Quantitative differential monitoring of the metabolic activity of Corynebacterium glutamicum cultures utilizing a light-addressable potentiometric sensor system.

Applying biosensors for evaluation of the extracellular acidification of microorganisms in various biotechnological fermentation processes is on demand. An early stage detection of disturbances in the production line would avoid costly interventions related to metabolically inactive microorganisms. Furthermore, the determination of the number of living cells through cell plating procedure after cultivations is known as time- and material-consuming. In this work, a differential light-addressable potentiometric sensor (LAPS) system was developed to monitor the metabolic activity of Corynebacterium glutamicum (C. glutamicum ATCC13032) as typical microorganism in fermentation processes. In this context, the number of living cells in suspensions was directly determined utilizing the read-out principle of the LAPS system. The planar sensor surface of the LAPS design allows to fixate 3D-printed multi-chamber structures, which enables differential measurements. In this way, undesirable external influences such as pH variations of the medium and sensor signal drift can be compensated.

Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication.

The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the ß-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-ß-clamp complex formed via a carboxy-terminal sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system.

Complete genome sequencing of newly isolated thermotolerant Corynebacterium glutamicum N24 provides a new insights into its thermotolerant phenotype.

To understand the genetic background of thermotolerance, we determined the complete genome sequence of a thermotolerant Corynebacterium glutamicum N24 strain isolated from soil. The whole genome based phylogenetic analysis between N24 and other related species revealed that N24 diverged from other C. glutamicum strains at earlier stages. Comparisons of thermotolerance between N24 and its related species showed that N24 and Corynebacterium efficiens YS-314 have a higher thermotolerance than Corynebacterium callunae DSM 20147 and C. glutamicum KY9002. In order to understand the link between a particular genetic background and thermotolerance, we compared the genomes of these four strains by mapping their genomes onto the N24 chromosome. We then determined the genes that were conserved in the thermotolerant species. Our results indicated the specific presence of glutathione-dependent aldehyde dehydrogenase, two sortase homologs, and lipid synthesis-related genes in the genomes of thermotolerant strains, YS-314 and N24. Therefore, these genes may be responsible for the differences in their thermotolerant phenotypes.

Proteomics of FACS-sorted heterogeneous Corynebacterium glutamicum populations.

The metabolic status of individual cells in microbial cultures can differ, being relevant for biotechnology, environmental and medical microbiology. However, it is hardly understood in molecular detail due to limitations of current analytical tools. Here, we demonstrate that FACS in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. A previously established GFP reporter system was used to detect and sort single Corynebacterium glutamicum cells based on the concentration of branched chain amino acids (BCAA) using FACS. A proteomics workflow optimized for small cell numbers was used to quantitatively compare proteomes of a ΔaceE mutant, lacking functional pyruvate dehydrogenase (PD), and the wild type. About 800 proteins could be quantified from 1,000,000 cells. In the ΔaceE mutant BCAA production was coordinated with upregulation of the glyoxylate cycle and TCA cycle to counter the lack of acetyl CoA resulting from the deletion of aceE. BIOLOGICAL SIGNIFICANCE: Metabolic pathways in C. glutamicum WT and ΔaceE, devoid of functional pyruvate dehydrogenase, were compared to understand proteome changes that contribute to the high production of branched chain amino acids (BCAA) in the ΔaceE strain. The data complements previous metabolome studies and corroborates the role of malate provided by the glyoxylate cycle and increased activity of glycolysis and pyruvate carboxylase reaction to replenish the TCA cycle. A slight increase in acetohydroxyacid synthase (ILV subunit B) substantiates the previously reported increased pyruvate pool in C. glutamicumΔaceE, and the benefit of additional ilv gene cluster overexpression for BCAA production.

Picoliter nDEP traps enable time-resolved contactless single bacterial cell analysis in controlled microenvironments.

We present a lab-on-a-chip device, the Envirostat 2.0, which allows for the first time contactless cultivation of a single bacterial cell by negative dielectrophoresis (nDEP) in a precisely controllable microenvironment. Stable trapping in perfusing growth medium was achieved by a miniaturization of octupole electrode geometries, matching the dimensions of bacteria. Temperature sensitive fluorescent measurements showed that these reductions of microelectrode distances led to reduced Joule heating during cell manipulation. The presented miniaturization is not possible with conventional manufacturing processes. Therefore, we present a novel bonding technology, which permits miniaturization of 3D octupole electrode geometry with biocompatible materials. To exclude the influence of other cells and to enable sampling of perfusion medium from the isolated living bacterium under study, computer aided flow simulations were used to develop a microfluidic nDEP isolation procedure. The developed microchannel and microelectrode design integrates for the first time well characterized nDEP cell sorting mechanisms and time-resolved contactless single bacterial cell cultivation in a 1.7 picoliter bioreactor system. The cell type independent trapping is demonstrated with singularized Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum and other industrially relevant microbes. The static and precisely controlled microenvironment resulted in a consistent and significant faster growth compared to maximal growth rates observed on population level. Preventing the influence of surfaces and cell-cell interactions, the Envirostat 2.0 chip permits total microenvironmental control by the experimenter and therefore provides major opportunities for microfluidic based cell analysis of bacteria and small eukaryotes.

Size exclusion chromatography: an improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites.

The efficient separation of Corynebacterium glutamicum cells from culture medium by size exclusion chromatography (SEC) is presented. Residue analysis demonstrated that this method effectively depletes extracellular compounds. For evaluation, SEC was compared with the common methods cold methanol treatment, fast centrifugation and fast filtration. For this purpose, samples of C. glutamicum cells from fermenter cultures were harvested and subjected to a metabolome analysis. In particular, the wild type strain C. glutamicum ATCC13032 and the lysine production strain C. glutamicum DM1730 were grown in a minimal or in a complex medium. Comparison of metabolite pool sizes after harvesting C. glutamicum cells by the methods mentioned above by gas chromatography coupled to mass spectrometry (GC-MS) revealed that SEC is the most suitable method when intracellular metabolite pools are to be measured during growth in complex media or in the presence of significant amounts of secreted metabolites. In contrast to the other methods tested, the SEC method turned out to be fast and able to remove extracellular compounds almost completely.

Distribution of aerobic motile and non-motile bacteria within the capillary fringe of silica sand.

Retention of bacterial cells as "particles" by silica sand during formation of a capillary fringe (CF) and the influence of motility was examined with motile Pseudomonas putida and non-motile Corynebacterium glutamicum suspensions in the absence of nutrients. The fractional retention of C. glutamicum cells at all regions of the CF was higher than for P. putida cells, most probably due to the motility of P. putida. Only about 5% of P. putida cells and almost no C. glutamicum cells reached the upper end of a CF of 10 cm height. With cell suspensions of P. putida and C. glutamicum in nutrient broth the development of a CF in silica sand fractions of 355-710 microm and 710-1000 microm respectively, was finished after about 6 h. Growth of cells proceeded for about 6 days. P. putida formed a biofilm on silica grains, whereas no attachment of C. glutamicum on silica sand occurred. Relative cell densities of C. glutamicum on the bottom and in the upper regions of the CF were always lower than those of P. putida and were also lower than those reached in suspended cultures with the same medium. In coarse sand the motile P. putida cells reached significantly higher cell densities in upper CF regions than in fine sand. Growth of C. glutamicum in the CF apparently was slower and a higher proportion of the energy was required for maintenance. Whereas cell densities of P. putida, in CFs of both sand fractions, varied less than one order of magnitude, those of C. glutamicum varied in a wider range from the basis to the top of the CF. Analyses of the esterase activity of P. putida and C. glutamicum with fluorescein diacetate (FDA) revealed that the cells in higher CF regions were significantly more active than those at the bottom of the CF. Furthermore, a significant correlation (r = 0.66, p < 0.01) between cells ml(-1) and the FDA conversion to fluorescein was found.

SAMPI: protein identification with mass spectra alignments.

BACKGROUND: Mass spectrometry based peptide mass fingerprints (PMFs) offer a fast, efficient, and robust method for protein identification. A protein is digested (usually by trypsin) and its mass spectrum is compared to simulated spectra for protein sequences in a database. However, existing tools for analyzing PMFs often suffer from missing or heuristic analysis of the significance of search results and insufficient handling of missing and additional peaks. RESULTS: We present an unified framework for analyzing Peptide Mass Fingerprints that offers a number of advantages over existing methods: First, comparison of mass spectra is based on a scoring function that can be custom-designed for certain applications and explicitly takes missing and additional peaks into account. The method is able to simulate almost every additive scoring scheme. Second, we present an efficient deterministic method for assessing the significance of a protein hit, independent of the underlying scoring function and sequence database. We prove the applicability of our approach using biological mass spectrometry data and compare our results to the standard software Mascot. CONCLUSION: The proposed framework for analyzing Peptide Mass Fingerprints shows performance comparable to Mascot on small peak lists. Introducing more noise peaks, we are able to keep identification rates at a similar level by using the flexibility introduced by scoring schemes.