Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803

BACKGROUND: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product. RESULTS: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level. CONCLUSION: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription

The biosynthetic routes for carbon monoxide and cyanide in the Ni-Fe active site of hydrogenases are different

The incorporation of carbon into the carbon monoxide and cyanide ligands of [NiFe]-hydrogenases has been investigated by using (13)C labelling in infrared studies of the Allochromatium vinosum enzyme and by (14)C labelling experiments with overproduced Hyp proteins from Escherichia coli. The results suggest that the biosynthetic routes of the carbon monoxide and cyanide ligands in [NiFe]-hydrogenases are different

Influence des propriétésdu graphite sur le premier cycle d'intercalation du lithium

Les batteries à ions lithium alimentent la plupart des petits appareils électriques, portables. Elles font usage du graphite comme électrode négative. Pour optimiser celle-ci, il faut réduire la perte spécifique de charge du premier cycle d intercalation du lithium. Cette perte est principalement due à la formation d une couche passive par décomposition de l électrolyte. Les propriétés du graphite qui l influencent sont partiellement connues. En particulier, une meilleure compréhension de l exfoliation du graphite, responsable d une grande perte spécifique de charge, est souhaitée. Ce phénomène est provoqué par la co-intercalation de solvant à travers les plans de bord des particules. Des paramètres comme la cristallinité, la chimie de surface et la réactivité du graphite semblent jouer un rôle. Une étude systématique a été entreprise afin de déterminer leur influence sur le premier cycle dans un électrolyte standard à base de carbonate d éthylène et de diméthyle. Il apparaît que les complexes de surface oxygénés ne jouent pas de rôle particulier tandis que les complexes hydrogénés favorisent la co-intercalation de solvant. De plus, les graphites ayant une faible teneur en sites actifs constituant l Active Surface Area (ASA), mesurée par chimisorption d oxygène, sont plus enclins à exfolier. Comme les atomes de bord à deux voisins sont les plus réactifs en raison de la présence d un électron célibataire, la plus grande sensibilité à la co-intercalation de solvant des graphites de faible ASA peut s expliquer par la formation d une couche passive inappropriée sur des plans de bord peu réactifs laissant passer le solvant.In the field of small portable electrical devices, lithium-ion batteries are common. Graphite is used as the negative electrode. To improve its electrochemical performances, the first cycle specific charge loss must be decreased. It is predominantly attributed to the electrolyte reduction into a passivation layer. The graphite properties which influence this charge loss are not clearly identified. In particular, the graphite exfoliation which is responsible for a huge specific charge loss must be better understood. This dramatic phenomenon is due to solvent co-intercalation through the particle edge planes. Many graphite parameters such as crystallinity, surface chemistry and reactivity are thought to play a role. A systematic study was carried out in which the influence of each parameter on the first cycle was assessed in a standard ethylene and dimethyl carbonate based electrolyte. It appears that the presence of oxygen surface complexes does not have any influence whereas C6H bonds cause slight exfoliation. In addition, graphite samples containing low amount of active sites : the so-called Active Surface Area (ASA), quantified by oxygen chemisorption, are more likely to exfoliate. Since graphite active sites are mainly the edge atoms because of unpaired electron presence, low ASA graphite exfoliation can be explained by the formation of inappropriate passivation layer on the edge planes letting solvent molecules co-intercalate.MULHOUSE-SCD Sciences (682242102) / SudocSudocFranceF

Changes in the Spore Proteome of Bacillus cereus in Response to Introduction of Plasmids

Fluorescent fusion proteins were expressed in Bacillus cereus to visualize the germinosome by introducing a plasmid that carries fluorescent fusion proteins of germinant receptor GerR subunits or germinosome scaffold protein GerD. The effects of plasmid insertion and recombinant protein expression on the spore proteome were investigated. Proteomic analysis showed that overexpression of the target proteins had negligible effects on the spore proteome. However, plasmid-bearing spores displayed dramatic abundance changes in spore proteins involved in signaling and metabolism. Our findings indicate that the introduction of a plasmid alone alters the spore protein composition dramatically, with 993 proteins significantly down-regulated and 415 proteins significantly up-regulated among 3323 identified proteins. This shows that empty vector controls are more appropriate to compare proteome changes due to plasmid-encoded genes than is the wild-type strain, when using plasmid-based genetic tools. Therefore, researchers should keep in mind that molecular cloning techniques can alter more than their intended targets in a biological system, and interpret results with this in mind

Integrative Metabolomics and Proteomics Allow the Global Intracellular Characterization of <i>Bacillus subtilis</i> Cells and Spores

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis (B. subtilis) is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular data sets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol) and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition, and the quality of proteomics profiles. The 60% EtOH protocol proved to be the easiest in sample processing and was more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrate that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. The results obtained by the 60% EtOH protocol provide comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores

Integrative Metabolomics and Proteomics Allow the Global Intracellular Characterization of <i>Bacillus subtilis</i> Cells and Spores

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis (B. subtilis) is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular data sets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol) and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition, and the quality of proteomics profiles. The 60% EtOH protocol proved to be the easiest in sample processing and was more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrate that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. The results obtained by the 60% EtOH protocol provide comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores

Integrative Metabolomics and Proteomics Allow the Global Intracellular Characterization of <i>Bacillus subtilis</i> Cells and Spores

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis (B. subtilis) is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular data sets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol) and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition, and the quality of proteomics profiles. The 60% EtOH protocol proved to be the easiest in sample processing and was more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrate that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. The results obtained by the 60% EtOH protocol provide comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores

Integrative Metabolomics and Proteomics Allow the Global Intracellular Characterization of <i>Bacillus subtilis</i> Cells and Spores

Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis (B. subtilis) is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular data sets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol) and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition, and the quality of proteomics profiles. The 60% EtOH protocol proved to be the easiest in sample processing and was more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrate that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. The results obtained by the 60% EtOH protocol provide comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores