RESUMO
Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B ⢠Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control ⢠Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.
Assuntos
Corynebacterium glutamicum , Aminoácidos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , AlemanhaRESUMO
Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on "The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes". On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.
Assuntos
Bioquímica , Bioengenharia , Biotecnologia , HumanosRESUMO
Microbially derived surfactants, so-called biosurfactants, have drawn much attention in recent years and are expected to replace current petrochemical surfactants, owing to their environmental and toxicological benefits. One strategy to support that goal is to reduce production costs by replacing relatively expensive sugars with cheaper raw materials, such as short-chain alkanes. Herein, we report the successful one-pot total synthesis of rhamnolipids, a class of biosurfactants with 12â stereocenters, from butane as sole carbon and energy source through the design of a tailored whole-cell biocatalyst.
RESUMO
Sphingolipids are not only essential components of biological membranes but also play numerous other vital functions in living cells. Moreover, they are major constituents of the outermost layer of human epidermis which acts as permeability barrier of the skin. Therefore, they have a high potential to be used in a wide variety of application fields such as antibacterial and antifungal agents, active pharmaceutical ingredients of therapeutics as well as active ingredients in cosmeceutical or nutraceutical formulations. However, their chemical synthesis is a complex and cost-intensive process. As the yeast Wickerhamomyces ciferrii has been found to be a natural producer of acetylated sphingoid bases, it provides a promising alternative for their biotechnological synthesis. In the last years, this yeast has been established by classical strain improvements as well as modern genetic engineering for the industrial production of phytosphingosine. Moreover, routes for the synthesis of sphinganine and sphingosine have been implemented. This mini-review summarizes the current knowledge about biosynthesis of sphingoid bases, genetic engineering of W. ciferrii for their biotechnological production, as well as their applications in cosmetic formulations.
Assuntos
Biotecnologia , Esfingolipídeos/biossíntese , Engenharia Genética , Pichia/genética , Pichia/metabolismo , Esfingolipídeos/metabolismoRESUMO
A modified synthetic acetone operon was constructed. It consists of two genes from Clostridium acetobutylicum (thlA coding for thiolase and adc coding for acetoacetate decarboxylase) and one from Bacillus subtilis or Haemophilus influenzae (teII(srf) or ybgC, respectively, for thioesterase). Expression of this operon in Escherichia coli resulted in the production of acetone starting from the common metabolite acetyl-CoA via acetoacetyl-CoA and acetoacetate. The thioesterases do not need a CoA acceptor for acetoacetyl-CoA hydrolysis. Thus, in contrast to the classic acetone pathway of Clostridium acetobutylicum and related microorganisms which employ a CoA transferase, the new pathway is acetate independent. The genetic background of the host strains was crucial. Only E. coli strains HB101 and WL3 were able to produce acetone via the modified plasmid based pathway, up to 64mM and 42mM in 5-ml cultures, respectively. Using glucose fed-batch cultures the concentration could be increased up to 122mM acetone with HB101 carrying the recombinant plasmid pUC19ayt (thioesterase from H. influenzae). The formation of acetone led to a decreased acetate production by E. coli.
Assuntos
Acetona/metabolismo , Aciltransferases/genética , Carboxiliases/genética , Escherichia coli/fisiologia , Engenharia Metabólica/métodos , Transdução de Sinais/genética , Tioléster Hidrolases/genética , Acetona/isolamento & purificaçãoRESUMO
Wickerhamomyces ciferrii is a microorganism characterized by the production and secretion of large amounts of acetylated sphingoid bases, in particular tetraacetyl phytosphingosine. Here, we present the 15.90-Mbp draft genome sequence of W. ciferrii NRRL Y-1031 F-60-10 generated by pyrosequencing and de novo assembly. The draft genome sequence comprising 364 contigs in 150 scaffolds was annotated and covered 6,702 protein-coding sequences. This information will contribute to the metabolic engineering of this yeast to improve the yield and spectrum of acetylated sphingoid bases in biotechnological production.
Assuntos
Genoma Fúngico , Pichia/genética , Sequência de Bases , Mapeamento de Sequências Contíguas , Bases de Dados Genéticas , Anotação de Sequência Molecular , Dados de Sequência MolecularRESUMO
The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase(PcSYR2), as well as structural genes for dihydroceramide Δ(4)-desaturase (PcDES1) and sphingolipid Δ(8)-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts. In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240mgL(-1) triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890mgkg(-1) triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications.
Assuntos
Engenharia Metabólica/métodos , Pichia/enzimologia , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Ceramidase Alcalina/genética , Ceramidase Alcalina/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Pichia/genética , Esfingosina/genéticaRESUMO
Plant polyphenols have been the subject of several recent scientific investigations since many of the molecules in this class have been found to be highly active in the human body, with a plethora of health-promoting activities against a variety of diseases, including heart disease, diabetes, and cancer, and with even the potential to slow aging. Further development of these potent natural therapeutics hinges on the formation of robust industrial production platforms designed using specifically selected as well as engineered protein sources along with the construction of optimal expression platforms. In this work, we first report the investigation of various stilbene synthases from an array of plant species considering structure-activity relationships, their expression efficiency in microorganisms, and their ability to synthesize resveratrol. Second, we looked into the construct environment of recombinantly expressed stilbene synthases, including different promoters, construct designs, and host strains, to create an Escherichia coli strain capable of producing superior resveratrol titers sufficient for commercial usage. Further improvement of metabolic capabilities of the recombinant strain aimed at improving the intracellular malonyl-coenzyme A pool, a resveratrol precursor, resulted in a final improved titer of 2.3 g/liter resveratrol.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Redes e Vias Metabólicas/genética , Estilbenos/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Cromatografia Líquida de Alta Pressão , Expressão Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ResveratrolRESUMO
Resveratrol synthesis from p-coumarate was analyzed in different Saccharomyces cerevisiae strains expressing the 4-coumaroyl-coenzyme A ligase (4CL1) from Arabidopsis thaliana and the stilbene synthase (STS) from Vitis vinifera and compared between yeast cultures growing in rich or synthetic medium. The use of rich medium considerably improved resveratrol production, and resveratrol yields of up to 391 mg/liter could be achieved with an industrial Brazilian sugar cane-fermenting yeast.
Assuntos
Microbiologia Industrial/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estilbenos , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brasil , Meios de Cultura , DNA Recombinante/genética , Resveratrol , Vitis/genética , Vitis/metabolismoRESUMO
In this work, the molecular basis of aerobic citrate utilization by the gram-positive bacterium Corynebacterium glutamicum was studied. Genome analysis revealed the presence of two putative citrate transport systems. The permease encoded by citH belongs to the citrate-Mg(2+):H(+)/citrate-Ca(2+):H(+) symporter family, whereas the permease encoded by the tctCBA operon is a member of the tripartite tricarboxylate transporter family. The expression of citH or tctCBA in Escherichia coli enabled this species to utilize citrate aerobically, indicating that both CitH and TctABC are functional citrate transporters. Growth tests with the recombinant E. coli strains indicated that CitH is active with Ca(2+) or Sr(2+) but not with Mg(2+) and that TctABC is active with Ca(2+) or Mg(2+) but not with Sr(2+). We could subsequently show that, with 50 mM citrate as the sole carbon and energy source, the C. glutamicum wild type grew best when the minimal medium was supplemented with CaCl(2) but that MgCl(2) and SrCl(2) also supported growth. Each of the two transporters alone was sufficient for growth on citrate. The expression of citH and tctCBA was activated by citrate in the growth medium, independent of the presence or absence of glucose. This activation was dependent on the two-component signal transduction system CitAB, composed of the sensor kinase CitA and the response regulator CitB. CitAB belongs to the CitAB/DcuSR family of two-component systems, whose members control the expression of genes that are involved in the transport and catabolism of tricarboxylates or dicarboxylates. C. glutamicum CitAB is the first member of this family studied in Actinobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Ácido Cítrico/metabolismo , Corynebacterium glutamicum/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas Quinases/genética , Regulação para Cima , Proteínas de Bactérias/genética , Transporte Biológico , Proteínas de Transporte/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases/metabolismoRESUMO
We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL 21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 310 nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26 min. The K(m) and V(max) values were determined as follows: l-alanine to d-alanine, K(m) (l-alanine) 5.01 mM and V(max) 306 U/mg; d-alanine to l-alanine, K(m) (d-alanine) 5.24 mM and V(max) 345 U/mg. The K(eq) value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C. glutamicum ATCC 13032 is expected to be a biocatalyst for d-alanine production from the l-counter part.
Assuntos
Alanina Racemase/genética , Alanina Racemase/metabolismo , Corynebacterium glutamicum/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Alanina/metabolismo , Alanina Racemase/química , Alanina Racemase/isolamento & purificação , Clonagem Molecular , Escherichia coli/enzimologia , Expressão Gênica , Genes Bacterianos , Filogenia , Subunidades Proteicas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Transformação BacterianaRESUMO
In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.
Assuntos
Acetatos/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica , Transativadores/metabolismo , Transcrição Gênica , Proteínas de Bactérias/genética , Sítios de Ligação , Regiões Promotoras Genéticas , Ligação Proteica , Transativadores/genéticaRESUMO
Corynebacterium glutamicum contains genes for 13 two-component signal transduction systems. In order to test for their essentiality and involvement in the adaptive response to phosphate (Pi) starvation, a set of 12 deletion mutants was constructed. One of the mutants was specifically impaired in its ability to grow under Pi limitation, and therefore the genes lacking in this strain were named phoS (encoding the sensor kinase) and phoR (encoding the response regulator). DNA microarray analyses with the C. glutamicum wild type and the DeltaphoRS mutant supported a role for the PhoRS system in the adaptation to Pi starvation. In contrast to the wild type, the DeltaphoRS mutant did not induce the known Pi starvation-inducible (psi) genes within 1 hour after a shift from Pi excess to Pi limitation, except for the pstSCAB operon, which was still partially induced. This indicates an activator function for PhoR and the existence of at least one additional regulator of the pst operon. Primer extension analysis of selected psi genes (pstS, ugpA, phoR, ushA, and nucH) confirmed the microarray data and provided evidence for positive autoregulation of the phoRS genes.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteínas de Bactérias/genética , Sequência de Bases , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Meios de Cultura , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Óperon , Fosfatos , Proteínas Quinases/genética , RNA Mensageiro/genéticaRESUMO
Expression of the structural genes encoding the ATP-dependent proteases ClpCP and Lon in Corynebacterium glutamicum and Streptomyces lividans is activated by the transcriptional regulator ClgR in response to yet unknown environmental stimuli. As it was not known whether ClgR controls expression of additional genes we used DNA microarrays in order to comprehensively define the ClgR regulon in C. glutamicum. The mRNA levels of 16 genes decreased >/= 2-fold in a DeltaclgRDeltaclpC mutant (ClgR absent) compared with a DeltaclpC mutant (ClgR present). For five genes in four operons (NCgl0748, ptrB, hflX and NCgl0240-recR) regulation by ClgR could be independently verified by primer extension analyses and confirmation of binding of purified ClgR to the regulatory regions of these operons. ptrB encodes an endopeptidase, which is consistent with the proteolytic functions of the genes already known to be under ClgR control. However, RecR is unrelated to proteolysis but required for recombinational repair of UV-induced DNA damage. Possibly ClgR-dependent activation of gene expression is triggered by environmental stresses damaging both proteins and nucleic acids, although DNA damage induced by UV radiation and mitomycin C treatment did not result in ClgR-dependent transcriptional activation of any of the newly identified ClgR regulon members. In order to functionally analyse the NCgl0748 and hflX genes we have constructed C. glutamicum strains with deletions in these genes. The DeltaNCgl0748 mutant displayed reduced growth rates in minimal and rich media. The NCgl0748 protein was shown to be localized in the cytoplasm only, while the HflX pool is equally distributed between cytoplasm and plasma membrane. In order to study the proposed degradation of ClgR by ClpCP we have constructed a conditional clpP1P2 mutant. Depletion of ClpP1 and ClpP2 in that strain resulted in the accumulation of ClgR, indicating that ClgR is in fact a substrate of the ClpCP1 and/or ClpCP2 protease in C. glutamicum.
Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Reparo do DNA/genética , Regulação Bacteriana da Expressão Gênica , Peptídeo Hidrolases/genética , Transativadores/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dano ao DNA , Pegada de DNA , DNA Bacteriano/metabolismo , Endopeptidase Clp/genética , Deleção de Genes , Mitomicina , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Protease La/genética , Ligação Proteica , RNA Bacteriano/análise , RNA Mensageiro/análise , Sequências Reguladoras de Ácido Nucleico , Regulon/genética , Transativadores/genética , Raios UltravioletaRESUMO
The MtrAB two-component signal transduction system is highly conserved in sequence and genomic organization in Mycobacterium and Corynebacterium species, but its function is completely unknown. Here, the role of MtrAB was studied with C. glutamicum as model organism. In contrast to M. tuberculosis, it was possible to delete the mtrAB genes in C. glutamicum. The mutant cells showed a radically different cell morphology and were more sensitive to penicillin, vancomycin and lysozyme but more resistant to ethambutol. In order to identify the molecular basis for this pleiotropic phenotype, the mRNA profiles of mutant and wild type were compared with DNA microarrays. Three genes showed a more than threefold increased RNA level in the mutant, i.e. mepA (NCgl2411) encoding a putative secreted metalloprotease, ppmA (NCgl2737 ) encoding a putative membrane-bound protease modulator, and lpqB encoding a putative lipoprotein of unknown function. Expression of plasmid-encoded mepA in Escherichia coli led to elongated cells that were hypersensitive to an osmotic downshift, supporting the idea that peptidoglycan is the target of MepA. The mRNA level of two genes was more than fivefold decreased in the mutant, i.e. betP and proP which encode transporters for the uptake of betaine and proline respectively. The microarray results were confirmed by primer extension and RNA dot blot experiments. In the latter, the transcript level of genes involved in osmoprotection was tested before and after an osmotic upshift. The mRNA level of betP, proP and lcoP was strongly reduced or undetectable in the mutant, whereas that of mscL (mechanosensitive channel) was increased. The changes in cell morphology, antibiotics susceptibility and the mRNA levels of betP, proP, lcoP, mscL and mepA could be reversed by expression of plasmid-encoded copies of mtrAB in the DeltamtrAB mutant, confirming that these changes occurred as a consequence of the mtrAB deletion.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Corynebacterium glutamicum , Farmacorresistência Bacteriana/fisiologia , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Equilíbrio Hidroeletrolítico/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Forma Celular , Corynebacterium glutamicum/efeitos dos fármacos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/ultraestrutura , Perfilação da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
P(II)-type signal transduction proteins play a central role in nitrogen regulation in many bacteria. In response to the intracellular nitrogen status, these proteins are rendered in their function and interaction with other proteins by modification/demodification events, e.g. by phosphorylation or uridylylation. In this study, we show that GlnK, the only P(II)-type protein in Corynebacterium glutamicum, is adenylylated in response to nitrogen starvation and deadenylylated when the nitrogen supply improves again. Both processes depend on the GlnD protein. As shown by mutant analyses, the modifying activity of this enzyme is located in the N-terminal part of the enzyme, while demodification depends on its C-terminal domain. Besides its modification status, the GlnK protein changes its intracellular localization in response to changes of the cellular nitrogen supply. While it is present in the cytoplasm during nitrogen starvation, the GlnK protein is sequestered to the cytoplasmic membrane in response to an ammonium pulse following a nitrogen starvation period. About 2-5% of the GlnK pool is located at the cytoplasmic membrane after ammonium addition. GlnK binding to the cytoplasmic membrane depends on the ammonium transporter AmtB, which is encoded in the same transcriptional unit as GlnK and GlnD, the amtB-glnK-glnD operon. In contrast, the structurally related methylammonium/ammonium permease AmtA does not bind GlnK. The membrane-bound GlnK protein is stable, most likely to inactivate AmtB-dependent ammonium transport in order to prevent a detrimental futile cycle under post-starvation ammonium-rich conditions, while the majority of GlnK is degraded within 2-4 min. Proteolysis in the transition period from nitrogen starvation to nitrogen-rich growth seems to be specific for GlnK; other proteins of the nitrogen metabolism, such as glutamine synthetase, or proteins unrelated to ammonium assimilation, such as enolase and ATP synthase subunit F(1)beta, are stable under these conditions. Our analyses of different mutant strains have shown that at least three different proteases influence the degradation of GlnK, namely FtsH, the ClpCP and the ClpXP protease complex.
Assuntos
Corynebacterium/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Corynebacterium/genética , Endopeptidase Clp/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência MolecularRESUMO
The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.
Assuntos
Acetatos/metabolismo , Proteínas de Bactérias/fisiologia , Corynebacterium/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/fisiologia , Acetato Quinase/genética , Corynebacterium/genética , Corynebacterium/crescimento & desenvolvimento , Isocitrato Liase/genética , Malato Sintase/genética , Óperon , Fosfato Acetiltransferase/genética , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The ATP-dependent protease Clp plays important roles in the cell's protein quality control system and in the regulation of cellular processes. In Corynebacterium glutamicum, the levels of the proteolytic subunits ClpP1 and ClpP2 as well as of the corresponding mRNAs were drastically increased upon deletion of the clpC gene, coding for a Clp ATPase subunit. We identified a regulatory protein, designated ClgR, binding to a common palindromic sequence motif in front of clpP1P2 as well as of clpC. Deletion of clgR in the DeltaclpC background completely abolished the increased transcription of both operons, indicating that ClgR activates transcription of these genes. ClgR activity itself is probably controlled via ClpC-dependent regulation of its stability, as ClgR is only present in DeltaclpC and not in wild-type cells, whereas the levels of clgR mRNA are comparable in both strains. clpC, clpP1P2 and clgR expression is induced upon severe heat stress, however, independently of ClgR. Identification of the heat-responsive transcriptional start sites in front of these genes revealed the presence of sequence motifs typical for sigmaECF-dependent promoters. The ECF sigma factor sigmaH could be identified as being required for transcriptional activation of clpC, clpP1P2 and clgR in response to severe heat stress. A second heat-responsive but sigmaH-independent promoter in front of clgR could be identified that is subject to negative regulation by the transcriptional repressor HspR. Taken together, these results show that clpC and clpP1P2 expression in C. glutamicum is subject to complex regulation via both independent and hierarchically organized pathways, allowing for the integration of multiple environmental stimuli. Both the ClgR- and sigmaH-dependent regulation of clpC and clpP1P2 expression appears to be conserved in other actinomycetes.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Repressoras/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fator sigma/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Corynebacterium/fisiologia , Endopeptidase Clp , Deleção de Genes , Genes Bacterianos , Resposta ao Choque Térmico , Dados de Sequência Molecular , Óperon , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Regulon , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sítio de Iniciação de Transcrição , Transcrição Gênica , Ativação TranscricionalRESUMO
In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.