Project description:The influence of different nitrogen sources on transcriptome of Purple non sulfur bacterium R. Capsulatus was investigated by comparing expression profile on 5mM ammonium chloride and 2 mM glutamate. Carbon source was 40mM acetate on both conditions. To study the effect of different acetate concentrations, 40mM and 80mM acetate were used with 2 mM glutamate as nitrogen source.
Project description:The influence of different nitrogen sources on transcriptome of Purple non sulfur bacterium R. Capsulatus was investigated by comparing expression profile on 5mM ammonium chloride and 2 mM glutamate. Carbon source was 40mM acetate on both conditions. To study the effect of different acetate concentrations, 40mM and 80mM acetate were used with 2 mM glutamate as nitrogen source. Bacteria were grown anaerobically under continuous illumination (200 W/m2) in 150 ml volume photobioreactors at 30M-BM-0C. For microarray analysis, RNA samples were collected after 16h of inoculation from 1.5x109 cells. A microarray chip for Rhodobacter capsulatus was not commercially available. For that reason, a GeneChipM-BM-. array was custom designed and manufactured by Affymetrix, Santa Clara, California. The nucleotide sequence of the bacterium is available in GenBank with the accession numbers CP001312 for the chromosome and CP001313 for the plasmid pRCB133. The feature size of the antisense DNA array was 11 micron and the format was 100-3660. A total of 4,052 probe sets for open reading frames (ORFs) and 200 intergenic sequences with longer than 300bp were placed on the microarray. The cDNA synthesis, labeling, and hybridization protocols were performed according to the Affymetrix Expression Analysis Technical Manual given for prokaryotic samples
Project description:Transcriptomic data has been previously used to determine expression patterns in various R. capsulatus strains using custom made Affymetrix microarrays. Additional expression analyses have since been carried out to obtain preliminary data on certain wild type and mutant strains that have not been reported on. We used all current microarray expression data available and constructed an R. capsulatus gene co-expression network and performed functional analysis of identified gene modules. RNA was harvested from various wild type and mutant R. capsulatus strains grown under photoheterotrophic conditions to different growth phases. The samples were hybridized to custom made R. capsulatus Affymetrix microarrays according to manufacturers recommendations. The raw data was RMA normalized and log transformed.
Project description:Global transcriptome analyses at growth before and after 10 min of photooxidative stress were applied to monitor stress dependent gene expression in the alpha-proteobacterium Rhodobacter capsulatus. Transcriptome profiles of pigmented cultures with high aeration were monitored before and after the onset of singlet oxygen stress.
Project description:Transcriptomic data has been previously used to determine expression patterns in various R. capsulatus strains using custom made Affymetrix microarrays. Additional expression analyses have since been carried out to obtain preliminary data on certain wild type and mutant strains that have not been reported on. We used all current microarray expression data available and constructed an R. capsulatus gene co-expression network and performed functional analysis of identified gene modules.
Project description:Nitrogenases are the only enzymes able to ‘fix’ gaseous nitrogen into bioavailable ammonia and, hence, are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited, with electron transport to the iron (Fe)-nitrogenase having hardly been investigated. Here, we characterised the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain (nifD), compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterised by monitoring diazotrophic growth and nitrogenase activity in vivo. Only deletion of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double-deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviours of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.
Project description:Gene Co-Expression Network Analysis in Rhodobacter capsulatus and Application to Comparative Expression Analysis of Rhodobacter sphaeroides
Project description:The diazotrophic bacterium Rhodobacter capsulatus synthesizes a molybdenum nitrogenase and an alternative iron-only nitrogenase, enabling growth with molecular dinitrogen as sole nitrogen source. Regulation of nitrogen fixation was analyzed by proteome profiling of wild-type and mutant strains lacking the transcriptional regulators NifA, AnfA, and MopAB.
