RESUMO
Brassinosteroids (BRs) are a class of steroid molecules perceived at the cell surface that act as plant hormones. The BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) offers a model to understand receptor-mediated signaling in plants and the role of post-translational modifications. Here we identify SUMOylation as a new modification targeting BRI1 to regulate its activity. BRI1 is SUMOylated in planta on two lysine residues, and the levels of BRI1 SUMO conjugates are controlled by the Desi3a SUMO protease. Loss of Desi3a leads to hypersensitivity to BRs, indicating that Desi3a acts as a negative regulator of BR signaling. Besides, we demonstrate that BRI1 is deSUMOylated at elevated temperature by Desi3a, leading to increased BRI1 interaction with the negative regulator of BR signaling BIK1 and to enhanced BRI1 endocytosis. Loss of Desi3a or BIK1 results in increased response to temperature elevation, indicating that BRI1 deSUMOylation acts as a safety mechanism necessary to keep temperature responses in check. Altogether, our work establishes BRI1 deSUMOylation as a molecular crosstalk mechanism between temperature and BR signaling, allowing plants to translate environmental inputs into growth response.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Brassinosteroides/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Temperatura , Reguladores de Crescimento de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
A key function of photoreceptor signaling is the coordinated regulation of a large number of genes to optimize plant growth and development. The basic helix loop helix (bHLH) transcription factor MYC2 is crucial for regulating gene expression in Arabidopsis thaliana during development in blue light. Here we demonstrate that blue light induces the SUMOylation of MYC2. Non-SUMOylatable MYC2 is less effective in suppressing blue light-mediated photomorphogenesis than wild-type (WT) MYC2. MYC2 interacts physically with the SUMO proteases SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2. Blue light exposure promotes the degradation of SPF1 and SPF2 and enhances the SUMOylation of MYC2. Phenotypic analysis revealed that SPF1/SPF2 function redundantly as positive regulators of blue light-mediated photomorphogenesis. Our data demonstrate that SUMO conjugation does not affect the dimerization of MYC transcription factors but modulates the interaction of MYC2 with its cognate DNA cis-element and with the ubiquitin ligase Plant U-box 10 (PUB10). Finally, we show that non-SUMOylatable MYC2 is less stable and interacts more strongly with PUB10 than the WT. Taken together, we conclude that SUMO functions as a counterpoint to the ubiquitin-mediated degradation of MYC2, thereby enhancing its function in blue light signaling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Plântula/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinas/genéticaRESUMO
In plants, exoribonuclease-resistant RNAs (xrRNAs) are produced by many viruses. Whereas xrRNAs contribute to the pathogenicity of these viruses, the role of xrRNAs in the virus infectious cycle remains elusive. Here, we show that xrRNAs produced by a benyvirus (a multipartite RNA virus with four genomic segments) in plants are involved in the formation of monocistronic coat protein (CP)-encoding chimeric RNAs. Naturally occurring chimeric RNAs, we discovered, are composed of 5'-end of RNA 2 and 3'-end of either RNA 3 or RNA 4 bearing conservative exoribonuclease-resistant 'coremin' region. Using computational tools and site-directed mutagenesis, we show that de novo formation of chimeric RNAs requires intermolecular base-pairing interaction between 'coremin' and 3'-proximal part of the CP gene of RNA 2 as well as a stem-loop structure immediately adjacent to the CP gene. Moreover, knockdown of the expression of the XRN4 gene, encoding 5'â3' exoribonuclease, inhibits biogenesis of both xrRNAs and chimeric RNAs. Our findings suggest a novel mechanism involving a unique tropology of the intermolecular base-pairing complex between xrRNAs and RNA2 to promote formation of chimeric RNAs in plants. XrRNAs, essential for chimeric RNA biogenesis, are generated through the action of cytoplasmic Xrn 4 5'â3' exoribonuclease conserved in all plant species.
Assuntos
Exorribonucleases , RNA Viral , RNA Viral/genética , RNA Viral/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , RNA não Traduzido/genéticaRESUMO
Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.
Assuntos
Acetato-CoA Ligase , Escherichia coli , Acetato Quinase/genética , Acetato Quinase/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Escherichia coli/metabolismo , Óperon , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismoRESUMO
Klebsiella pneumoniae (NCTC, CL687/80) harbors a large indigenous plasmid (p(C3)), which in addition to encoding for citrate utilization, proline synthesis and glutamate excretion, it uniquely carries the structural gene (icd); encoding isocitrate dehydrogenase (ICDH). Flux analysis revealed that ICDH, despite its role in the generation of NADPH required for glutamate dehydrogenase, is not rate-limiting (controlling) in central metabolism as evidenced by a negative flux control coefficient and an adverse effect of overexpression (14-fold) on glutamate excretion. More significantly, however, this paper presents, for the first time, clear evidence that the accumulation of glutamate and its subsequent excretion is associated with the C3 plasmid-encoded regulatory elements, which trigger a shift-down in the activity of α-ketoglutarate dehydrogenase, both in the K. pneumoniae parental strain as well as in the E. coli exconjugants strains. This finding opens the door for the exploitation of regulatory elements as a tool for manipulating flux in microbial cell factories.
Assuntos
Ciclo do Carbono , Ácido Glutâmico/metabolismo , Isocitrato Desidrogenase/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Plasmídeos/genética , Reatores Biológicos , Ciclo do Carbono/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Isocitrato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Klebsiella pneumoniae/enzimologia , NADP/metabolismo , Plasmídeos/metabolismoRESUMO
Understanding metabolic networks' architecture is central to successfully manipulating metabolic fluxes in microbial cell factories. The transition of central metabolism's architecture from acetogenic to gluconeogenic and from the canonical monocyclic architecture of the Krebs tricarboxylic acids (TCA) cycle to a bicyclic architecture in which the TCA and the dicarboxylic acids (DCA) cycles work in unison, with the glyoxylate bypass fulfilling the anaplerotic function, has been the subject of much debate and remains elusive. In this article, the author sheds light on the intricacies surrounding the transition of central metabolism from one architecture to another and shows that the transition from the monocyclic architecture to the bicyclic architecture is triggered in response to a minimum threshold signal of growth rate (â²0.40h-1) and is a consequence of competitions, on the one hand. between phosphotransacetylase (PTA) and α-ketoglutarate dehydrogenase (α-KGDH) for their common co-factor, free HS-CoA, and, on the other hand, between catabolic and anaplerotic routs for acetyl phosphate. Further restriction of carbon supply in the bioreactor to the point of starvation forces E. coli to further modify its central metabolism to the PEP-glyoxylate architecture to maintain the redox balance. Interestingly the sudden change from hunger ('famine') to carbon excess ('feast') leads to yet another architecture in which the methylglyoxal pathway figure prominently to maintain the adenylate energy charge. Moreover, the author sheds light on the biochemical implications of each architecture.
Assuntos
Escherichia coli , Glioxilatos , Escherichia coli/metabolismo , Glioxilatos/metabolismo , Ciclo do Ácido Cítrico , Carbono/metabolismo , Redes e Vias MetabólicasRESUMO
The signal that triggers the expression of the ace operon and, in turn, the transition of central metabolism's architecture from acetogenic to gluconeogenic in Escherichia coli remains elusive despite extensive research both in vivo and in vitro. Here, with the aid of flux analysis together with measurements of the enzymic activity of isocitrate lyase (ICL) and its aceA-messenger ribonucleuc acid (mRNA) transcripts, we provide credible evidence suggesting that the expression of the ace operon in E. coli is triggered in response to growth rate-dependent threshold flux-signal of adenosine triphosphate (ATP). Flux analysis revealed that the shortfall in ATP supply observed as the growth rate ($\mu $) diminishes from µmax to ≤ 0.43h-1 ($ \pm 0.02;n4)\ $is partially redressed by up-regulating flux through succinyl CoA synthetase. Unlike glycerol and glucose, pyruvate cannot feed directly into the two glycolytic ATP-generating reactions catalyzed by phosphoglycerokinase and pyruvate kinase. On the other hand, glycerol, which upon its conversion to D-glyceraldehyde, feeds into the phosphorylation and dephosphorylation parts of glycolysis including the substrate-level phosphorylation-ATP generating reactions, thus preventing ATP flux from dropping to the critical threshold signal required to trigger the acetate-diauxic switch until glycerol is fully consumed. The mRNA transcriptional patterns of key gluconeogenic enzymes, namely, ackA, acetate kinase; pta, phosphotransacetylase; acs, acetyl CoA synthetase and aceA, ICL, suggest that the pyruvate phenotype is better equipped than the glycerol phenotype for the switch from acetogenic to gluconeogenic metabolism.
Assuntos
Trifosfato de Adenosina/metabolismo , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Flux analysis is central to understanding cellular metabolism and successful manipulation of metabolic fluxes in microbial cell-factories. Isocitrate dehydrogenase (ICDH) deletion conferred contrasting effects on fluxes through substrate-level phosphorylation (SLP) reactions. While significantly increasing flux through pyruvate kinase, it diminishes flux through succinyl CoA synthetase and upregulates phosphotransacetylase (PTA) and acetate kinase (AK). In addition to acetate, the ICDH-less strain excretes pyruvate, citrate and isocitrate. While efflux to acetate excretion by the Escherichia coli parental strain and its ICDH-less derivative is a reflection of high throughput of glycolytic intermediates, excretion of pyruvate is a reflection of high throughput via pyruvate kinase. On the other hand, citrate and isocitrate excretion is a reflection of truncating the Krebs cycle at the level of ICDH. Furthermore, another striking finding is the inability of the ICDH-less cultures to utilize acetate as a source of carbon despite the availability of an adequate supply of extracellular glutamate (for biosynthesis) and elevated levels of AK and PTA (for acetate uptake). This striking observation is now explicable in the light of the newly proposed hypothesis that the expression of the ace operon enzymes is controlled in response to a minimum threshold signal (ATP), which could not be achieved in the ICDH-less strain.
Assuntos
Acetatos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Isocitrato Desidrogenase/genética , Análise do Fluxo Metabólico , Ciclo do Ácido Cítrico/genética , Glicólise , Deleção de SequênciaRESUMO
During aerobic growth of Escherichia coli on acetate, the component parts of the 'acetate switch' are turned-on as a consequence of direct competition, on the one hand, between phosphotransacetylase (PTA) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) for their common co-factor free-CoA (HS-CoA) and, on the other hand, between isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) for their common substrate isocitrate. Flux analysis revealed that competitions at both junctions in central metabolism are resolved in a precise way, so that the fraction of HS-CoA flux processed through PTA for biosynthesis relative to that processed through alpha-KGDH for energy generation, matches that observed for isocitrate flux through ICL relative to ICDH at the junction of isocitrate. Whereas the mechanism involved in the partition of carbon flux at the level of HS-CoA in central metabolism remains to be unravelled, the competition at the junction of isocitrate is resolved by the reversible phosphorylation/inactivation of ICDH and the operation of the glyoxylate bypass, the expression of which is subject to regulation at the transcriptional and translational levels as well as being dependent on growth rate.
Assuntos
Carbono/metabolismo , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Meios de Cultura , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glioxilatos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/metabolismo , Isocitrato Liase/metabolismo , Isocitratos/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Fosfato Acetiltransferase/metabolismo , FosforilaçãoRESUMO
During growth of Escherichia coli on acetate, phosphotransacetylase and alpha-ketoglutarate dehydrogenase are in direct competition for their common co-factor, HS-CoA. Such competition is resolved in favour of phosphotransacetylase, thus rendering alpha-ketoglutarate dehydrogenase rate-limiting (controlling) and, in turn, creating a bottleneck at the level of alpha-ketoglutarate in the Krebs cycle. Accumulation of alpha-ketoglutarate is then balanced by its excretion. Addition of pyruvate, glucose or any glycolytic intermediate to acetate-grown culture relieves such a bottleneck by reversing carbon flow through phosphotransacetylase to give acetyl phosphate and much-needed HS-CoA. The urgent need for HS-CoA by the primordial organism might therefore have provided the selective pressure that led to the co-evolution of phosphotransacetylase and the two-malate synthase isoenzymes.
Assuntos
Acetatos/metabolismo , Coenzima A/metabolismo , Escherichia coli/metabolismo , Ácidos Cetoglutáricos/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Aerobiose , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologiaRESUMO
During aerobic growth of Escherichia coli on acetate as sole source of carbon and energy, the organism requires the operation of the glyoxylate bypass enzymes, namely isocitrate lyase (ICL) and the anaplerotic enzyme malate synthase (MS). Under these conditions, the glyoxylate bypass enzyme ICL is in direct competition with the Krebs cycle enzyme isocitrate dehydrogenase (ICDH) for their common substrate and although ICDH has a much higher affinity for isocitrate, flux of carbon through ICL is assured by virtue of high intracellular level of isocitrate and the reversible phosphorylation/inactivation of a large fraction of ICDH. Reversible inactivation is due to reversible phosphorylation catalysed by ICDH kinase/phosphatase, which harbours both catalytic activities on the same polypeptide. The catalytic activities of ICDH kinase/phosphatase constitute a moiety conserved cycle, require ATP and exhibit 'zero-order ultrasensitivity'. The structural gene encoding ICDH kinase/phosphatase (aceK) together with those encoding ICL (aceA) and MS (aceB) form an operon (aceBAK; otherwise known as the ace operon) the expression of which is intricately regulated at the transcriptional level by IclR, FadR, FruR and IHF. Although ICDH, an NADP(+)-dependent, non-allosteric dimer, can be phosphorylated at multiple sites, it is the phosphorylation of the Ser-113 residue that renders the enzyme catalytically inactive as it prevents isocitrate from binding to the active site, which is a consequence of the negative charge carried on phosphoserine 113 and the conformational change associated with it. The ICDH molecule readily undergo domain shifts and/or induced-fit conformational changes to accommodate the binding of ICDH kinase/phosphatase, the function of which has now been shown to be central to successful adaptation and growth of E. coli and related genera on acetate and fatty acids.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Isocitrato Desidrogenase/metabolismo , Processamento de Proteína Pós-Traducional , Adaptação Fisiológica , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Isocitrato Desidrogenase/genética , Óperon , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The efficiency of carbon conversion to biomass and desirable end products in industrial fermentations is diminished by the diversion of carbon to acetate and lactate excretions. In this study, the use of prototrophic and mutant strains of Escherichia coli, as well as enzyme active site directed inhibitors, revealed that flux to acetate excretion is physiologically advantageous to the organism as it facilitates a faster growth rate (mu) and permits growth to high cell densities. Moreover, the abolition of flux to acetate excretion was balanced by the excretion of lactate as well as 2-oxoglutarate, isocitrate and citrate, suggesting a 'bottle-neck' effect at the level of 2-oxoglutarate in the Krebs cycle. It is proposed that the acetate excreting enzymes, phosphotransacetylase and acetate kinase, constitute an anaplerotic loop or by-pass, the primary function of which is to replenish the Krebs cycle with reduced CoA, thus relieving the bottle-neck effect at the level of 2-oxoglutarate dehydrogenase. Furthermore, flux to lactate excretion plays a central role in regenerating proton gradient and maintaining the redox balance within the cell. The long-held view that flux to acetate and lactate excretions is merely a function of an 'over-flow' in central metabolism should, therefore, be re-evaluated.