Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glucosyltransferase

A recombinant B. megaterium strain was used for the heterologous production of a glucosyltransferase (dextransucrase). To better understand the physiological and metabolic responses of the host cell to cultivation and induction conditions, proteomic analysis was carried out by combined use of two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) for protein separation and identification. 2-DE method was optimized for the separation of intracellular proteins. Since the genome of B. megaterium is not yet available, peptide sequencing using peptide fragment information obtained from nanoelectrospray ionization quadrupole-time-of-flight tandem mass spectrometry (ESI-QqTOF MS/MS) was applied for protein identification. 167 protein spots were identified as 149 individual proteins, including most enzymes involved in the central carbon metabolic pathways and many enzymes related to amino acid synthesis and protein synthesis. Based on the results a 2-DE reference map and a corresponding protein database were constructed for further proteomic approaches on B. megaterium. For the first time it became possible to perform comparative proteomic analysis on B. megaterium in a batch culture grown on glucose with xylose induction for dextrasucrase production. No significant differences were observed in the expression changes of enzymes of the glycolysis and TCA cycle, indicating that dextransucrase production, which amounted to only 2 % of the entire protein production, did not impose notable metabolic or energetic burdens on the central carbon metabolic pathway of the cells. However, a short-term up-regulation of aspartate aminotransferase, an enzyme closely related to dextransucrase production, in the induced culture demonstrated the feasibility to use 2-DE method for monitoring dextransucrase production. It was also observed that under the cultivation conditions used in this study B. megaterium tended to channel acetyl-CoA into pathways of polyhydroxybutyrate production. No expression increases were found with cytosolic chaperones such as GroEL and DnaK during dextransucrase production and secretion, whereas a strong up-regulation of the oligopeptide-binding protein OppA was observed in correlation with an increased secretion of dextransucrase into the culture medium

Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis, two members of the marine Roseobacter clade

<p>Abstract</p> <p>Background</p> <p>In the present work the central carbon metabolism of <it>Dinoroseobacter shibae </it>and <it>Phaeobacter gallaeciensis </it>was studied at the level of metabolic fluxes. These two strains belong to the marine <it>Roseobacter </it>clade, a dominant bacterial group in various marine habitats, and represent surface-associated, biofilm-forming growth (<it>P. gallaeciensis</it>) and symbiotic growth with eukaryotic algae (<it>D. shibae</it>). Based on information from recently sequenced genomes, a rich repertoire of pathways has been identified in the carbon core metabolism of these organisms, but little is known about the actual contribution of the various reactions <it>in vivo</it>.</p> <p>Results</p> <p>Using ¹³C labelling techniques in specifically designed experiments, it could be shown that glucose-grown cells of <it>D. shibae </it>catabolise the carbon source exclusively via the Entner-Doudoroff pathway, whereas alternative routes of glycolysis and the pentose phosphate pathway are obviously utilised for anabolic purposes only. Enzyme assays confirmed this flux pattern and link the lack of glycolytic flux to the absence of phosphofructokinase activity. The previously suggested formation of phosphoenolpyruvate from pyruvate during mixotrophic CO₂assimilation was found to be inactive under the conditions studied. Moreover, it could be shown that pyruvate carboxylase is involved in CO₂assimilation and that the <it>cyclic </it>respiratory mode of the TCA cycle is utilised. Interestingly, the use of intracellular pathways was highly similar for <it>P. gallaeciensis</it>.</p> <p>Conclusion</p> <p>The present study reveals the first insight into pathway utilisation within the <it>Roseobacter </it>group. Fluxes through major intracellular pathways of the central carbon metabolism, which are closely linked to the various important traits found for the <it>Roseobacter </it>clade, could be determined. The close similarity of fluxes between the two physiologically rather different species might provide the first indication of more general key properties among members of the <it>Roseobacter </it>clade which may explain their enormous success in the marine realm.</p