RESUMEN
BACKGROUND: C. glutamicum has traditionally been grown in neutral-pH media for amino acid production, but in a previous article we reported that this microorganism is a moderate alkaliphile since it grows optimally at pH 7.0-9.0, as shown in fermentor studies under tightly controlled pH conditions. We determined the best pH values to study differential expression of several genes after acidic or basic pH conditions (pH 6.0 for acidic expression and pH 9.0 for alkaline expression). Thus, it was interesting to perform a detailed analysis of the pH-adaptation response of the proteome of C. glutamicum ATCC 13032 to clarify the circuits involved in stress responses in this bacterium. In this paper we used the above indicated pH conditions, based on transcriptional studies, to confirm that pH adaptation results in significant changes in cytoplasmatic and membrane proteins. RESULTS: The cytoplasmatic and membrane proteome of Corynebacterium glutamicum ATCC 13032 at different pH conditions (6.0, 7.0 and 9.0) was analyzed by classical 2D-electrophoresis, and by anion exchange chromatography followed by SDS-PAGE (AIEC/SDS-PAGE). A few cytoplasmatic proteins showed differential expression at the three pH values with the classical 2D-technique including a hypothetical protein cg2797, L-2.3-butanediol dehydrogenase (ButA), and catalase (KatA). The AIEC/SDS-PAGE technique revealed several membrane proteins that respond to pH changes, including the succinate dehydrogenase complex (SdhABCD), F0F1-ATP synthase complex subunits b, alpha and delta (AtpF, AtpH and AtpA), the nitrate reductase II alpha subunit (NarG), and a hypothetical secreted/membrane protein cg0752. Induction of the F0F1-ATP synthase complex beta subunit (AtpD) at pH 9.0 was evidenced by Western analysis. By contrast, L-2.3-butanediol dehydrogenase (ButA), an ATPase with chaperone activity, the ATP-binding subunit (ClpC) of an ATP-dependent protease complex, a 7 TMHs hypothetical protein cg0896, a conserved hypothetical protein cg1556, and the dihydrolipoamide acyltransferase SucB, were clearly up-regulated at pH 6.0. CONCLUSION: The observed protein changes explain the effect of the extracellular pH on the growth and physiology of C. glutamicum. Some of the proteins up-regulated at alkaline pH respond also to other stress factors suggesting that they serve to integrate the cell response to different stressing conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/fisiología , Citoplasma/metabolismo , Proteoma/metabolismo , Proteínas Bacterianas/genética , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Citoplasma/química , Citoplasma/genética , Electroforesis en Gel Bidimensional , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteoma/química , Proteoma/genética , Estrés FisiológicoRESUMEN
Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. To characterize the citrate utilization in C. glutamicum on a genomewide scale, a comparative analysis was carried out by combining transcriptome and proteome analysis. In cells grown on citrate, transcriptome analysis revealed highest expression changes for two different citrate-uptake systems encoded by citM and tctCBA, whereas genes encoding uptake systems for the glucose- (ptsG), sucrose- (ptsS) and fructose- (ptsF) specific PTS components and permeases for gluconate (gntP) and glutamate (gluC) displayed decreased mRNA levels in citrate-grown cells. This pattern was also observed when cells grown in Luria-Bertani (LB) medium plus citrate were compared with cells grown in LB medium, indicating some kind of catabolite repression. Genes encoding enzymes of the tricarboxylic acid cycle (aconitase, succinyl-CoA synthetase, succinate dehydrogenase and fumarase), malic enzyme, PEP carboxykinase, gluconeogenic glyceraldehyde-3-phosphate dehydrogenase and ATP synthase displayed increased expression in cells grown on citrate. Accordingly, proteome analysis revealed elevated protein levels of these enzymes and showed a good correlation with the mRNA levels. In conclusion, this study revealed the citrate stimulon in C. glutamicum and the regulated central metabolic genes when grown on citrate.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Ácido Cítrico/metabolismo , Corynebacterium glutamicum/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteoma/análisis , ARN Mensajero/análisis , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Perfilación de la Expresión Génica , Redes y Vías Metabólicas/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The membrane proteomes of a wild-type Corynebacterium glutamicum and an L-lysine-producing strain were quantitatively analyzed by two complementary proteomics techniques -- anion exchange chromatography AIEC/SDS-PAGE and 16BAC-PAGE/SDS-PAGE -- and the results were compared. Although both techniques allow for the fast screening of differences in protein abundance, AIEC/SDS-PAGE was superior to 16BAC-PAGE/SDS-PAGE with respect to protein separation, it was more suitable for relative protein quantification, and allowed more differentially regulated proteins to be detected (the succinate dehydrogenase complex, an ABC-type cobalamin/Fe(3+) siderophore transport system, the maltose binding protein, and a subunit of the cytochrome bc-aa(3) supercomplex were upregulated, while a periplasmic component of an ABC-type transporter and an iron-regulated ABC-type transporter were downregulated in the producer). The results indicate the important role of tricarboxylic acid cycle enzymes as well as the adaptation of transport processes in L-lysine-producing cells. Since the only genetic differences between the wild type and the L-lysine producer occur between four central metabolic enzymes in the cytoplasm, our study illustrates the complex effects of metabolic engineering on cell physiology and the power of the new AIEC/SDS-PAGE proteomics approach to detect these effects.
Asunto(s)
Corynebacterium glutamicum/metabolismo , Lisina/biosíntesis , Proteínas de la Membrana/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Betaína/análogos & derivados , Betaína/química , Cromatografía por Intercambio Iónico/métodos , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Alcoholes Grasos , Genotipo , Proteínas de la Membrana/análisis , Modelos Biológicos , Proteoma/análisis , Compuestos de Amonio Cuaternario/química , Reproducibilidad de los Resultados , Succinato Deshidrogenasa/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis. RESULTS: In contrast to the situation in other bacteria, deletion of C. glutamicum ftsH has no significant effect on growth in standard minimal medium or response to heat or osmotic stress. On the proteome level, deletion of the ftsH gene resulted in a strong increase of ten cytoplasmic and membrane proteins, namely biotin carboxylase/biotin carboxyl carrier protein (accBC), glyceraldehyde-3-phosphate dehydrogenase (gap), homocysteine methyltransferase (metE), malate synthase (aceB), isocitrate lyase (aceA), a conserved hypothetical protein (NCgl1985), succinate dehydrogenase A (sdhA), succinate dehydrogenase B (sdhB), succinate dehydrogenase CD (sdhCD), and glutamate binding protein (gluB), while 38 cytoplasmic and membrane-associated proteins showed a decreased abundance. The decreasing amount of succinate dehydrogenase A (sdhA) in the cytoplasmic fraction of the ftsH mutant compared to the wild type and its increasing abundance in the membrane fraction indicates that FtsH might be involved in the cleavage of a membrane anchor of this membrane-associated protein and by this changes its localization. CONCLUSION: The data obtained hint to an involvement of C. glutamicum FtsH protease mainly in regulation of energy and carbon metabolism, while the protease is not involved in stress response, as found in other bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/enzimología , Metaloproteasas/metabolismo , Proteómica , Aminoácidos/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Carbono/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crecimiento & desarrollo , Corynebacterium glutamicum/metabolismo , Citoplasma/química , Electroforesis en Gel Bidimensional , Metabolismo Energético , Eliminación de Gen , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas , Metaloproteasas/genéticaRESUMEN
In order to avoid the specific problems with intrinsic membrane proteins in proteome analysis, a new procedure was developed which is superior to the classical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method in terms of intrinsic membrane proteins. For analysis of the membrane proteome from Corynebacterium glutamicum, we replaced the first separation dimension, i.e., the isoelectric focusing step, by anion-exchange chromatography, followed by sodium dodecyl sulfate (SDS)-PAGE in the second separation dimension. Enrichment of the membrane intrinsic subproteome was achieved by washing with 2.5 M NaBr which removed more than 35% of the membrane-associated soluble proteins. For the extraction and solubilization of membrane proteins, the detergent amidosulfobetaine 14 (ASB-14) was most efficient in a detailed screening procedure and proved also suitable for chromatography. 356 gel bands were spotted, and out of 170 different identified proteins, 50 were membrane-integral. Membrane proteins with one up to 13 transmembrane helices were found. Careful analysis revealed that this new procedure covers proteins from a wide pI range (3.7-10.6) and a wide mass range of 10-120 kDa. About 50% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, protein translocation, and proteolysis while for the others a function is not yet known, indicating the potential of the developed method for elucidation of membrane proteomes in general.
Asunto(s)
Proteínas Bacterianas/análisis , Membrana Celular/química , Corynebacterium glutamicum/química , Proteínas de la Membrana/análisis , Proteoma/análisis , Fraccionamiento Celular , Cromatografía por Intercambio Iónico , Detergentes/química , Electroforesis en Gel Bidimensional/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803. PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis. For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities. Therefore we cloned the corresponding genes into expression vectors. To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms. Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies. Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC). BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions. An optimized refolding protocol was established that yielded pure BtpA. In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity. Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins. Both Ycf3 and BtpA show homo-oligomerization in vitro. In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.