Discovery of novel replication proteins for large plasmids in cyanobacteria and their potential applications in genetic engineering.

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid." Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.

Label-free visualization of photosynthetic microbial biofilms using mid-infrared photothermal and autofluorescence imaging.

The formation of photosynthetic microbial biofilms comprising multispecies biomolecules, such as extracellular polymeric substances (EPSs), and microbial cells play pivotal roles in maintaining or stimulating their biological functions. Although there are numerous studies on photosynthetic microbial biofilms, the spatial distribution of EPS components that are vital for microbial biofilm formation, such as exopolysaccharides and proteins, is not well understood. Visualization of photosynthetic microbial biofilms requires label-free methods, because labelling EPSs results in structural changes or aggregation. Raman spectroscopy is useful for label-free visualization of biofilm constituents based on chemical contrast. However, interference resulting from the bright autofluorescence of photosynthetic molecules and the low detection efficiency of Raman scattering make visualization a challenge. Herein, we visualized photosynthetic microbial biofilms in a label-free manner using a super-resolution optical infrared absorption imaging technique, called mid-infrared photothermal (MIP) microscopy. By leveraging the advantages of MIP microscopy, such as its sub-micrometer spatial resolution, autofluorescence-free features, and high detection sensitivity, the distribution of cyanobacteria and their extracellular polysaccharides in the biofilm matrix were successfully visualized. This showed that cyanobacterial cells were aligned along acidic/sulfated polysaccharides in the extracellular environment. Furthermore, spectroscopic analyses elucidated that during formation of biofilms, sulfated polysaccharides initially form linear structures followed by entrapment of cyanobacterial cells. The present study provides the foundation for further studies on the formation, structure, and biological functions of microbial biofilms.

Characterization of a cyanobacterial rep protein with broad-host range and its utilization for expression vectors.

Owing to their photosynthetic capabilities, cyanobacteria are regarded as ecologically friendly hosts for production of biomaterials. However, compared to other bacteria, tools for genetic engineering, especially expression vector systems, are limited. In this study, we characterized a Rep protein, exhibiting replication activity in multiple cyanobacteria and established an expression vector using this protein. Our comprehensive screening using a genomic library of Synechocystis sp. PCC 6803 revealed that a certain region encoding a Rep-related protein (here named Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. A reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, GFP expression in the pYS-based system was tightly regulated by IPTG, achieving 10-fold higher levels than in the chromosome-based system. Furthermore, pYS could be used together with the conventional vector pEX, which was constructed from an endogenous plasmid in S. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

Biosynthesis of a sulfated exopolysaccharide, synechan, and bloom formation in the model cyanobacterium Synechocystis sp. strain PCC 6803.

Extracellularpolysaccharides of bacteria contribute to biofilm formation, stress tolerance, and infectivity. Cyanobacteria, the oxygenic photoautotrophic bacteria, uniquely produce sulfated extracellular polysaccharides among bacteria to support phototrophic biofilms. In addition, sulfated polysaccharides of cyanobacteria and other organisms have been focused as beneficial biomaterial. However, very little is known about their biosynthesis machinery and function in cyanobacteria. Here, we found that the model cyanobacterium, Synechocystis sp. strain PCC 6803, formed bloom-like cell aggregates embedded in sulfated extracellular polysaccharides (designated as synechan) and identified whole set of genes responsible for synechan biosynthesis and its transcriptional regulation, thereby suggesting a model for the synechan biosynthesis apparatus. Because similar genes are found in many cyanobacterial genomes with wide variation, our findings may lead elucidation of various sulfated polysaccharides, their functions, and their potential application in biotechnology.

Bacteria are single-cell microorganisms that can form communities called biofilms, which stick to surfaces such as rocks, plants or animals. Biofilms confer protection to bacteria and allow them to colonize new environments. The physical scaffold of biofilms is a viscous matrix made of several molecules, the main one being polysaccharides, complex carbohydrates formed by many monosaccharides (single sugar molecules) joined together. Cyanobacteria, also known as blue-green algae, are a type of bacteria that produce oxygen and use sunlight as an energy source, just as plants and algae do. Cyanobacteria produce extracellular polysaccharides that contain sulfate groups. These sulfated polysaccharides are also produced by animals and algae but are not common in other bacteria or plants. One possible role of sulfated, extracellular polysaccharides in cyanobacteria is keeping cells together in the floating aggregates found in cyanobacterial blooms. These are visible discolorations of the water caused by an overgrowth of cyanobacteria that occur in lakes, estuaries and coastal waters. However, little is known about how these polysaccharides are synthesized in cyanobacteria and what their natural role is. Maeda et al. found a strain of cyanobacteria that formed bloom-like aggregates that were embedded in sulfated extracellular polysaccharides. Using genetic engineering techniques, the researchers identified a set of genes responsible for producing a sulfated extracellular polysaccharide and regulating its levels. They also found that cell aggregates of cyanobacteria can float without having intracellular gas vesicles, which was previously thought to enable blooms to float. The results of the present study could have applications for human health, since many sulfated polysaccharides have antiviral, antitumor or anti-inflammatory properties, and similar genes are found in many cyanobacteria. In addition, these findings could be useful for controlling toxic cyanobacterial blooms, which are becoming increasingly problematic for society.

Astaxanthin production in a model cyanobacterium Synechocystis sp. PCC 6803.

Heterologous production of a useful carotenoid astaxanthin was achieved in a cyanobacterium Synechocystis sp. PCC 6803 with the aid of marine bacterial genes. Astaxanthin and its intermediates emerged at high levels, whereas ß-carotene and zeaxanthin disappeared in the strain. Total carotenoid accumulation was nearly two fold compared with wild type. The astaxanthin-producing strain was capable of only growing heterotrophically, which was likely due to the absence of ß-carotene. Further enhanced accumulation was pursued by gene overexpression for possible rate-limiting steps in the biosynthesis pathway.

Acclimation process of the chlorophyll d-bearing cyanobacterium Acaryochloris marina to an orange light environment revealed by transcriptomic analysis and electron microscopic observation.

The cyanobacterium Acaryochloris marina MBIC 11017 (A. marina 11017) possesses chlorophyll d (Chl. d) peaking at 698 nm as photosystem reaction center pigments, instead of chlorophyll a (Chl. a) peaking at 665 nm. About 95% of the total chlorophylls is Chl. d in A. marina 11017. In addition, A. marina 11017 possesses phycobilisome (PBS) supercomplex to harvest orange light and to transfer the absorbing energy to the photosystems. In this context, A. marina 11017 utilizes both far-red and orange light as the photosynthetic energy source. In the present study, we incubated A. marina 11017 cells under monochromatic orange and far-red light conditions and performed transcriptional and morphological studies by RNA-seq analysis and electron microscopy. Cellular absorption spectra, transcriptomic profiles, and microscopic observations demonstrated that PBS was highly accumulated under an orange light condition relative to a far-red light condition. Notably, transcription of one cpcBA operon encoding the phycobiliprotein of the phycocyanin was up-regulated under the orange light condition, but another operon was constitutively expressed under both conditions, indicating functional diversification of these two operons for light harvesting. Taking the other observations into consideration, we could illustrate the photoacclimation processes of A. marina 11017 in response to orange and far-red light conditions in detail.

Functional diversification of two bilin reductases for light perception and harvesting in unique cyanobacterium Acaryochloris marina MBIC 11017.

Bilin pigments play important roles for both light perception and harvesting in cyanobacteria by binding to cyanobacteriochromes (CBCRs) and phycobilisomes (PBS), respectively. Among various cyanobacteria, Acaryochloris marina MBIC 11017 (A. marina 11017) exceptionally uses chlorophyll d as the main photosynthetic pigment absorbing longer wavelength light than the canonical pigment, chlorophyll a, indicating existence of a system to sense longer wavelength light than others. On the other hand, A. marina 11017 has the PBS apparatus to harvest short-wavelength orange light, similar to most cyanobacteria. Thus, A. marina 11017 might sense longer wavelength light and harvest shorter wavelength light by using bilin pigments. Phycocyanobilin (PCB) is the main bilin pigment of both systems. Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes PCB synthesis from biliverdin via the intermediate 181 ,182 -dihydrobiliverdin (181 ,182 -DHBV), resulting in the stepwise shortening of the absorbing wavelengths. In this study, we found that A. marina 11017 exceptionally encodes two PcyA homologs, AmPcyAc and AmPcyAp. AmPcyAc is encoded on the main chromosome with most photoreceptor genes, whereas AmPcyAp is encoded on a plasmid with PBS-related genes. High accumulation of 181 ,182 -DHBV for extended periods was observed during the reaction catalyzed by AmPcyAc, whereas 181 ,182 -DHBV was transiently accumulated for a short period during the reaction catalyzed by AmPcyAp. CBCRs could sense longer wavelength far-red light through 181 ,182 -DHBV incorporation, whereas PBS could only harvest orange light through PCB incorporation, suggesting functional diversification of PcyA as AmPcyAc and AmPcyAp to provide 181 ,182 -DHBV and PCB to the light perception and harvesting systems, respectively.

Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system.

Cells of the thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN (NIES-2134) aggregate and produce extracellular cellulose under induced conditions of blue light and low temperature, and both aggregation and cellulose production require the cellulose synthase Tll0007 (XcsA) and photosensory diguanylate cyclases. However, overexpression of both the cellulose synthase and a constitutively active diguanylate cyclase was not sufficient to induce cellulose-mediated cell aggregation under normal growth conditions. Synteny analysis and gene knockout revealed that two putative genes, hlyD-like tlr0903 (xcsB) and endoglucanase-like tlr1902 (xcsC), are linked to tll0007, although they are located apart from tll0007 in the T. vulcanus genome. Gene knockdown revealed that tlr1605 (tolC) was essential for the cellulose-mediated cell aggregation. Low temperature induced marked upregulation of tlr0903, and overexpression of both tlr0903 (but not tlr1902) and diguanylate cyclase resulted in the strong cell aggregation and cellulose accumulation under normal conditions. Based on these and phylogenetic analysis, we propose that the cyanobacterial extracellular cellulose production is due to a novel variant of the bacterial tripartite secretion system.

CugP is a novel ubiquitous non-GalU-type bacterial UDP-glucose pyrophosphorylase found in cyanobacteria.

UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and glucose 1-phosphate and exists in almost all species. Most bacteria possess a GalU-type UDP-glucose pyrophosphorylase, whereas many cyanobacteria species do not. In certain cyanobacteria, UDP-glucose is used as a substrate for synthesis of exopolysaccharide cellulose in spite of the absence of GalU-type UDP-glucose pyrophosphorylase. Therefore, there should be an uncharacterized UDP-glucose pyrophosphorylase in cyanobacteria. Here, we show that all cyanobacteria possess a non-GalU-type bacterial UDP-glucose pyrophosphorylase, i.e., CugP, a novel family in the nucleotide triphosphate transferase superfamily. The expressed recombinant Synechocystis sp. strain PCC 6803 CugP had pyrophosphorylase activity that was highly specific for UTP and glucose 1-phosphate. The fact that the CugP gene cannot be deleted completely in Synechocystis sp. PCC 6803 suggests its central role as the substrate supplier for galactolipid synthesis. Galactolipids are major constituents of the photosynthetic thylakoid membrane and important for photosynthetic activity. Based on phylogenetic analysis, this CugP-type UDP-glucose pyrophosphorylase may have recently been horizontally transferred to certain noncyanobacteria.