Quantification of proteome dynamics in Corynebacterium glutamicum by 15N-labeling and selected reaction monitoring

Voges, Raphael; Noack, Stephan

doi:10.1016/j.jprot.2012.03.020

Cited by 25 publications

(21 citation statements)

References 51 publications

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“…DNA microarray and quantitative reverse transcription-PCR experiments confirmed an acetate-dependent transcriptional regulation of the pck gene (38). In C. glutamicum ATCC 13032, a growth phase-dependent regulation of pck gene expression was not observed by either enzyme activity (23) or quantitative protein measurements (39), whereas in C. glutamicum R, growth-phase-dependent differences in the pck transcript level on glucose were reported, with maxima in the exponential and early stationary phases (40). The presence of a binding site for the CRP homologue GlxR in the pck promoter region and the fact that GlxR binds to the pck promoter in a cAMP-dependent manner in vitro (40,41) indicate an involvement of GlxR in regulation of pck transcription.…”

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confidence: 80%

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

Klaffl¹,

Brocker²,

Kalinowski³

et al. 2013

Journal of Bacteriology

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c DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2, and IolR. Determination of the phosphoenolpyruvate carboxykinase activity of the ⌬ramA, ⌬gntR1 ⌬gntR2, and ⌬iolR deletion mutants indicated that RamA represses pck during growth on glucose about 2-fold, whereas GntR1, GntR2, and IolR activate pck expression about 2-fold irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myoinositol uptake and degradation. Comparative DNA microarray analysis of the ⌬iolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5=-KGWCHTRACA-3=) which corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.

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confidence: 80%

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

Klaffl¹,

Brocker²,

Kalinowski³

et al. 2013

Journal of Bacteriology

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“…Further processing and analysis was performed as described previously (Voges and Noack, 2012). For each time point, nine samples were taken for the reference processes (three technical replicates (n T ¼ 3) for each of the three biological replicates (n B ¼ 3) and six samples for the scale-down processes (n B ¼ 2 and n T ¼ 3).…”

Section: Proteome Analysismentioning

confidence: 99%

“…The targeted quantification method via LC-MS/MS was developed by Voges and Noack (2012) with triple quadrupole MS and incorporates 23 specific enzymes from the central carbon metabolism of C. glutamicum. Relative quantification was done for three characteristic peptides per protein.…”

Section: Proteome Analysismentioning

confidence: 99%

Metabolic profile of 1,5‐diaminopentane producing Corynebacterium glutamicum under scale‐down conditions: Blueprint for robustness to bioreactor inhomogeneities

Limberg

Schulte

Aryani

et al. 2016

Biotech & Bioengineering

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Performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. Especially, Corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. This study focuses on the identification of metabolic key mechanisms for this robustness to get a deeper insight and provide future targets for process orientated strain development. A 1,5-diaminopentane producing C. glutamicum strain was cultivated in a two compartment scale-down device to create short-term environmental changes simulating industrial scale cultivation conditions. Using multi omics based methods, it is shown, that central metabolism is flexibly rearranged under short-term oxygen depletion and carbon source excess to overcome shortage in NAD recycling. In order to balance the redox state, key enzymes for the non-oxygen dependent fermentative NAD regeneration were significantly up-regulated while parts of non-essential pathways were down-regulated. The transfer of the cells back into the well aerated zones with low substrate concentration triggers an additional upregulation of genes for the re-assimilation of previously formed side products, showing L-lactate forming and utilizing reactions being active at the same time. Especially L-lactate as reversible and flexible external buffer for carbon and redox equivalents puts C. glutamicum in a robust position to deal with inhomogeneity in large scale processes. Biotechnol. Bioeng. 2017;114: 560-575. © 2016 Wiley Periodicals, Inc.

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“…Predominantly, quantitative mass spectrometry is being employed to capture a snapshot of the cellular proteome at any given time-point – in that a relative and/or absolute quantification are carried out to determine alterations in abundance or absolute protein copy-number. There are, currently, several publications existing that deal with protein turnover in four different species belonging to the order Actinomycetales : (i) transition from exponential growth to stationary phase in antibiotics producer Streptomyces coelicolor , (ii) iron starvation and acid shock in model strain Mycobacterium smegmatis , (iii) iron starvation in the human pathogen Mycobacterium tuberculosis and (iv) salt stress, growth on different carbon sources as well as heat adaptation in the amino acid producer C. glutamicum (Jayapal et al ., 2008; 2010; Rao et al ., 2008a,b; Franzel et al ., 2010; Trotschel et al ., 2012; Voges and Noack, 2012). All these species belong to the same class, Actinobacteria , a very large and diverse group of Gram-positive, high G + C bacteria (Ventura et al ., 2012).…”

Section: Case Studiesmentioning

confidence: 99%

Proteome turnover in bacteria: current status for Corynebacterium glutamicum and related bacteria

Trötschel

Albaum

Poetsch

2013

Microbial Biotechnology

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With the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and degradation rates (i.e. protein turnover) on a proteome-wide scale by employing stable isotope-labelled amino acids has become feasible. These dynamic data provide a deeper understanding of protein homeostasis and stress response mechanisms in microorganisms than well-established ‘steady state’ proteomics approaches. In this article, we summarize the technological challenges and solutions both on the biochemistry/mass spectrometry and bioinformatics level for turnover proteomics with a focus on chromatographic techniques. Although the number of available case studies for Corynebacterium glutamicum and related actinobacteria is still very limited, our review illustrates the potential of protein turnover studies for an improved understanding of questions in the area of biotechnology and biomedicine. Here, new insights from investigations of growth phase transition and different stress dynamics including iron, acid and heat stress for pathogenic but also for industrial actinobacteria are presented. Finally, we will comment on the advantages of integrated software solutions for biologists and briefly discuss the remaining technical challenges and upcoming possibilities for protein turnover analysis.

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Quantification of proteome dynamics in Corynebacterium glutamicum by 15N-labeling and selected reaction monitoring

Cited by 25 publications

References 51 publications

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

Metabolic profile of 1,5‐diaminopentane producing Corynebacterium glutamicum under scale‐down conditions: Blueprint for robustness to bioreactor inhomogeneities

Proteome turnover in bacteria: current status for Corynebacterium glutamicum and related bacteria

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