Project description:Whole genome gene expression analysis was examined with Ralstonia eutropha strain H16 cultures grown in PHB production medium (recipe per Peoples and Sinskey, 1989) containing fructose or trioleate as the main carbon source. The goal of this analysis was to determine the identity of the triacylglycerol and fatty acid breakdown genes in R. eutropha strain H16.
Project description:Whole genome gene expression analysis was examined with Ralstonia eutropha strain H16 cultures grown in PHB production medium (recipe per Peoples and Sinskey, 1989) containing fructose or trioleate as the main carbon source. The goal of this analysis was to determine the identity of the triacylglycerol and fatty acid breakdown genes in R. eutropha strain H16. In the study presented here, triplicates of R. eutropha strain H16 were examined for changes in expression of 6702 genes during growth and PHB production on each carbon source.
Project description:Ralstonia eutropha H16 is well-known to produce poly(3-hydroxybutyrate) [P(3HB)], a kind of polyhydroxyalkanoates attracted as bio-based biodegradable plastics, efficiently as an energy storage material under unbalanced growth conditions. To obtain further extended knowledge of PHA biosynthesis, this study employed quantitative transcriptome analysis based on deep sequencing of complementary DNA generated from RNA (RNA-seq) for R. eutropha H16. Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined by using Illumina high-throughput sequencer. The RNA-seq results supported induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in growth phase, and repression trends for genes involved in central metabolisms in PHA production phase. Interestingly, transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several selected genes for ?-oxidation were significantly induced in PHA production phase even when the cells were grown on fructose. mRNA profiles of R. eutropha H16 grown on fructose at different phases were generated by deep sequencing, in duplicate, using Illumina GAIIx.
Project description:Ralstonia eutropha H16 is well-known to produce poly(3-hydroxybutyrate) [P(3HB)], a kind of polyhydroxyalkanoates attracted as bio-based biodegradable plastics, efficiently as an energy storage material under unbalanced growth conditions. To obtain further extended knowledge of PHA biosynthesis, this study employed quantitative transcriptome analysis based on deep sequencing of complementary DNA generated from RNA (RNA-seq) for R. eutropha H16. Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined by using Illumina high-throughput sequencer. The RNA-seq results supported induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in growth phase, and repression trends for genes involved in central metabolisms in PHA production phase. Interestingly, transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several selected genes for β-oxidation were significantly induced in PHA production phase even when the cells were grown on fructose.
Project description:Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, cell appendages formation, DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D-gel-LC and conventional 2D-gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the abundance pattern of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations of the central metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase, respectively. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on proteomic and transcriptomic level and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions.
Project description:The aim of this study was to understand how autotrophic (CO2-fixing) bacteria balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment.To this end, the proteome of the model chemolithoautotroph Ralstonia eutropha a.k.a. Cupriavidus necator was studied in different environmental conditions (four limiting substrates, and five different growth rates). Cupriavidus was cultivated in substrate-limited chemostats with fructose, formate, succinate and ammonium limitation to obtain steady state cell samples. The dilution rate/growth rate was increased step-wise from 0.05 to 0.25 1/h in 0.05 steps. Protein quantity was determined by LC-MS, and enzyme utilization was investigated by resource balance analysis modeling.
Project description:C. necator H16 can grow on aspartate as the sole carbon source. Using RNA-seq analysis here, we tried to identify genes that are overexpressed when the culture medium is supplemented with aspartate.
Project description:In order to identify genes which contribute on the uptake of glucose into cells of the mutant R. eutropha G+1, a genome wide transcription analyses was done. Transcripts of strain H16 and the glucose-utilizing mutant R. eutropha G+1, cultivated in mineral salts medium supplemented with either fructose or glucose were compared.