Saheed Imam

iRsp1095: A genome-scale reconstruction of the Rhodobacter sphaeroides metabolic network

BackgroundRhodobacter sphaeroides is one of the best studied purple non-sulfur photosynthetic bacteria and serves as an excellent model for the study of photosynthesis and the metabolic capabilities of this and related facultative organisms. The ability of R. sphaeroides to produce hydrogen (H2), polyhydroxybutyrate (PHB) or other hydrocarbons, as well as its ability to utilize atmospheric carbon dioxide (CO2) as a carbon source under defined conditions, make it an excellent candidate for use in a wide variety of biotechnological applications. A genome-level understanding of its metabolic capabilities should help realize this biotechnological potential.ResultsHere we present a genome-scale metabolic network model for R. sphaeroides strain 2.4.1, designated iRsp1095, consisting of 1,095 genes, 796 metabolites and 1158 reactions, including R. sphaeroides-specific biomass reactions developed in this study. Constraint-based analysis showed that iRsp1095 agreed well with experimental observations when modeling growth under respiratory and phototrophic conditions. Genes essential for phototrophic growth were predicted by single gene deletion analysis. During pathway-level analyses of R. sphaeroides metabolism, an alternative route for CO2 assimilation was identified. Evaluation of photoheterotrophic H2 production using iRsp1095 indicated that maximal yield would be obtained from growing cells, with this predicted maximum ~50% higher than that observed experimentally from wild type cells. Competing pathways that might prevent the achievement of this theoretical maximum were identified to guide future genetic studies.ConclusionsiRsp1095 provides a robust framework for future metabolic engineering efforts to optimize the solar- and nutrient-powered production of biofuels and other valuable products by R. sphaeroides and closely related organisms.

Convergence of the Transcriptional Responses to Heat Shock and Singlet Oxygen Stresses

Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoHI and RpoHII using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoHI and RpoHII regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the −35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoHI promoter sequence logo, is critical for RpoHI–dependent transcription; and that several bases in the predicted −10 element were important for activity with either RpoHII or both RpoH homologs. Genes that are transcribed by both RpoHI and RpoHII are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoHI regulon are associated with a classic heat shock response, while those specific to RpoHII are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.

Pathways Involved in Reductant Distribution during Photobiological H2 Production by Rhodobacter sphaeroides

ABSTRACT We used global transcript analyses and mutant studies to investigate the pathways that impact H2 production in the photosynthetic bacterium Rhodobacter sphaeroides. We found that H2 production capacity is related to the levels of expression of the nitrogenase and hydrogenase enzymes and the enzymes of the Calvin-Benson-Bassham pathway.

Global insights into energetic and metabolic networks in Rhodobacter sphaeroides

BackgroundImproving 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.ResultsWe 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 ΔpntAB mutant. We then used a combination of metabolic flux predictions and mutational analysis to identify flux redistribution patterns utilized in the ΔpntAB 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.ConclusionsThese 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.

Global Analysis of Photosynthesis Transcriptional Regulatory Networks

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

An integrated approach to reconstructing genome-scale transcriptional regulatory networks

Transcriptional regulatory networks (TRNs) program cells to dynamically alter their gene expression in response to changing internal or environmental conditions. In this study, we develop a novel workflow for generating large-scale TRN models that integrates comparative genomics data, global gene expression analyses, and intrinsic properties of transcription factors (TFs). An assessment of this workflow using benchmark datasets for the well-studied γ-proteobacterium Escherichia coli showed that it outperforms expression-based inference approaches, having a significantly larger area under the precision-recall curve. Further analysis indicated that this integrated workflow captures different aspects of the E. coli TRN than expression-based approaches, potentially making them highly complementary. We leveraged this new workflow and observations to build a large-scale TRN model for the α-Proteobacterium Rhodobacter sphaeroides that comprises 120 gene clusters, 1211 genes (including 93 TFs), 1858 predicted protein-DNA interactions and 76 DNA binding motifs. We found that ~67% of the predicted gene clusters in this TRN are enriched for functions ranging from photosynthesis or central carbon metabolism to environmental stress responses. We also found that members of many of the predicted gene clusters were consistent with prior knowledge in R. sphaeroides and/or other bacteria. Experimental validation of predictions from this R. sphaeroides TRN model showed that high precision and recall was also obtained for TFs involved in photosynthesis (PpsR), carbon metabolism (RSP_0489) and iron homeostasis (RSP_3341). In addition, this integrative approach enabled generation of TRNs with increased information content relative to R. sphaeroides TRN models built via other approaches. We also show how this approach can be used to simultaneously produce TRN models for each related organism used in the comparative genomics analysis. Our results highlight the advantages of integrating comparative genomics of closely related organisms with gene expression data to assemble large-scale TRN models with high-quality predictions.

Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides

Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival. ABSTRACT Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. IMPORTANCE Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival. Author Video: An author video summary of this article is available.

CceR and AkgR Regulate Central Carbon and Energy Metabolism in Alphaproteobacteria

ABSTRACT  Many pathways of carbon and energy metabolism are conserved across the phylogeny, but the networks that regulate their expression or activity often vary considerably among organisms. In this work, we show that two previously uncharacterized transcription factors (TFs) are direct regulators of genes encoding enzymes of central carbon and energy metabolism in the alphaproteobacterium Rhodobacter sphaeroides. The LacI family member CceR (RSP_1663) directly represses genes encoding enzymes in the Entner-Doudoroff pathway, while activating those encoding the F1F0 ATPase and enzymes of the tricarboxylic acid (TCA) cycle and gluconeogenesis, providing a direct transcriptional network connection between carbon and energy metabolism. We identified bases that are important for CceR DNA binding and showed that DNA binding by this TF is inhibited by 6-phosphogluconate. We also showed that the GntR family TF AkgR (RSP_0981) directly activates genes encoding several TCA cycle enzymes, and we identified conditions where its activity is increased. The properties of single and double ΔCceR and ΔAkgR mutants illustrate that these 2 TFs cooperatively regulate carbon and energy metabolism. Comparative genomic analysis indicates that CceR and AkgR orthologs are found in other alphaproteobacteria, where they are predicted to have a conserved function in regulating central carbon metabolism. Our characterization of CceR and AkgR has provided important new insight into the networks that control central carbon and energy metabolism in alphaproteobacteria that can be exploited to modify or engineer new traits in these widespread and versatile bacteria. IMPORTANCE To extract and conserve energy from nutrients, cells coordinate a set of metabolic pathways into integrated networks. Many pathways that conserve energy or interconvert metabolites are conserved across cells, but the networks regulating these processes are often highly variable. In this study, we characterize two previously unknown transcriptional regulators of carbon and energy metabolism that are conserved in alphaproteobacteria, a group of abundant, environmentally and biotechnologically important organisms. We identify the genes they regulate, the DNA sequences they recognize, the metabolite that controls the activity of one of the regulators, and conditions where they are required for growth. We provide important new insight into conserved cellular networks that can also be used to improve a variety of hosts for converting feedstock into valuable products. To extract and conserve energy from nutrients, cells coordinate a set of metabolic pathways into integrated networks. Many pathways that conserve energy or interconvert metabolites are conserved across cells, but the networks regulating these processes are often highly variable. In this study, we characterize two previously unknown transcriptional regulators of carbon and energy metabolism that are conserved in alphaproteobacteria, a group of abundant, environmentally and biotechnologically important organisms. We identify the genes they regulate, the DNA sequences they recognize, the metabolite that controls the activity of one of the regulators, and conditions where they are required for growth. We provide important new insight into conserved cellular networks that can also be used to improve a variety of hosts for converting feedstock into valuable products.

Electron Partitioning in Anoxic Phototrophic Bacteria

Photosynthetic cells make major contributions to many important processes on this planet, including solar energy capture, nitrogen or carbon dioxide sequestration and production of useful biocommodities. The sheer number of photosynthetic cells also makes them significant contributors to global nutrient cycling, especially in aquatic ecosystems. For each of these activities, photosynthetic cells need efficient systems for production and distribution of reducing power among the myriad of cellular pathways that depend on reductant. This chapter focuses on the partitioning of reductant in purple nonsulfur photosynthetic bacteria. It summarizes known membrane and cytoplasmic enzymes and pathways that need the reductant produced via photochemical activity (quinol in these organisms). These observations illustrate that quinol is used to provide reducing power to a variety of crucial processes (cellular biosynthesis, maintenance of a proton motive force) and key assimilatory pathways (carbon dioxide and nitrogen fixation), depending on the availability of nutrients. We also summarize data illustrating that cells use a variety of pathways to recycle excess reductant. Finally, we illustrate how the recent use of genomic and computational approaches to the analysis of these and other photosynthetic organisms has provided testable predictions and considerable new insight into the partitioning of reductant that is produced from solar energy capture.