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Global insights into energetic and metabolic networks in Rhodobacter sphaeroides.

ABSTRACT: BACKGROUND: Improving our understanding of processes at the core of cellular lifestyles can be aided by combining information from genetic analyses, high-throughput experiments and computational predictions. RESULTS: We combined data and predictions derived from phenotypic, physiological, genetic and computational analyses to dissect the metabolic and energetic networks of the facultative photosynthetic bacterium Rhodobacter sphaeroides. We focused our analysis on pathways crucial to the production and recycling of pyridine nucleotides during aerobic respiratory and anaerobic photosynthetic growth in the presence of an organic electron donor. In particular, we assessed the requirement for NADH/NADPH transhydrogenase enzyme, PntAB during respiratory and photosynthetic growth. Using high-throughput phenotype microarrays (PMs), we found that PntAB is essential for photosynthetic growth in the presence of many organic electron donors, particularly those predicted to require its activity to produce NADPH. Utilizing the genome-scale metabolic model iRsp1095, we predicted alternative routes of NADPH synthesis and used gene expression analyses to show that transcripts from a subset of the corresponding genes were conditionally increased in a ?pntAB mutant. We then used a combination of metabolic flux predictions and mutational analysis to identify flux redistribution patterns utilized in the ?pntAB mutant to compensate for the loss of this enzyme. Data generated from metabolic and phenotypic analyses of wild type and mutant cells were used to develop iRsp1140, an expanded genome-scale metabolic reconstruction for R. sphaeroides with improved ability to analyze and predict pathways associated with photosynthesis and other metabolic processes. CONCLUSIONS: These analyses increased our understanding of key aspects of the photosynthetic lifestyle, highlighting the added importance of NADPH production under these conditions. It also led to a significant improvement in the predictive capabilities of a metabolic model for the different energetic lifestyles of a facultative organism.

SUBMITTER: Imam S

PROVIDER: S-EPMC3849096 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Global insights into energetic and metabolic networks in Rhodobacter sphaeroides.

Imam Saheed S Noguera Daniel R DR Donohue Timothy J TJ

BMC systems biology 20130913

<h4>Background</h4>Improving our understanding of processes at the core of cellular lifestyles can be aided by combining information from genetic analyses, high-throughput experiments and computational predictions.<h4>Results</h4>We combined data and predictions derived from phenotypic, physiological, genetic and computational analyses to dissect the metabolic and energetic networks of the facultative photosynthetic bacterium Rhodobacter sphaeroides. We focused our analysis on pathways crucial t ...[more]

PMID: 24034347

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Project description:BACKGROUND:Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. RESULTS:Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. CONCLUSIONS:In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

Project description:We report the role of a gene (rpoH) from the facultative phototroph Rhodobacter sphaeroides that encodes a protein (sigma37) similar to Escherichia coli sigma32 and other members of the heat shock family of eubacterial sigma factors. R. sphaeroides sigma37 controls genes that function during environmental stress, since an R. sphaeroides deltaRpoH mutant is approximately 30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells. However, the deltaRpoH mutant lacks several phenotypes characteristic of E. coli cells lacking sigma32. For example, an R. sphaeroides deltaRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent. In characterizing the response of R. sphaeroides to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis. However, growth of the deltaRpoH mutant is comparable to that of a wild-type strain under each of these conditions. The deltaRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42 degrees C, but it exhibited altered induction kinetics of approximately 120-, 85-, 75-, and 65-kDa proteins. There was also reduced accumulation of several presumed heat shock transcripts (rpoD P(HS), groESL1, etc.) when aerobically grown deltaRpoH cells were placed at 42 degrees C. Under aerobic conditions, it appears that another sigma factor enables the deltaRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an deltaRpoH mutant, reconstituted Esigma37, or a holoenzyme containing a 38-kDa protein (sigma38) each transcribed E. coli Esigma32-dependent promoters. The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the deltaRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37 degrees C.

Dataset Information

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides.

Publications

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides.

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