Size exclusion chromatography—An improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites

Persicke, Marcus; Plassmeier, Jens; Neuweger, Heiko; Rückert, Christian; Pühler, Alfred; Kalinowski, Jörn

doi:10.1016/j.jbiotec.2010.08.016

Cited by 9 publications

(5 citation statements)

References 34 publications

(40 reference statements)

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“…The chromatographic separation was accomplished utilizing a 30 m × 0.25 mm OPTIMA ® 5 MS Accent GC column with a 0.25 μm silarylene phase (Macherey-Nagel, Düren, Germany). Compound detection and normalization to the internal standard ribitol was performed as described in Persicke et al (2011), using the Xcalibur TM software (Version 1.4, Thermo Fisher, Dreieich, Germany) and the MeltDB software (Neuweger et al, 2008).…”

Section: Methodsmentioning

confidence: 99%

Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum

et al. 2019

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The alarmone species ppGpp and pppGpp are elementary components of bacterial physiology as they both coordinate the bacterial stress response and serve as fine-tuners of general metabolism during conditions of balanced growth. Since the regulation of (p)ppGpp metabolism and the effects of (p)ppGpp on cellular processes are highly complex and show massive differences between bacterial species, the underlying molecular mechanisms have so far only been insufficiently investigated for numerous microorganisms. In this study, (p)ppGpp physiology in the actinobacterial model organism Corynebacterium glutamicum was analyzed by phenotypic characterization and RNAseq-based transcriptome analysis. Total nutrient starvation was identified as the most effective method to induce alarmone production, whereas traditional induction methods such as the addition of serine hydroxamate (SHX) or mupirocin did not show a strong accumulation of (p)ppGpp. The predominant alarmone in C. glutamicum represents guanosine tetraphosphate, whose stress-associated production depends on the presence of the bifunctional RSH enzyme Rel. Interestingly, in addition to ppGpp, another substance yet not identified accumulated strongly under inducing conditions. A C. glutamicum triple mutant (Δrel,ΔrelS,ΔrelH) unable to produce alarmones [(p)ppGpp0 strain] exhibited unstable growth characteristics and interesting features such as an influence of illumination on its physiology, production of amino acids as well as differences in vitamin and carotenoid production. Differential transcriptome analysis using RNAseq provided numerous indications for the molecular basis of the observed phenotype. An evaluation of the (p)ppGpp-dependent transcriptional regulation under total nutrient starvation revealed a complex interplay with the involvement of ribosome-mediated transcriptional attenuation, the stress-responsive sigma factors σB and σH and transcription factors such as McbR, the master regulator of sulfur metabolism. In addition to the differential regulation of genes connected with various cell functions, the transcriptome analysis revealed conserved motifs within the promoter regions of (p)ppGpp-dependently and independently regulated genes. In particular, the representatives of translation-associated genes are both (p)ppGpp-dependent transcriptionally downregulated and show a highly conserved and so far unknown TTTTG motif in the −35 region, which is also present in other actinobacterial genera.

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Section: Methodsmentioning

confidence: 99%

Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum

et al. 2019

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“…Cell harvesting and metabolite extraction were performed as previously [ 48 , 49 ]. Two milliliters of bacterial culture were transferred into a 2 mL reaction tube with screw cap and centrifuged at 20,000 × g for 15 s. The supernatant was discarded and the cell pellet was immediately frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

et al. 2013

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BackgroundArginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, “classical” ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene.ResultsArginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families.ConclusionsThe N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising amongst others the genera Corynebacterium, Mycobacterium, and Nocardia. Therefore, the name C-NAGS (Corynebacterineae-type NAGS) is proposed for this new family.

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“…After the removal of the supernatant, cell pellets were immediately frozen at -80°C [28,29]. Subsequently, cell pellets were extracted three times by boiling at 90°C for 5 min as described previously [30].…”

Section: 1mentioning

confidence: 99%

Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production

Lai

Zhang

Liu

et al. 2012

Sci. China Life Sci.

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L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH r ). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22±1.41) μmol g CDM 1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH r improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C 1 units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.

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Size exclusion chromatography—An improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites

Cited by 9 publications

References 34 publications

Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum

Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum

A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production

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