RESUMO
Polyhydroxybutyrate (PHB) is a common carbon storage polymer among heterotrophic bacteria. It is also accumulated in some photoautotrophic cyanobacteria; however, the knowledge of how PHB accumulation is regulated in this group is limited. PHB synthesis in Synechocystis sp. PCC 6803 is initiated once macronutrients like phosphorus or nitrogen are limiting. We have previously reported a mutation in the gene sll0783 that impairs PHB accumulation in this cyanobacterium upon nitrogen starvation. In this study we present data which explain the observed phenotype. We investigated differences in intracellular localization of PHB synthase, metabolism, and the NADPH pool between wild type and mutant. Localization of PHB synthase was not impaired in the sll0783 mutant; however, metabolome analysis revealed a difference in sorbitol levels, indicating a more oxidizing intracellular environment than in the wild type. We confirmed this by directly measuring the NADPH/NADP ratio and by altering the intracellular redox state of wild type and sll0783 mutant. We were able to physiologically complement the mutant phenotype of diminished PHB synthase activity by making the intracellular environment more reducing. Our data illustrate that the NADPH pool is an important factor for regulation of PHB biosynthesis and metabolism, which is also of interest for potential biotechnological applications.
RESUMO
Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.
Assuntos
Oxirredutases do Álcool/metabolismo , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Oxigenases de Função Mista/metabolismo , Nostoc/enzimologia , Nostoc/genética , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Respiração Celular , Chlamydomonas reinhardtii/fisiologia , Chlamydomonas reinhardtii/efeitos da radiação , Cianobactérias/enzimologia , Cianobactérias/genética , Cianobactérias/fisiologia , Cianobactérias/efeitos da radiação , Glicolatos/metabolismo , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Mutação , Fixação de Nitrogênio/fisiologia , Nitrogenase/genética , Nitrogenase/metabolismo , Nostoc/fisiologia , Nostoc/efeitos da radiação , Oxirredução , Oxigênio/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
The amount of inorganic carbon is one of the main limiting environmental factors for photosynthetic organisms such as cyanobacteria. Using Synechococcus elongatus PCC 7942, we characterized metabolic and transcriptomic changes in cells that had been shifted from high to low CO(2) levels. Metabolic phenotyping indicated an activation of glycolysis, the oxidative pentose phosphate cycle, and glycolate metabolism at lowered CO(2) levels. The metabolic changes coincided with a general reprogramming of gene expression, which included not only increased transcription of inorganic carbon transporter genes but also genes for enzymes involved in glycolytic and photorespiratory metabolism. In contrast, the mRNA content for genes from nitrogen assimilatory pathways decreased. These observations indicated that cyanobacteria control the homeostasis of the carbon-nitrogen ratio. Therefore, results obtained from the wild type were compared with the MP2 mutant of Synechococcus 7942, which is defective for the carbon-nitrogen ratio-regulating PII protein. Metabolites and genes linked to nitrogen assimilation were differentially regulated, whereas the changes in metabolite concentrations and gene expression for processes related to central carbon metabolism were mostly similar in mutant and wild-type cells after shifts to low-CO(2) conditions. The PII signaling appears to down-regulate the nitrogen metabolism at lowered CO(2), whereas the specific shortage of inorganic carbon is recognized by different mechanisms.