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The methylerythritol phosphate pathway in photosynthetic cyanobacteria was engineered to allow highly efficient production of isoprene from CO2.

Living organisms have evolved mechanisms for adjusting their metabolism to adapt to environmental nutrient availability. Terrestrial animals utilize the ornithine-urea cycle to dispose of excess nitrogen derived from dietary protein. Here, we identified an active ornithine-ammonia cycle (OAC) in cyanobacteria through an approach combining dynamic N andC tracers, metabolomics, and mathematical modeling. The pathway starts with carbamoyl phosphate synthesis by the bacterial- and plant-type glutamine-dependent enzyme and ends with conversion of arginine to ornithine and ammonia by a novel arginine dihydrolase. An arginine dihydrolase-deficient mutant showed disruption of OAC and severely impaired cell growth when nitrogen availability oscillated. We demonstrated that the OAC allows for rapid remobilization of nitrogen reserves under starvation and a high rate of nitrogen assimilation and storage after the nutrient becomes available. Thus, the OAC serves as a conduit in the nitrogen storage-and-remobilization machinery in cyanobacteria and enables cellular adaptation to nitrogen fluctuations.

c Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD ؉ ratio, has recently been found to play a role in the solventogenic shift of Clostridium acetobutylicum. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO 2 /CO fixation. The predicted Rex-binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with purified Rex protein. Novel members of the C. acetobutylicum Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and rex-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H 2 O 2 on intracellular NADH and NAD ؉ concentrations, expression of Rex regulon genes, and physiology of the wild type and rex-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD ؉ ratio in vivo to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in C. acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD ؉ homeostasis in clostridia.

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Background: The σ 54 factor controls unique promoters and interacts with a specialized activator (enhancer binding proteins [EBP]) for transcription initiation. Although σ 54 is present in many Clostridiales species that have great importance in human health and biotechnological applications, the cellular processes controlled by σ 54 remain unknown. Results: For systematic analysis of the regulatory functions of σ 54 , we performed comparative genomic reconstruction of transcriptional regulons of σ 54 in 57 species from the Clostridiales order. The EBP-binding DNA motifs and regulated genes were identified for 263 EBPs that constitute 39 distinct groups. The reconstructed σ 54 regulons contain the genes involved in fermentation and amino acid catabolism. The predicted σ 54 binding sites in the genomes of Clostridiales spp. were verified by in vitro binding assays. To our knowledge, this is the first report about direct regulation of the Stickland reactions and butyrate and alcohols synthesis by σ 54 and the respective EBPs. Considerable variations were demonstrated in the sizes and gene contents of reconstructed σ 54 regulons between different Clostridiales species. It is proposed that σ 54 controls butyrate and alcohols synthesis in solvent-producing species, regulates autotrophic metabolism in acetogenic species, and affects the toxin production in pathogenic species. Conclusions: This study reveals previously unrecognized functions of σ 54 and provides novel insights into the regulation of fermentation and amino acid metabolism in Clostridiales species, which could have potential applications in guiding the treatment and efficient utilization of these species.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domain (PRD)-containing enhancer binding proteins (EBPs) are an important class of σ(54) -interacting transcriptional activators. Although PRD-containing EBPs are present in many Firmicutes, most of their regulatory functions remain unclear. In this study, the transcriptional regulons of about 50 PRD-containing EBPs in diverse Firmicutes species are reconstructed by using a comparative genomic approach, which contain the genes associated with utilization of β-glucosides, fructose/levan, mannose/glucose, pentitols, and glucosamine/fructosamine. We then present experimental evidence that the cel operon involved in cellobiose utilization is directly regulated by CelR and σ(54) (SigL) in Clostridium acetobutylicum. The predicted three CelR-binding sites and σ(54) promoter elements upstream of the cel operon are verified by in vitro binding assays. We show that CelR has an ATPase activity, which is strongly stimulated by the presence of DNA containing the CelR-binding sites. Moreover, mutations in any one of the three CelR-binding sites significantly decreased the cel promoter activity probably due to the need for all three DNA sites for maximal ATPase activity of CelR. It is suggested that CelR is regulated by PTS-mediated phosphorylation at His-551 and His-829, which exerts a positive effect and an inhibitory effect, respectively, on the CelR activity.

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