Cyanobacteria belonging to the Synechocystis genus have been isolated from diverse aquatic environments. This announcement reports the complete genome sequence of Synechocystis sp. LKSZ1 newly isolated from a pond in a university campus in Yokohama, Japan. The genome sequencing was performed using the PacBio Revio HiFi long-read technology.

The circadian clock of cyanobacteria, which predicts daily environmental changes, typically includes a standard oscillator consisting of proteins KaiA, KaiB, and KaiC. However, several cyanobacteria have diverse Kai protein homologs of unclear function. In particular, Synechocystis sp. PCC 6803 harbours, in addition to a canonical kaiABC gene cluster (named kaiAB1C1), two further kaiB and kaiC homologs (kaiB2, kaiB3, kaiC2, kaiC3). Here, we identify a chimeric KaiA homolog, named KaiA3, encoded by a gene located upstream of kaiB3. At the N-terminus, KaiA3 is similar to response-regulator receiver domains, whereas its C-terminal domain resembles that of KaiA. Homology analysis shows that a KaiA3-KaiB3-KaiC3 system exists in several cyanobacteria and other bacteria. Using the Synechocystis sp. PCC 6803 homologs, we observe circadian oscillations in KaiC3 phosphorylation in vitro in the presence of KaiA3 and KaiB3. Mutations of kaiA3 affect KaiC3 phosphorylation, leading to growth defects under both mixotrophic and chemoheterotrophic conditions. KaiC1 and KaiC3 exhibit phase-locked free-running phosphorylation rhythms. Deletion of either system (∆kaiAB1C1 or ∆kaiA3B3C3) alters the period of the cellular backscattering rhythm. Furthermore, both oscillators are required to maintain high-amplitude, self-sustained backscatter oscillations with a period of approximately 24 h, indicating their interconnected nature.

The hox operon in Synechocystis sp. PCC 6803, encoding bidirectional hydrogenase responsible for H2 production, is transcriptionally upregulated under microoxic conditions. Although several regulators for hox transcription have been identified, their dynamics and higher-order DNA structure of hox region in microoxic conditions remain elusive. We focused on key regulators for the hox operon: cyAbrB2, a conserved regulator in cyanobacteria, and SigE, an alternative sigma factor. Chromatin immunoprecipitation sequencing revealed that cyAbrB2 binds to the hox promoter region under aerobic conditions, with its binding being flattened in microoxic conditions. Concurrently, SigE exhibited increased localization to the hox promoter under microoxic conditions. Genome-wide analysis revealed that cyAbrB2 binds broadly to AT-rich genome regions and represses gene expression. Moreover, we demonstrated the physical interactions of the hox promoter region with its distal genomic loci. Both the transition to microoxic conditions and the absence of cyAbrB2 influenced the chromosomal interaction. From these results, we propose that cyAbrB2 is a cyanobacterial nucleoid-associated protein (NAP), modulating chromosomal conformation, which blocks RNA polymerase from the hox promoter in aerobic conditions. We further infer that cyAbrB2, with altered localization pattern upon microoxic conditions, modifies chromosomal conformation in microoxic conditions, which allows SigE-containing RNA polymerase to access the hox promoter. The coordinated actions of this NAP and the alternative sigma factor are crucial for the proper hox expression in microoxic conditions. Our results highlight the impact of cyanobacterial chromosome conformation and NAPs on transcription, which have been insufficiently investigated.

Cyanobacteria show promise as hosts for whole-cell biocatalysis. Their photoautotrophic metabolism can be leveraged for a sustainable production process. Despite advancements, performance still lags behind heterotrophic hosts. A key challenge is the limited ability to overexpress recombinant enzymes, which also hinders their biocatalytic efficiency. To address this, we generated large-scale expression libraries and developed a high-throughput method combining fluorescence-activated cell sorting (FACS) and deep sequencing in Synechocystis sp. PCC 6803 (Syn. 6803) to screen and optimize its genetic background. We apply this approach to enhance expression and biocatalyst performance for three enzymes: the ketoreductase LfSDR1M50, enoate reductase YqjM, and Baeyer-Villiger monooxygenase (BVMO) CHMOmut. Diverse genetic combinations yielded significant improvements: optimizing LfSDR1M50 expression showed a 17-fold increase to 39.2 U gcell dry weight (CDW)-1. In vivo activity of Syn. YqjM was improved 16-fold to 58.7 U gCDW-1 and, for Syn. CHMOmut, a 1.5-fold increase to 7.3 U gCDW-1 was achieved by tailored genetic design. Thus, this strategy offers a pathway to optimize cyanobacteria as expression hosts, paving the way for broader applications in other cyanobacteria strains and larger libraries.

SynDLP, a dynamin-like protein (DLP) encoded in the cyanobacterium Synechocystis sp. PCC 6803, has recently been identified to be structurally highly similar to eukaryotic dynamins. To elucidate structural changes during guanosine triphosphate (GTP) hydrolysis, we solved the cryoelectron microscopy (cryo-EM) structures of oligomeric full-length SynDLP after addition of guanosine diphosphate (GDP) at 4.1 Å and GTP at 3.6-Å resolution as well as a GMPPNP-bound dimer structure of a minimal G-domain construct of SynDLP at 3.8-Å resolution. In comparison with what has been seen in the previously resolved apo structure, we found that the G-domain is tilted upward relative to the stalk upon GTP hydrolysis and that the G-domain dimerizes via an additional extended dimerization domain not present in canonical G-domains. When incubated with lipid vesicles, we observed formation of irregular tubular SynDLP assemblies that interact with negatively charged lipids. Here, we provide the structural framework of a series of different functional SynDLP assembly states during GTP turnover.

Polyphosphate is prevalent in living organisms. To obtain insights into polyphosphate synthesis and its physiological significance in cyanobacteria, we characterize sll0290, a homolog of the polyphosphate-kinase-1 gene, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. The Sll0290 protein structure reveals characteristics of Ppk1. A Synechocystis sll0290 disruptant and sll0290-overexpressing Escherichia coli transformant demonstrated loss and gain of polyphosphate synthesis ability, respectively. Accordingly, sll0290 is identified as ppk1. The disruptant (Δppk1) grows normally with aeration of ordinary air (0.04% CO2), consistent with its photosynthesis comparable to the wild type level, which contrasts with a previously reported high-CO2 (5%) requirement for Δppk1 in an alkaline hot spring cyanobacterium, Synechococcus OS-B\'. Synechocystis Δppk1 is defective in polyphosphate hyperaccumulation and survival competence at the stationary phase, and also under sulfur-starvation conditions, implying that sulfur limitation is one of the triggers to induce polyphosphate hyperaccumulation in stationary cells. Furthermore, Δppk1 is defective in the enhancement of total phosphorus contents under sulfur-starvation conditions, a phenomenon that is only partially explained by polyphosphate hyperaccumulation. This study therefore demonstrates that in Synechocystis, ppk1 is not essential for low-CO2 acclimation but plays a crucial role in dynamic P-metabolic regulation, including polyP hyperaccumulation, to maintain physiological fitness under sulfur-starvation conditions.

Cyanobacteria are important primary producers, contributing to 25% of the global carbon fixation through photosynthesis. They serve as model organisms to study the photosynthesis, and are important cell factories for synthetic biology. To enable efficient genetic dissection and metabolic engineering in cyanobacteria, effective and accurate genetic manipulation tools are required. However, genetic manipulation in cyanobacteria by the conventional homologous recombination-based method and the recently developed CRISPR-Cas gene editing system require complicated cloning steps, especially during multi-site editing and single base mutation. This restricts the extensive research on cyanobacteria and reduces its application potential. In this study, a highly efficient and convenient cytosine base editing system was developed which allows rapid and precise C → T point mutation and gene inactivation in the genomes of Synechocystis and Anabaena. This base editing system also enables efficient multiplex editing and can be easily cured after editing by sucrose counter-selection. This work will expand the knowledge base regarding the engineering of cyanobacteria. The findings of this study will encourage the biotechnological applications of cyanobacteria.

Singlet oxygen (1O2) is an important reactive oxygen species whose formation by the type-II, light-dependent, photodynamic reaction is inevitable during photosynthetic processes. In the last decades, the recognition that 1O2 is not only a damaging agent, but can also affect gene expression and participates in signal transduction pathways has received increasing attention. However, contrary to several other taxa, 1O2-responsive genes have not been identified in the important cyanobacterial model organism Synechocystis PCC 6803. By using global transcript analysis we have identified a large set of Synechocystis genes, whose transcript levels were either enhanced or repressed in the presence of 1O2. Characteristic 1O2 responses were observed in several light-inducible genes of Synechocystis, especially in the hli (or scp) family encoding HLIP/SCP proteins involved in photoprotection. Other important 1O2-induced genes include components of the Photosystem II repair machinery (psbA2 and ftsH2, ftsH3), iron homeostasis genes isiA and idiA, the group 2 sigma factor sigD, some components of the transcriptomes induced by salt-, hyperosmotic and cold-stress, as well as several genes of unknown function. The most pronounced 1O2-induced upregulation was observed for the hliB and the co-transcribed lilA genes, whose deletion induced enhanced sensitivity against 1O2-mediated light damage. A bioreporter Synechocystis strain was created by fusing the hliB promoter to the bacterial luciferase (lux), which showed its utility for continuous monitoring of 1O2 concentrations inside the cell.

Polyhydroxyalkanoates (PHAs) are biopolymers produced by microorganisms under nutrient limiting conditions and in the presence of excess carbon source. PHAs have gained popularity as a sustainable alternative to traditional plastics. However, large scale production of PHAs is economically challenging due to the relatively high costs of organic carbon. Alternative options include using organisms capable of phototrophic or mixotrophic growth. This study aimed at the production of poly(3-hydroxybutyrate) P(3HB), a type of PHA, at pilot scale using the freshwater cyanobacterium Synechocystis sp. PCC6803. First, to identify optimal conditions for P(3HB) production from Synechocystis sp. PCC6803, different supplemental carbon source concentrations and salinity levels were tested at laboratory scale. The addition of 4 g/L acetate with no added NaCl led to P(3HB) accumulation of 10.7 % dry cell weight on the 28th day of cultivation. Although acetate additions were replicated in an outdoor 400 L serpentine photobioreactor, P(3HB) content was lower, implying uncontrolled conditions impact on biopolymer production efficiency. An optimized P(3HB) extraction methodology was developed to remove pigments, and the biopolymer was characterized and subjected to 3D printing (fused deposition modelling) to confirm its processability. This study thus successfully led to the large-scale production of P(3HB) using sustainable and environmentally friendly cyanobacterial fermentation.

Co-culturing fungi and microalgae may effectively remediate wastewater containing Cd and harvest microalgae. Nevertheless, a detailed study of the mechanisms underlying the synergistic interactions between fungi and microalgae under Cd(II) exposure is lacking. In this study, Cd(II) exposure resulted in a significant enhancement of antioxidants, such as glutathione (GSH), malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide dismutase (SOD) compared to the control group, suggesting that the cellular antioxidant defense response was activated. Extracellular proteins and extracellular polysaccharides of the symbiotic system were increased by 60.61 % and ,24.29 %, respectively, after Cd(II) exposure for 72 h. The adsorption behavior of Cd(II) was investigated using three-dimensional fluorescence excitation-emission matrix (3D-EEM), fourier transform infrared spectroscopy (FTIR), and scanning electron microscope (SEM). Metabolomics results showed that the TCA cycle provided effective material and energy supply for the symbiotic system to resist the toxicity of Cd(II); Proline, histidine, and glutamine strengthened the synergistic adsorption capacity of the fungus and microalgae. Overall, the theoretical foundation for a deep comprehension of the beneficial interactions between fungi and microalgae under Cd(II) exposure and the role of the fungal-algal symbiotic system in the management of heavy metal pollution is provided by this combined physiological and metabolomic investigation.