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Further unraveling the regulatory twist by elucidating metabolic coinducer-mediated CbbR-cbbI promoter interactions in Rhodopseudomonas palustris CGA010.

ABSTRACT: The cbb(I) region of Rhodopseudomonas palustris (Rp. palustris) contains the cbbLS genes encoding form I ribulose-1,5-bisphosphate (RuBP) carboxylase oxygenase (RubisCO) along with a divergently transcribed regulator gene, cbbR. Juxtaposed between cbbR and cbbLS are the cbbRRS genes, encoding an unusual three-protein two-component (CbbRRS) system that modulates the ability of CbbR to influence cbbLS expression. The nature of the metabolic signals that Rp. palustris CbbR perceives to regulate cbbLS transcription is not known. Thus, in this study, the CbbR binding region was first mapped within the cbbLS promoter by the use of gel mobility shift assays and DNase I footprinting. In addition, potential metabolic coinducers (metabolites) were tested for their ability to alter the cbbLS promoter binding properties of CbbR. Gel mobility shift assays and surface plasmon resonance analyses together indicated that biosynthetic intermediates such as RuBP, ATP, fructose 1,6-bisphosphate, and NADPH enhanced DNA binding by CbbR. These coinducers did not yield identical CbbR-dependent DNase I footprints, indicating that the coinducers caused significant changes in DNA structure. These in vitro studies suggest that cellular signals such as fluctuating metabolite concentrations are perceived by and transduced to the cbbLS promoter via the master regulator CbbR.

SUBMITTER: Joshi GS

PROVIDER: S-EPMC3294837 | biostudies-other | 2012 Mar

REPOSITORIES: biostudies-other

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Further unraveling the regulatory twist by elucidating metabolic coinducer-mediated CbbR-cbbI promoter interactions in Rhodopseudomonas palustris CGA010.

Joshi Gauri S GS Zianni Michael M Bobst Cedric E CE Tabita F Robert FR

Journal of bacteriology 20120113 6

The cbb(I) region of Rhodopseudomonas palustris (Rp. palustris) contains the cbbLS genes encoding form I ribulose-1,5-bisphosphate (RuBP) carboxylase oxygenase (RubisCO) along with a divergently transcribed regulator gene, cbbR. Juxtaposed between cbbR and cbbLS are the cbbRRS genes, encoding an unusual three-protein two-component (CbbRRS) system that modulates the ability of CbbR to influence cbbLS expression. The nature of the metabolic signals that Rp. palustris CbbR perceives to regulate cbb ...[more]

PMID: 22247506

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Project description:BackgroundLiving organisms need to allocate their limited resources in a manner that optimizes their overall fitness by simultaneously achieving several different biological objectives. Examination of these biological trade-offs can provide invaluable information regarding the biophysical and biochemical bases behind observed cellular phenotypes. A quantitative knowledge of a cell system's critical objectives is also needed for engineering of cellular metabolism, where there is interest in mitigating the fitness costs that may result from human manipulation.ResultsTo study metabolism in photoheterotrophs, we developed and validated a genome-scale model of metabolism in Rhodopseudomonas palustris, a metabolically versatile gram-negative purple non-sulfur bacterium capable of growing phototrophically on various carbon sources, including inorganic carbon and aromatic compounds. To quantitatively assess trade-offs among a set of important biological objectives during different metabolic growth modes, we used our new model to conduct an 8-dimensional multi-objective flux analysis of metabolism in R. palustris. Our results revealed that phototrophic metabolism in R. palustris is light-limited under anaerobic conditions, regardless of the available carbon source. Under photoheterotrophic conditions, R. palustris prioritizes the optimization of carbon efficiency, followed by ATP production and biomass production rate, in a Pareto-optimal manner. To achieve maximum carbon fixation, cells appear to divert limited energy resources away from growth and toward CO2 fixation, even in the presence of excess reduced carbon. We also found that to achieve the theoretical maximum rate of biomass production, anaerobic metabolism requires import of additional compounds (such as protons) to serve as electron acceptors. Finally, we found that production of hydrogen gas, of potential interest as a candidate biofuel, lowers the cellular growth rates under all circumstances.ConclusionsPhotoheterotrophic metabolism of R. palustris is primarily regulated by the amount of light it can absorb and not the availability of carbon. However, despite carbon's secondary role as a regulating factor, R. palustris' metabolism strives for maximum carbon efficiency, even when this increased efficiency leads to slightly lower growth rates.

Project description:Rhodopseudomonas palustris, a nonsulphur purple photosynthetic bacteria, has been extensively investigated for its metabolic versatility including ability to produce hydrogen gas from sunlight and biomass. The availability of the finished genome sequences of six R. palustris strains (BisA53, BisB18, BisB5, CGA009, HaA2 and TIE-1) combined with online bioinformatics software for integrated analysis presents new opportunities to determine the genomic basis of metabolic versatility and ecological lifestyles of the bacteria species. The purpose of this investigation was to compare the functional annotations available for multiple R. palustris genomes to identify annotations that can be further investigated for strain-specific or uniquely shared phenotypic characteristics. A total of 2,355 protein family Pfam domain annotations were clustered based on presence or absence in the six genomes. The clustering process identified groups of functional annotations including those that could be verified as strain-specific or uniquely shared phenotypes. For example, genes encoding water/glycerol transport were present in the genome sequences of strains CGA009 and BisB5, but absent in strains BisA53, BisB18, HaA2 and TIE-1. Protein structural homology modeling predicted that the two orthologous 240 aa R. palustris aquaporins have water-specific transport function. Based on observations in other microbes, the presence of aquaporin in R. palustris strains may improve freeze tolerance in natural conditions of rapid freezing such as nitrogen fixation at low temperatures where access to liquid water is a limiting factor for nitrogenase activation. In the case of adaptive loss of aquaporin genes, strains may be better adapted to survive in conditions of high-sugar content such as fermentation of biomass for biohydrogen production. Finally, web-based resources were developed to allow for interactive, user-defined selection of the relationship between protein family annotations and the R. palustris genomes.

Project description:Rhodopseudomonas palustris is an alphaproteobacterium that has served as a model organism for studies of photophosphorylation, regulation of nitrogen fixation, production of hydrogen as a biofuel, and anaerobic degradation of aromatic compounds. This bacterium is able to transition between anaerobic photoautotrophic growth, anaerobic photoheterotrophic growth, and aerobic heterotrophic growth. As a starting point to explore the genetic basis for the metabolic versatility of R. palustris, we used transposon mutagenesis and Tn-seq to identify 552 genes as essential for viability in cells growing aerobically on semirich medium. Of these, 323 have essential gene homologs in the alphaproteobacterium Caulobacter crescentus, and 187 have essential gene homologs in Escherichia coli. There were 24 R. palustris genes that were essential for viability under aerobic growth conditions that have low sequence identity but are likely to be functionally homologous to essential E. coli genes. As expected, certain functional categories of essential genes were highly conserved among the three organisms, including translation, ribosome structure and biogenesis, secretion, and lipid metabolism. R. palustris cells divide by budding in which a sessile cell gives rise to a motile swarmer cell. Conserved cell cycle genes required for this developmental process were essential in both C. crescentus and R. palustris. Our results suggest that despite vast differences in lifestyles, members of the alphaproteobacteria have a common set of essential genes that is specific to this group and distinct from that of gammaproteobacteria like E. coli.Essential genes in bacteria and other organisms are those absolutely required for viability. Rhodopseudomonas palustris has served as a model organism for studies of anaerobic aromatic compound degradation, hydrogen gas production, nitrogen fixation, and photosynthesis. We used the technique of Tn-seq to determine the essential genes of R. palustris grown under heterotrophic aerobic conditions. The transposon library generated in this study will be useful for future studies to identify R. palustris genes essential for viability under specialized growth conditions and also for survival under conditions of stress.

Project description:The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 uses the three-carbon dicarboxylic acid malonate as the sole carbon source under phototrophic conditions. However, this bacterium grows extremely slowly on this compound and does not have operons for the two pathways for malonate degradation that have been detected in other bacteria. Many bacteria grow on a spectrum of carbon sources, some of which are classified as poor growth substrates because they support low growth rates. This trait is rarely addressed in the literature, but slow growth is potentially useful in biotechnological applications where it is imperative for bacteria to divert cellular resources to value-added products rather than to growth. This prompted us to explore the genetic and physiological basis for the slow growth of R. palustris with malonate as a carbon source. There are two unlinked genes annotated as encoding a malonyl coenzyme A (malonyl-CoA) synthetase (MatB) and a malonyl-CoA decarboxylase (MatA) in the genome of R. palustris, which we verified as having the predicted functions. Additionally, two tripartite ATP-independent periplasmic transporters (TRAP systems) encoded by rpa2047 to rpa2049 and rpa2541 to rpa2543 were needed for optimal growth on malonate. Most of these genes were expressed constitutively during growth on several carbon sources, including malonate. Our data indicate that R. palustris uses a piecemeal approach to growing on malonate. The data also raise the possibility that this bacterium will evolve to use malonate efficiently if confronted with an appropriate selection pressure.IMPORTANCE There is interest in understanding how bacteria metabolize malonate because this three-carbon dicarboxylic acid can serve as a building block in bioengineering applications to generate useful compounds that have an odd number of carbons. We found that the phototrophic bacterium Rhodopseudomonas palustris grows extremely slowly on malonate. We identified two enzymes and two TRAP transporters involved in the uptake and metabolism of malonate, but some of these elements are apparently not very efficient. R. palustris cells growing with malonate have the potential to be excellent biocatalysts, because cells would be able to divert cellular resources to the production of value-added compounds instead of using them to support rapid growth. In addition, our results suggest that R. palustris is a candidate for directed evolution studies to improve growth on malonate and to observe the kinds of genetic adaptations that occur to make a metabolic pathway operate more efficiently.

Dataset Information

Further unraveling the regulatory twist by elucidating metabolic coinducer-mediated CbbR-cbbI promoter interactions in Rhodopseudomonas palustris CGA010.

Publications

Further unraveling the regulatory twist by elucidating metabolic coinducer-mediated CbbR-cbbI promoter interactions in Rhodopseudomonas palustris CGA010.

Similar Datasets

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