Endogenous clock-mediated regulation of intracellular oxygen dynamics is essential for diazotrophic growth of unicellular cyanobacteria.

The discovery of nitrogen fixation in unicellular cyanobacteria provided the first clues for the existence of a circadian clock in prokaryotes. However, recalcitrance to genetic manipulation barred their use as model systems for deciphering the clock function. Here, we explore the circadian clock in the now genetically amenable Cyanothece 51142, a unicellular, nitrogen-fixing cyanobacterium. Unlike non-diazotrophic clock models, Cyanothece 51142 exhibits conspicuous self-sustained rhythms in various discernable phenotypes, offering a platform to directly study the effects of the clock on the physiology of an organism. Deletion of kaiA, an essential clock component in the cyanobacterial system, impacted the regulation of oxygen cycling and hindered nitrogenase activity. Our findings imply a role for the KaiA component of the clock in regulating the intracellular oxygen dynamics in unicellular diazotrophic cyanobacteria and suggest that its addition to the KaiBC clock was likely an adaptive strategy that ensured optimal nitrogen fixation as microbes evolved from an anaerobic to an aerobic atmosphere under nitrogen constraints.

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here, we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR-Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55-kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the wild type (WT). This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome-to-phenome relationships.IMPORTANCEGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.