of growth retardation of MFT mutants in cholesterol. Finally, MSMEG_6242 was found to be indispensable for ethanol assimilation and is a candidate catalytic interactor with MFT. The function of MFT appears to resemble pyrroloquinoline quinone cofactor, similar to their biosynthetic steps. While our results serve as a salutary reminder that gene essentiality is dependent on a context in which it is probed, they also establish MFT as a redox cofactor, along with flavin or nicotinamide, to enable the function of its dependent enzymes in mycobacteria.
2019-04-25 | GSE121398 | GEO
Project description:Mycofactocin is a redox cofactor essential for the alcohol metabolism of Mycobacteria. While the biosynthesis of mycofactocin is well established, the mftG gene, which encodes an oxidoreductase of the glucose-methanol-choline superfamily remained functionally uncharacterized. Here, we show that MftG enzymes strictly require mft biosynthetic genes, and are found in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin re-oxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.
2024-03-01 | GSE250373 | GEO
Project description:Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many ActinobacteriaMycobacterium tuberculosis assimilates cholesterol during chronic infection, and its in vitro growth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA, mftB, mftC, mftD, mftE, and mftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in Mycobacterium smegmatis, M. marinum, and M. tuberculosis We found that the growth deficit of mft deletion mutants in medium containing cholesterol-a phenotypic basis for gene essentiality prediction-depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, MSMEG_6242 was indispensable for M. smegmatis ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in M. tuberculosis During ethanol treatment, mftC mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mftC mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems.IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways-e.g., cholesterol assimilation-that are critical for persistence and for pathogenesis of M. tuberculosis In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for M. tuberculosisin vitro growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.
| S-EPMC6529628 | biostudies-literature
Project description:Nontuberculous mycobacteria Metagenome
| PRJNA779755 | ENA
Project description:The tumor suppressor p53 is critical for tumor suppression and other biological events. Yet, the regulatory role of p53 in alcohol-induced fatty liver remains unclear. Here, we show a role for p53 in regulating the ethanol metabolism via acetaldehyde dehydrogenase 2 (ALDH2), a key enzyme responsible for oxidization of alcohol. Through repressing ethanol oxidization, p53 suppresses intracellular levels of acetyl-CoA and histone acetylation, leading to the inhibition of the stearoyl-CoA desaturase-1 (SCD1) gene expression. Mechanistically, p53 directly binds to ALDH2 and prevents the formation of its active tetramer, and indirectly limits the production of pyruvate that promotes the activity of ALDH2. Notably, p53 deficient mice exhibit increased lipid accumulation, which can be reversed by ALDH2 depletion. Moreover, hepatic specific knockdown of SCD1 diminishes ethanol-induced fatty liver caused by p53 loss. By contrast, overexpression of SCD1 in liver promotes ethanol-induced fatty liver development in wildtype mice, while has mild effect on p53-/- or ALDH2-/- mice. Overall, our findings reveal a previously unrecognized function of p53 in alcohol-induced fatty liver, and uncover pyruvate as a natural regulator of ALDH2.
2023-02-08 | GSE222743 | GEO
Project description:nontuberculosis mycobacteria Raw sequence reads
| PRJNA974445 | ENA
Project description:Cell cycle-associated expression patterns predict gene function in mycobacteria
| PRJNA613916 | ENA
Project description:Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-H+ symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9 % higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5’ coding sequences of SUC2, resulting in relocation of invertase to the cytosol. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 95 generations of anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11 % higher ethanol yield on sucrose in chemostat and batch cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.
2011-09-06 | GSE30535 | GEO
Project description:Complete genome sequence of environmental Mycobacteria isolated from water
| PRJNA767558 | ENA