Expression data of FMN supplementation from Saccharomyces cerevisiae
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE128905 | GEO | 2020/02/27
REPOSITORIES: GEO
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Project description:Riboflavin-derived molecules such as flavin mononucleotide and flavin adenine dinucleotide comprise the most important redox coenzymes all across life kingdoms. Vibrio cholerae, a pandemic diarrheagenic bacterium, is able to synthesize riboflavin through the riboflavin biosynthetic pathway (RBP) and to internalize exogenous riboflavin using the RibN importer. This bacterium thrives in different environments such as estuarine waters and the human intestinal tract. The presence of two independent riboflavin supply pathways in this species may be related to the variable riboflavin availability and requirements across different niches. In order to gain insights into the role of the riboflavin provision pathways in the physiology of V. cholerae, we analyzed the bacterial transcriptomics response to extracellular riboflavin and to the deletions of ribD (RBP-deficient strain) or ribN. Notably, many genes responding to riboflavin have been previously reported to belong to the iron regulon. Real time PCR analysis confirmed this effect and further documented that reciprocally, iron regulates RBP and ribN genes in a riboflavin-dependent way. A subset of genes were responding to both ribD and ribN deletions. Nonetheless, a group of genes was specifically affected in the ∆ribD strain, on which the functional terms protein folding and oxidation reduction process were enriched. Also, a subset of genes was affected specifically in the ∆ribN strain, on which the cytochrome complex assembly functional term was enriched. Results indicate that iron and riboflavin interrelate to regulate its respective provision genes and suggest that both common and specific effects of biosynthesized and internalized riboflavin exist.
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Project description:Respiratory complex I plays a crucial role in the mitochondrial electron transport chain and shows promise as a therapeutic target for various human diseases. While most studies focus on inhibiting complex I at the Q-site, little is known about inhibitors targeting other sites within the complex. In this study, we demonstrate that diphenyleneiodonium (DPI), a known N-site inhibitor, uniquely affects the stability of complex I by inhibiting its flavin cofactor FMN. Treatment with DPI blocks the final stage of complex I assembly, leading to the accumulation of assembly intermediates that lack the N-module. Remarkably, high-dose DPI treatment can completely and reversibly degrade complex I in different cellular models. The mechanism involves the accumulation of DPI in mitochondria, where it reacts with FMN, depleting the effective pool of FMN and halting complex I maturation. Consistently, growing cells in medium lacking the FMN precursor riboflavin or knocking out the mitochondrial FAD carrier gene SLC25A32 results in complex I degradation and impairs complex I-dependent respiration. Overall, our findings establish a direct connection between mitochondrial flavin homeostasis and complex I stability and assembly, paving the way for novel pharmacological strategies to regulate respiratory complex I.
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Project description:The pathogenic bacterium Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis worldwide yet it does not grow in the aerobic environment. The paralogues RrpA and RrpB which are members of MarR family of DNA binding proteins have been shown to be important for the survival of C. jejuni under aerobic and redox stress. We report that RrpA is a positive regulator of mdaB, encoding a flavin-dependent quinone reductase. MdaB confers protection to the cell from redox stress mediated by structurally diverse quinones. RrpB negatively regulates the expression of nfrA (Cj1555c), a flavin reductase. NfrA reduces riboflavin at a much higher rate than flavin mononucleotide (FMN),suggesting exogenous free flavins are the natural substrate. Enzymatic activity of MdaB and NfrA towards their substrates revealed both reductases preferred NADPH as an electron donor. DNA-binding and post translational modification analyses showed that the mechanism of RrpA and RrpB DNA binding is likely a cysteine-based redox switch. Complete genome sequences analysis indicated that MdaB is predominant in Campylobacter spp. and the related Helicobacter spp., whilst NfrA is more often found in C. jejuni strains. Quinones and flavins are antimicrobial redox cycling agents secreted by a wide range of cell-types that can form damaging superoxide by one-electron reactions. We propose that MdaB and NfrA production allows a two-electron reduction mechanism to the less toxic quinol forms. These enzymes thus aid the survival and persistence of C. jejuni in the face of toxic compounds from competing microbes.
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Project description:Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing all annotated sigma factor genes on C. glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. HPLC analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA Microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes of the pentose phosphate pathway and for enzymes dependent on FMN, FAD or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. Endogenous sigma factor gene, sigH, was overexpressed in C. glutamicum ATCC13032 from IPTG inducible vector, pEKEx3. Two different concentration of IPTG (10 μM and 15 μM) was used for induction of SigH expression.
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Project description:Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing all annotated sigma factor genes on C. glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. HPLC analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA Microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes of the pentose phosphate pathway and for enzymes dependent on FMN, FAD or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production.
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