Implications of various phosphoenolpyruvate-carbohydrate phosphotransferase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16

The enhanced global biodiesel production is also yielding increased quantities of glycerol as main coproduct. An effective application of glycerol, for example, as low-cost substrate for microbial growth in industrial fermentation processes to specific products will reduce the production costs for biodiesel. Our study focuses on the utilization of glycerol as a cheap carbon source during cultivation of the thermoplastic producing bacterium Ralstonia eutropha H16, and on the investigation of carbohydrate transport proteins involved herein. Seven open reading frames were identified in the genome of strain H16 to encode for putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS). Although the core components of PEP-PTS, enzyme I (ptsI) and histidine phosphocarrier protein (ptsH), are available in strain H16, a complete PTS-mediated carbohydrate transport is lacking. Growth experiments employing several PEP-PTS mutants indicate that the putative ptsMHI operon, comprising ptsM (a fructose-specific EIIA component of PTS), ptsH, and ptsI, is responsible for limited cell growth and reduced PHB accumulation (53%, w/w, less PHB than the wild type) of this strain in media containing glycerol as a sole carbon source. Otherwise, the deletion of gene H16_A0384 (ptsN, nitrogen regulatory EIIA component of PTS) seemed to largely compensate the effect of the deleted ptsMHI operon (49%, w/w, PHB). The involvement of the PTS homologous proteins on the utilization of the non-PTS sugar alcohol glycerol and its effect on cell growth as well as PHB and carbon metabolism of R. eutropha will be discussed

Impact of the Core Components of the Phosphoenolpyruvate-Carbohydrate Phosphotransferase System, HPr and EI, on Differential Protein Expression in <i>Ralstonia eutropha</i> H16

In <i>Ralstonia eutropha</i> H16, seven genes encoding proteins being involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified. In order to provide more insights into the poly­(3-hydroxybutyrate) (PHB)-leaky phenotype of the HPr/EI deletion mutants H16Δ<i>ptsH</i>, H16Δ<i>ptsI</i>, and H16Δ<i>ptsHI</i> when grown on the non-PTS substrate gluconate, parallel fermentations for comparison of their growth behavior were performed. Samples from the exponential, the early stationary, and late stationary growth phases were investigated by microscopy, gas chromatography and (phospho-) proteome analysis. A total of 71 differentially expressed proteins were identified using 2D-PAGE, Pro-Q Diamond and Coomassie staining, and MALDI-TOF analysis. Detected proteins were classified into five major functional groups: carbon metabolism, energy metabolism, amino acid metabolism, translation, and membrane transport/outer membrane proteins. Proteome analyses revealed enhanced expression of proteins involved in the Entner–Doudoroff pathway and in subsequent reactions in cells of strain H16 compared to the mutant H16Δ<i>ptsHI</i>. Furthermore, proteins involved in PHB accumulation showed increased abundance in the wild-type. This expression pattern allowed us to identify proteins affecting carbon metabolism/PHB biosynthesis in strain H16 and translation/amino acid metabolism in strain H16Δ<i>ptsHI</i>, and to gain insight into the molecular response of <i>R. eutropha</i> to the deletion of HPr/EI

Analysis of the endophytic lifestyle and plant growth promotion of Burkholderia terricola ZR2-12

Members of the genus Burkholderia are highly versatile bacteria that can be beneficial as well as pathogenic for their eukaryotic hosts. Furthermore, many strains exhibit a remarkable biotechnological potential. To study the ecosystem function and lifestyle of B. terricola, we analysed the interactions with plants and survival in soil as well as the mechanisms behind it. We used a combination of in vitro and ad planta assays to study Burkholderia-plant interaction and assess the role of poly-beta-hydroxybutyrate (PHB). Additionally, DsRed-labelled bacteria were analysed by confocal laser scanning microscopy (CLSM) to study root colonisation. B. terricola ZR2-12 treatment resulted in enhanced growth of sugar beet plants with a more than doubled biomass relative to the non-treated control. The strain was a remarkable good root coloniser, which was found in rhizosphere as well as endorhiza of sugar beet up to 10 log(10) CFU g(-1). Using CLSM, we observed that ZR2-12 cells form large micro-colonies along the apoplastic spaces of the root. Xylem vessels were colonised by smaller aggregates and single cells, whereas in root tips mainly single cells were present. The colonisation patterns differed strongly between older and younger parts of the roots. PHB production of ZR2-12 (up to 70% (w/w) of cell dry mass) provided a competitive advantage for rhizosphere colonisation. B. terricola ZR2-12 belongs to the plant-associated Burkholderia cluster with biotechnological potential due to its excellent root colonisation and plant growth promotion