RESUMEN
Detyrosination of α-tubulin seems to be conserved in all eukaryotes. However, its biological function in plants has remained obscure. A conserved C-terminal tyrosine is removed by a still unidentified tubulin-tyrosine carboxypeptidase (TTC) and can be religated by a tubulin-tyrosine ligase (TTL). To obtain insight into the still elusive biological function of this detyrosination-tyrosination cycle, the effects of the TTC inhibitor parthenolide were analysed in BY-2 tobacco cells. Parthenolide caused a depletion of detyrosinated α-tubulin, whereas the level of tyrosinated tubulin was elevated. This biochemical effect was accompanied by growth inhibition in cycling BY-2 cells and alteration of microtubule-dependent events that define division and expansion geometry such as cell plate alignment or axial expansion. Furthermore, parthenolide triggered an apoplastic alkalinization indicative of activation of defence-related calcium influx channels. At the same time, parthenolide promoted the association of the plant-specific kinesin KCH with cortical microtubules. These observations are integrated into a working model, where detyrosination acts as signal to modulate the binding of kinesin motors involved in structural and sensory functions of the microtubular cytoskeleton.
Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Línea Celular , Células Cultivadas , Genes Reporteros , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Nicotiana/citología , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Tirosina/metabolismoRESUMEN
Viral infection is a stimulus for apoptosis, and in order to sustain viral replication many viruses are known to carry genes encoding apoptosis inhibitors. F1L, encoded by the orthopoxvirus modified vaccinia virus Ankara (MVA) has a Bcl-2-like structure. An MVA mutant lacking F1L (MVAΔF1L) induces apoptosis, indicating that MVA infection activates and F1L functions to inhibit the apoptotic pathway. In this study we investigated the events leading to apoptosis upon infection by MVAΔF1L. Apoptosis largely proceeded through the pro-apoptotic Bcl-2 family protein Bak with some contribution from Bax. Of the family of pro-apoptotic BH3-only proteins, only the loss of Noxa provided substantial protection, while the loss of Bim had a minor effect. In mice, MVA preferentially infected macrophages and DCs in vivo. In both cell types wt MVA induced apoptosis albeit more weakly than MVAΔF1L. The loss of Noxa had a significant protective effect in macrophages, DC and primary lymphocytes, and the combined loss of Bim and Noxa provided strong protection. Noxa protein was induced during infection, and the induction of Noxa protein and apoptosis induction required transcription factor IRF3 and type I interferon signalling. We further observed that helicases RIG-I and MDA5 and their signalling adapter MAVS contribute to Noxa induction and apoptosis in response to MVA infection. RNA isolated from MVA-infected cells induced Noxa expression and apoptosis when transfected in the absence of viral infection. We thus here describe a pathway leading from the detection of viral RNA during MVA infection by the cytosolic helicase-pathway, to the up-regulation of Noxa and apoptosis via IRF3 and type I IFN signalling.
Asunto(s)
Apoptosis/genética , ADN Helicasas/metabolismo , Factor 3 Regulador del Interferón/fisiología , Interferón beta/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Virus Vaccinia/fisiología , Células 3T3 , Animales , Células Cultivadas , Embrión de Pollo , Citosol/metabolismo , ADN Helicasas/genética , ADN Helicasas/fisiología , Células HeLa , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiología , Vaccinia/genética , Vaccinia/metabolismo , Vaccinia/patología , Virus Vaccinia/metabolismoRESUMEN
Vaccinia virus (VACV) infection induces phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), which inhibits cellular and viral protein synthesis. In turn, VACV has evolved the capacity to antagonize this antiviral response by expressing the viral host-range proteins K3 and E3. This study revealed that the host-range genes K1L and C7L also prevent eIF2alpha phosphorylation in modified VACV Ankara (MVA) infection of several human and murine cell lines. Moreover, C7L-deleted MVA (MVA-DeltaC7L) lacked late gene expression, which could be rescued by the function of host-range factor K1 or C7. It was demonstrated that viral gene expression was blocked after viral DNA replication and that it was independent of apoptosis induction. Furthermore, it was found that eIF2alpha phosphorylation in MVA-DeltaC7L-infected cells is mediated by protein kinase R (PKR) as shown in murine embryonic fibroblasts lacking PKR function, and it was shown that this was not due to reduced E3L gene expression. The block of eIF2alpha phosphorylation by C7 could be complemented by K1 in cells infected with MVA-DeltaC7L encoding a reinserted K1L gene (MVA-DeltaC7L-K1L). Importantly, these data illustrated that eIF2alpha phosphorylation by PKR is not responsible for the block of late viral gene expression. This suggests that other mechanisms targeted by C7 and K1 are essential for completing the MVA gene expression cycle and probably also for VACV replication in a diverse set of cell types.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Regulación Viral de la Expresión Génica , Virus Vaccinia/metabolismo , Vaccinia/metabolismo , Proteínas Virales/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Línea Celular , Cricetinae , Factor 2 Eucariótico de Iniciación/genética , Humanos , Ratones , Fosforilación , Vaccinia/enzimología , Vaccinia/genética , Vaccinia/virología , Virus Vaccinia/genética , Proteínas Virales/genética , eIF-2 Quinasa/genéticaRESUMEN
Vaccinia virus (VACV) replicates in mouse and human fibroblasts with comparable kinetics and efficiency, yielding similar titers of infectious progeny. Here we demonstrate that gamma interferon (IFN-gamma) but not IFN-alpha or IFN-beta pretreatment of mouse fibroblasts prior to VACV infection induces a long-lasting antiviral state blocking VACV replication. In contrast, high doses of IFN-gamma failed to establish an antiviral state in human fibroblasts. In mouse fibroblasts, IFN-gamma impeded the viral replication cycle at the level of late gene transcription and blocked the multiplication of VACV genomes. The IFN-gamma-induced antiviral state invariably prevented the growth of different VACV strains but was not effective against the replication of ectromelia virus. The IFN-gamma effect required intact IFN-gamma receptor signaling prior to VACV infection through Janus kinase 2 (Jak2) and signal transducer and activator of transcription 1 (STAT1). The permissive state of IFN-gamma-treated human cells was unrelated to the VACV-encoded IFN decoy receptors B8 and B18 and associated with a complete disruption of STAT1 homodimer formation and DNA binding. Unlike human fibroblasts, mouse cells responded with long-lasting STAT1 activation which was preserved after VACV infection. The deletion of the IFN regulatory factor 1 (IRF-1) gene from mouse cells rescued efficient VACV replication, demonstrating that IRF-1 target genes have a critical role in VACV control. These data have implications for the understanding of VACV pathogenesis and identify an incongruent IFN-gamma response between the human host and the mouse model.
Asunto(s)
Fibroblastos/virología , Factor 1 Regulador del Interferón/inmunología , Interferón gamma/inmunología , Virus Vaccinia/inmunología , Virus Vaccinia/fisiología , Replicación Viral , Animales , Células Cultivadas , Replicación del ADN , ADN Viral/biosíntesis , Virus de la Ectromelia/fisiología , Eliminación de Gen , Humanos , Factor 1 Regulador del Interferón/genética , Janus Quinasa 2/metabolismo , Ratones , Receptores de Interferón/metabolismo , Factor de Transcripción STAT1/metabolismo , Receptor de Interferón gammaRESUMEN
Poxviruses such as virulent vaccinia virus (VACV) strain Western Reserve encode a broad range of immune modulators that interfere with host responses to infection. Upon more than 570 in vitro passages in chicken embryo fibroblasts (CEF), chorioallantois VACV Ankara (CVA) accumulated mutations that resulted in highly attenuated modified vaccinia virus Ankara (MVA). MVA infection of mice and of dendritic cells (DC) induced significant type I interferon (IFN) responses, whereas infection with VACV alone or in combination with MVA did not. These results implied that VACV expressed an IFN inhibitor(s) that was functionally deleted in MVA. To further characterize the IFN inhibitor(s), infection experiments were carried out with CVA strains isolated after 152 (CVA152) and 386 CEF passages (CVA386). Interestingly, neither CVA152 nor CVA386 induced IFN-alpha, whereas the latter variant did induce IFN-beta. This pattern suggested a consecutive loss of inhibitors during MVA attenuation. Similar to supernatants of VACV- and CVA152-infected DC cultures, recombinantly expressed soluble IFN decoy receptor B18, which is encoded in the VACV genome, inhibited MVA-induced IFN-alpha but not IFN-beta. In the same direction, a B18R-deficient VACV variant triggered only IFN-alpha, confirming B18 as the soluble IFN-alpha inhibitor. Interestingly, VACV infection inhibited IFN responses induced by a multitude of different stimuli, including oligodeoxynucleotides containing CpG motifs, poly(I:C), and vesicular stomatitis virus. Collectively, the data presented show that VACV-mediated IFN inhibition is a multistep process involving secreted factors such as B18 plus intracellular components that cooperate to efficiently shut off systemic IFN-alpha and IFN-beta responses.
Asunto(s)
Interacciones Huésped-Patógeno , Interferón Tipo I/antagonistas & inhibidores , Proteínas/metabolismo , Virus Vaccinia/inmunología , Proteínas Virales/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
The highly attenuated Modified Vaccinia virus Ankara (MVA) lacks most of the known vaccinia virus (VACV) virulence and immune evasion genes. Today MVA can serve as a safety-tested next-generation smallpox vaccine. Yet, we still need to learn about regulatory gene functions preserved in the MVA genome, such as the apoptosis inhibitor genes F1L and E3L. Here, we tested MVA vaccine preparations on the basis of the deletion mutant viruses MVA-ΔF1L and MVA-ΔE3L for efficacy against ectromelia virus (ECTV) challenge infections in mice. In non-permissive human tissue culture the MVA deletion mutant viruses produced reduced levels of the VACV envelope antigen B5. Upon mousepox challenge at three weeks after vaccination, MVA-ΔF1L and MVA-ΔE3L exhibited reduced protective capacity in comparison to wildtype MVA. Surprisingly, however, all vaccines proved equally protective against a lethal ECTV infection at two days after vaccination. Accordingly, the deletion mutant MVA vaccines induced high levels of virus-specific CD8+ T cells previously shown to be essential for rapidly protective MVA vaccination. These results suggest that inactivation of the anti-apoptotic genes F1L or E3L modulates the protective capacity of MVA vaccination most likely through the induction of distinct orthopoxvirus specific immunity in the absence of these viral regulatory proteins.
Asunto(s)
Inmunomodulación , Proteínas de Unión al ARN/genética , Vacuna contra Viruela/inmunología , Viruela/inmunología , Viruela/prevención & control , Virus Vaccinia/genética , Proteínas Virales/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Regulación Viral de la Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Inmunización , Ratones , Vacuna contra Viruela/administración & dosificaciónRESUMEN
Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus strain undergoing clinical evaluation as a replication-deficient vaccine vector against various infections and tumor diseases. To analyze the basis of its high immunogenicity, we investigated the mechanism of how MVA induces type I interferon (IFN) responses. MVA stimulation of bone marrow-derived dendritic cells (DC) showed that plasmacytoid DC were main alpha IFN (IFN-alpha) producers that were triggered independently of productive infection, viral replication, or intermediate and late viral gene expression. Increased IFN-alpha levels were induced upon treatment with mildly UV-irradiated MVA, suggesting that a virus-encoded immune modulator(s) interfered with the host cytokine response. Mice devoid of Toll-like receptor 9 (TLR9), the receptor for double-stranded DNA, mounted normal IFN-alpha responses upon MVA treatment. Furthermore, mice devoid of the adaptors of TLR signaling MyD88 and TRIF and mice deficient in protein kinase R (PKR) showed IFN-alpha responses that were only slightly reduced compared to those of wild-type mice. MVA-induced IFN-alpha responses were critically dependent on autocrine/paracrine triggering of the IFN-alpha/beta receptor and were independent of IFN-beta, thus involving "one-half" of a positive-feedback loop. In conclusion, MVA-mediated type I IFN secretion was primarily triggered by non-TLR molecules, was independent of virus propagation, and critically involved IFN feedback stimulation. These data provide the basis to further improve MVA as a vaccine vector.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Interferón-alfa/metabolismo , Vacuna contra Viruela/inmunología , Virus Vaccinia/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Vacunas contra el Cáncer/genética , Expresión Génica , Interferón Tipo I/metabolismo , Interferón beta/metabolismo , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Vacuna contra Viruela/genética , Receptor Toll-Like 9/genética , Virus Vaccinia/genética , Virus Vaccinia/fisiología , Replicación ViralRESUMEN
Vector-infected dendritic cells (DC) are evaluated for antigen delivery in experimental therapy of cancer and infectious diseases. Here, we investigated infections of immature or mature, monocyte-derived human DC with recombinant vaccinia virus MVA producing human Her-2/neu, a candidate tumor-associated antigen. Assessment of the molecular virus life cycle in infected DC revealed a general arrest at the level of viral early gene expression. When monitoring the phenotype of MVA-infected DC, including expression of cell surface markers, we found immature cells readily undergoing apoptosis. Nevertheless, we detected significant populations of viable DC being characterized by high level Her-2/neu expression and unimpaired display of costimulatory molecules. While infected viable immature DC failed to undergo maturation despite cytokine treatment, both DC populations efficiently presented MVA-produced target antigen. These findings allow to better define the requirements for MVA-mediated antigen delivery to DC and help to derive optimized vectors for this advanced therapy option.
Asunto(s)
Células Dendríticas/virología , Transcripción Genética , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Apoptosis , Células Dendríticas/citología , Células Dendríticas/fisiología , Genes erbB-2 , Humanos , Receptor ErbB-2/genética , Recombinación Genética , Virus Vaccinia/fisiología , Replicación ViralRESUMEN
Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-DeltaE3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2'-5'-oligoadenylate synthetase (2'-5'OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2'-5'OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2alpha (eIF2alpha) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-DeltaE3L-infected mouse embryonic fibroblasts from Pkr(0/0) mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.
Asunto(s)
Proteínas de Unión al ARN/genética , Virus Vaccinia/fisiología , Proteínas Virales/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Células Cultivadas , Regulación Viral de la Expresión Génica , Humanos , Ratones , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , ARN Bicatenario/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Especificidad de la Especie , Factores de Transcripción/metabolismo , Virus Vaccinia/genética , Replicación ViralRESUMEN
The tricarboxylic acid (TCA) cycle is one of the major routes of carbon catabolism in Bacillus subtilis. The syntheses of the enzymes performing the initial reactions of the cycle, citrate synthase, and aconitase, are synergistically repressed by rapidly metabolizable carbon sources and glutamine. This regulation involves the general transcription factor CcpA and the specific repressor CcpC. In this study, we analyzed the expression and intracellular localization of CcpC. The synthesis of citrate, the effector of CcpC, requires acetyl-CoA. This metabolite is located at a branching point in metabolism. It can be converted to acetate in overflow metabolism or to citrate. Manipulations of the fate of acetyl-CoA revealed that efficient citrate synthesis is required for the expression of the citB gene encoding aconitase and that control of the two pathways utilizing acetyl-CoA converges in the control of citrate synthesis for the induction of the TCA cycle. The citrate pool seems also to be controlled by arginine catabolism. The presence of arginine results in a severe CcpC-dependent repression of citB. In addition to regulators involved in sensing the carbon status of the cell, the pleiotropic nitrogen-related transcription factor, TnrA, activates citB transcription in the absence of glutamine.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Citratos/metabolismo , Nitrógeno/metabolismo , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Arginina/farmacología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Northern Blotting , Western Blotting , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/genética , Redes y Vías Metabólicas/efectos de los fármacos , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The innate immune system recognizes nucleic acids during infection or tissue damage; however, the mechanisms of intracellular recognition of DNA have not been fully elucidated. Here we show that intracellular administration of double-stranded B-form DNA (B-DNA) triggered antiviral responses including production of type I interferons and chemokines independently of Toll-like receptors or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding interferon-beta through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-kappaB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of interferon-beta and NF-kappaB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders.
Asunto(s)
ADN/inmunología , Transducción de Señal/inmunología , Receptores Toll-Like/inmunología , Virosis/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Quimiocinas/metabolismo , ADN Bacteriano/inmunología , ADN Viral/inmunología , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/inmunología , Fibroblastos/metabolismo , Expresión Génica/inmunología , Humanos , Quinasa I-kappa B , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain being developed as a vaccine for delivery of viral and recombinant antigens. The MVA genome lacks functional copies of numerous genes interfering with host response to infection. The interferon resistance gene E3L encodes one important viral immune defense factor still made by MVA. Here we demonstrate an essential role of E3L to allow for completion of the MVA molecular life cycle upon infection of human HeLa cells. A deletion mutant virus, MVA-DeltaE3L, was found defective in late protein synthesis, viral late transcription, and viral DNA replication in infected HeLa cells. Moreover, we detected viral early and continuing intermediate transcription associated with degradation of rRNA, indicating rapid activation of 2'-5'-oligoadenylate synthetase/RNase L in the absence of E3L. Further molecular monitoring of E3L function by microarray analysis of host cell transcription in MVA- or MVA-DeltaE3L-infected HeLa cells revealed an overall significant down regulation of more than 50% of cellular transcripts expressed under mock conditions already at 5 h after infection, with a more prominent shutoff following MVA-DeltaE3L infection. Interestingly, a cluster of genes up regulated exclusively in MVA-DeltaE3L-infected cells could be identified, including transcripts for interleukin 6, growth arrest and DNA damage-inducible protein beta, and dual-specificity protein phosphatases. Our data indicate that lack of E3L inhibits MVA antigen production in human HeLa cells at the level of viral late gene expression and suggest that E3L can prevent activation of additional host factors possibly affecting the MVA molecular life cycle.
Asunto(s)
Interacciones Huésped-Parásitos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Unión al ARN/fisiología , Virus Vaccinia/fisiología , Vaccinia/genética , Proteínas Virales/fisiología , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Proteínas de Unión al ARN/farmacología , Virus Vaccinia/genética , Proteínas Virales/farmacologíaRESUMEN
Bacillus subtilis ccpA mutant strains exhibit two distinct phenotypes: they are defective in catabolite repression, and their growth on minimal media is strongly impaired. This growth defect is largely due to a lack of expression of the gltAB operon. However, growth is impaired even in the presence of glutamate. Here, we demonstrate that the ccpA mutant strain needs methionine and the branched-chain amino acids for optimal growth. The control of expression of the ilv-leu operon by CcpA provides a novel regulatory link between carbon and amino acid metabolism.
Asunto(s)
Aminoácidos de Cadena Ramificada/biosíntesis , Bacillus subtilis/enzimología , Proteínas Bacterianas , Proteínas de Unión al ADN/genética , Mutación , Operón/fisiología , Proteínas Represoras/genética , Aminoácidos de Cadena Ramificada/genética , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Medios de Cultivo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucosa/metabolismo , Glutamato Sintasa/genética , Glutamato Sintasa/metabolismo , Operón/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Transcripción Genética , Xilosa/metabolismoRESUMEN
Glycolysis is one of the central routes of carbon catabolism in Bacillus subtilis. Several glycolytic enzymes, including the key enzyme glyceraldehyde-3-phosphate dehydrogenase, are encoded in the hexacistronic gapA operon. Expression of this operon is induced by a variety of sugars and amino acids. Under non-inducing conditions, expression is repressed by the CggR repressor protein, the product of the promoter-proximal gene of the operon. Here, it is shown that the amount of glyceraldehyde-3-phosphate dehydrogenase encoded by the second gene of the operon exceeds that of the CggR repressor by about 100-fold. This differential synthesis was attributed to an mRNA processing event that takes place at the 3' end of the cggR open reading frame and to differential segmental stabilities of the resulting cleavage products. The mRNA specifying the truncated cggR gene is quickly degraded, whereas the downstream processing products encompassing gapA are quite stable. This increased stability is conferred by the presence of a stem-loop structure at the 5' end of the processed mRNAs. Mutations were introduced in the region of the cleavage site. A mutation affecting the stability of the stem-loop structure immediately downstream of the processing site had two effects. First, the steady-state transcript pattern was drastically shifted towards the primary transcripts; second, the stability of the processed mRNA containing the destabilized stem-loop structure was strongly decreased. This results in a reduction of the amount of glyceraldehyde-3-phosphate dehydrogenase in the cell. It is concluded that mRNA processing is involved in differential syntheses of the proteins encoded by the gapA operon.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glucólisis , Operón/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Datos de Secuencia Molecular , Mutación Puntual , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Bacillus subtilis assimilates ammonium by the concerted action of glutamine synthetase and glutamate synthase. The expression of the gltAB operon encoding the latter enzyme is impaired in B. subtilis ccpA mutant strains. CcpA is a pleiotropic transcriptional regulator that is the key factor in the regulation of carbon metabolism. However, in addition to their defect in catabolite repression ccpA mutants are unable to grow on minimal media with glucose and ammonium as the single sources of carbon and nitrogen, respectively. In this work, the expression of the gltAB operon was analysed and its role in growth on minimal sugar/ammonium media was studied. Expression of gltAB requires induction by glucose or other glycolytically catabolized carbon sources. In ccpA mutants, gltAB cannot be induced by glucose due to the low activity of the phosphotransferase sugar transport system in these mutants. A mutation that allowed phosphotransferase system activity in a ccpA background simultaneously restored glucose induction of gltAB and growth on glucose/ammonium medium. Moreover, artificial induction of the gltAB operon in the ccpA mutant allowed the mutant strain to grow on minimal medium with glucose and ammonium. It may be concluded that expression of the gltAB operon depends on the accumulation of glycolytic intermediates which cannot occur in the ccpA mutant. The lack of gltAB induction is the bottleneck that prevents growth of the ccpA mutant on glucose/ammonium media. The control of expression of the gltAB operon by CcpA provides a major regulatory link between carbon and amino acid metabolism.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas , Carbono/metabolismo , Proteínas de Unión al ADN/fisiología , Glutamato Sintasa/genética , Nitrógeno/metabolismo , Operón , Proteínas Represoras/fisiología , Bacillus subtilis/crecimiento & desarrollo , Medios de Cultivo , Mutación , FosforilaciónRESUMEN
Chemoheterotrophic bacteria use a few central metabolic pathways for carbon catabolism and energy production as well as for the generation of the main precursors for anabolic reactions. All sources of carbon and energy are converted to intermediates of these central pathways and then further metabolized. While the regulation of genes encoding enzymes used to introduce specific substrates into the central metabolism has already been studied to some detail, much less is known about the regulation of the central metabolic pathways. In this study, we investigated the responses of the Bacillus subtilis transcriptome to the presence of glucose and analyzed the role of the pleiotropic transcriptional regulator CcpA in these responses. We found that CcpA directly represses genes involved in the utilization of secondary carbon sources. In contrast, induction by glucose seems to be mediated by a variety of different mechanisms. In the presence of glucose, the genes encoding glycolytic enzymes are induced. Moreover, the genes responsible for the production of acetate from pyruvate with a concomitant substrate-level phosphorylation are induced by glucose. In contrast, the genes required for the complete oxidation of the sugar (Krebs cycle, respiration) are repressed if excess glucose is available for the bacteria. In the absence of glucose, the genes of the Krebs cycle as well as gluconeogenic genes are derepressed. The genes encoding enzymes of the pentose phosphate pathway are expressed both in the presence and the absence of glucose, as suggested by the central role of this pathway in generating anabolic precursors.
Asunto(s)
Bacillus subtilis/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Glucosa/metabolismo , Proteoma/genética , Proteoma/metabolismo , Transcripción Genética/fisiología , Acetatos/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ciclo del Ácido Cítrico/fisiología , Glucólisis/fisiología , Vía de Pentosa Fosfato/fisiología , Análisis por Matrices de Proteínas/métodos , Activación Transcripcional/fisiologíaRESUMEN
Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.