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Mutations in GlnA, NifA and NifK enabling photosynthetic growth of Rhodobacter sphaeroides in the absence of CO2 fixation

ABSTRACT: Previously isolated spontaneous mutants of Rhodobacter sphaeroides, which were initially compromised in their ability to assimilate carbon dioxide through the reductive pentose phosphate pathway, were found to exhibit abnormal nitrogenase gene regulation as well as altered patterns of nitrogenase enzymatic activity. The genomes of these strains were studied by whole genome pyrosequencing and whole genome microarray analysis to identify possible loci responsible for the observed phenotypes. 3 R.sphaeroides strains were grown in matching conditions to reveal strain-specific gene expression

ORGANISM(S): Rhodobacter sphaeroides

SUBMITTER: Oleg Moskvin

PROVIDER: E-GEOD-47149 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Mutations in GlnA, NifA and NifK enabling photosynthetic growth of Rhodobacter sphaeroides in the absence of CO2 fixation

Project description:Previously isolated spontaneous mutants of Rhodobacter sphaeroides, which were initially compromised in their ability to assimilate carbon dioxide through the reductive pentose phosphate pathway, were found to exhibit abnormal nitrogenase gene regulation as well as altered patterns of nitrogenase enzymatic activity. The genomes of these strains were studied by whole genome pyrosequencing and whole genome microarray analysis to identify possible loci responsible for the observed phenotypes.

2013-05-22 | GSE47149 | GEO

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus

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.

2024-02-05 | PXD046902 | Pride

Expression data from H2-producing Rhodobacter sphaeroides cultures fed different organic substrates

Project description:Rhodobacter sphaeroides produces hydrogen gas (H2) via its nitrogenase enzyme during photoheterotrophic growth under nitrogen-limited conditions. We find that cells produce different amounts of H2 and show different growth rates, depending on the organic substrate provided (lactate, succinate, glucose, xylose, or glycerol). We used global transcript analyses to determine what genes are involved in the onset of H2 production, and those that lead to different H2 production capacities in cells fed different organic substrates. Growth and real-time H2 production were followed for photoheterotrophically grown Rhodobacter sphaeroides cultures (19 mL test tube cultures) fed one of five different organic substrates (lactate, succinate, glucose, xylose, or glycerol) and provided glutamate as the sole nitrogen source. Cells were harvested for RNA extraction during early post-exponential growth, at points near the maximum H2 production rates of the cultures. Non-H2-producing cells (500 mL cultures) fed succinate and provided NH4+ as nitrogen source were also harvested for RNA extraction as reference samples. At least two samples for every given set of conditions were analyzed.

2011-08-21 | E-GEOD-29811 | biostudies-arrayexpress

Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris

2005-11-10 | GSE3030 | GEO

Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris

Project description:To address the question of how photosynthetic bacterium Rhodopseudomonas palustris differentially regulates gene expression of three nitrogenase isozymes (Mo, V, and Fe nitrogenases), we constructed Mo strain (Mo nitrogenase only strain), V strain (V nitrogenase only strain), and Fe strain (Fe nitrogenase only strain), and analyzed the whole genome transcriptome profiles of each mutant and wild-type cells grown under nitrogen-fixing conditions. RNA was isolated from various Rhodopseudomonas palustris strains that were grown to the mid-logarithmic phase of growth. Fluorescently labeled cDNA was prepared by direct incorporation of either Cy3-dCTP or Cy5-dCTP during a first-strand reverse transcription reaction. The hybridization mixtures containing the two labeled cDNA samples to be compared were applied to microarray slides that had been covered with Lifterslips (Erie Scientific Company, Portsmouth, NH). The slides were assembled with hybridization chambers (Corning, Corning, NY) and submerged in a 65ÂºC water bath. After 14-16 h of hybridization, the slides were washed and scanned with a ScanArray 4000XL scanner (PerkinElmer, Boston, MA). Images (Cy3 and Cy5) were captured as TIFF files and were analyzed with the image processing software ImaGene version 5.6 (BioDiscovery, Inc., El Segundo, CA). The software package lcDNA was used for data normalization and assessment of the statistical confidence intervals of gene expression. Duplicate calibration experiments and three comparative experiments using RNA from three separately grown cultures (three biological replicates) with duplicate slides for each (10 slides in total) were used to generate each data set.

2010-06-30 | E-GEOD-3030 | biostudies-arrayexpress

Not just an alternative nitrogenase: The Fe-nitrogenase system belongs to the NifA regulon in Rhodobacter capsulatus

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.

2019-07-19 | PXD013515 | Pride

Photosynthetic apparatus of Rhodobacter sphaeroides exhibits prolonged charge storage.

Project description:Photosynthetic proteins have been extensively researched for solar energy harvesting. Though the light-harvesting and charge-separation functions of these proteins have been studied in depth, their potential as charge storage systems has not been investigated to the best of our knowledge. Here, we report prolonged storage of electrical charge in multilayers of photoproteins isolated from Rhodobacter sphaeroides. Direct evidence for charge build-up within protein multilayers upon photoexcitation and external injection is obtained by Kelvin-probe and scanning-capacitance microscopies. Use of these proteins is key to realizing a 'self-charging biophotonic device' that not only harvests light and photo-generates charges but also stores them. In strong correlation with the microscopic evidence, the phenomenon of prolonged charge storage is also observed in primitive power cells constructed from the purple bacterial photoproteins. The proof-of-concept power cells generated a photovoltage as high as 0.45?V, and stored charge effectively for tens of minutes with a capacitance ranging from 0.1 to 0.2?F?m-2.

| S-EPMC6385238 | biostudies-literature

Expression data from H2-producing Rhodobacter sphaeroides cultures fed different organic substrates

2011-08-22 | GSE29811 | GEO

Transcriptome comparison of wild type Azoarcus sp. strain BH72 grown under nitrogen fixation (experiment) with cells grown with combined nitrogen (ammonia) as nitrogen source

Project description:Model endophyte Azoarcus sp. BH72 is known to contribute fixed nitrogen to its host Kallar grass by nitrogen fixation and also expresses nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. Therefore, we analysed global transcription in response to differences in the nitrogen source. Transcription profiles of cells grown microaerobically (0.6% oxygen) on minimal medium with nitrogen (N2-fixing) versus ammonium (combined nitrogen) were compared using a genome-wide microarray approach and differences in the gene expression profile were monitored.

2013-08-01 | GSE49394 | GEO

CRISPR/Cas12a-mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus.

Project description:Purple non-sulfur photosynthetic bacteria (PNSB) such as Rhodobacter capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for genome editing and transcriptional regulation in R. capsulatus. Template-free disruption method mediated by CRISPR/Cas12a reached ˜ 90% editing efficiency when targeting ccoO or nifH gene. When both genes were simultaneously edited, the multiplex editing efficiency reached > 63%. In addition, CRISPR interference (CRISPRi) using deactivated Cas12a was also evaluated using reporter genes egfp and lacZ, and the transcriptional repression efficiency reached ˜ 80%. In summary, our work represents the first report to develop CRISPR/Cas12a-mediated genome editing and transcriptional regulation in R. capsulatus, which would greatly accelerate PNSB-related researches.

| S-EPMC8601187 | biostudies-literature

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