Dynamics of the Synechococcus elongatus cytoskeletal GTPase FtsZ yields mechanistic and evolutionary insight into cyanobacterial and chloroplast FtsZs

The division of cyanobacteria and their chloroplast descendants is orchestrated by filamenting temperature-sensitive Z (FtsZ), a cytoskeletal GTPase that polymerizes into protofilaments that form a “Z ring” at the division site. The Z ring has both a scaffolding function for division-complex assembly and a GTPase-dependent contractile function that drives cell or organelle constriction. A single FtsZ performs these functions in bacteria, whereas in chloroplasts, they are performed by two copolymerizing FtsZs, called AtFtsZ2 and AtFtsZ1 in Arabidopsis thaliana, which promote protofilament stability and dynamics, respectively. To probe the differences between cyanobacterial and chloroplast FtsZs, we used light scattering to characterize the in vitro protofilament dynamics of FtsZ from the cyanobacterium Synechococcus elongatus PCC 7942 (SeFtsZ) and investigate how coassembly of AtFtsZ2 or AtFtsZ1 with SeFtsZ influences overall dynamics. SeFtsZ protofilaments assembled rapidly and began disassembling before GTP depletion, whereas AtFtsZ2 protofilaments were far more stable, persisting beyond GTP depletion. Coassembled SeFtsZ–AtFtsZ2 protofilaments began disassembling before GTP depletion, similar to SeFtsZ. In contrast, AtFtsZ1 did not alter disassembly onset when coassembled with SeFtsZ, but fluorescence recovery after photobleaching analysis showed it increased the turnover of SeFtsZ subunits from SeFtsZ–AtFtsZ1 protofilaments, mirroring its effect upon coassembly with AtFtsZ2. Comparisons of our findings with previous work revealed consistent differences between cyanobacterial and chloroplast FtsZ dynamics and suggest that the scaffolding and dynamics-promoting functions were partially separated during evolution of two chloroplast FtsZs from their cyanobacterial predecessor. They also suggest that chloroplasts may have evolved a mechanism distinct from that in cyanobacteria for promoting FtsZ protofilament dynamics.     

Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition

Capture and conversion of CO₂ to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry‐relevant chemicals from CO₂. However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO₂ to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP‐driven malonyl‐CoA synthesis pathway and heterologous phosphoketolase (PHK)‐phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl‐CoA directly from CO₂ in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl‐CoA‐derived biochemicals.     

Growth engineering of Synechococcus elongatus PCC 7942 for mixotrophy under natural light conditions for improved feedstock production

Synechococcus elongatus PCC 7942 has been widely explored as cyanobacterial cell factory through genetic modifications for production of various value‐added compounds. However, successful industrial scale‐ups have not been reported for the system predominantly due to its obligate photoautotrophic metabolism and use of artificial light in photobioreactors. Hence, engineering the organism to perform mixotrophy under natural light could serve as an effective solution. Thus, we applied a genetically engineered strain of Synechococcus elongatus PCC 7942 expressing heterologous hexose transporter gene (galP) to perform mixotrophy under natural light in a temperature controlled environmental chamber (EC). We systematically studied the comparative performances of these transformants using autotrophy and mixotrophy, which showed 3.4 times increase in biomass productivity of mixotrophically grown transformants over autotrophs in EC. Chlorophyll a yield was found to have decreased in mixotrophic conditions, possibly indicating reduced dependency on light for energy metabolism. Although pigment yield decreases under mixotrophy, titer was found to have improved due to increased biomass productivity. Carotenoid analysis showed that zeaxanthin is the major carotenoid produced by the species which is essential for photoprotection. Our work thus demonstrates that mixotrophy under temperature controlled natural light can serve as the viable solution to improve biomass productivity of Synechococcus elongatus PCC 7942 and for commercial production of natural or engineered value added compounds from the system. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1182–1192, 2017     

A thermophilic cyanobacterium Synechococcus elongatus has three different Class I prenyltransferase genes

Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C₅) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C₂₀) diphosphate synthase, another encodes a farnesyl (C₁₅) diphosphate synthase whose optimal reaction temperature is 60 °C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 °C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C₂₅), hexaprenyl (C₃₀), and heptaprenyl (C₃₅) diphosphates from dimethylallyl (C₅) diphosphate, geranyl (C₂₀) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).     

The Formation and Preservation of Synechococcus elongatus Cell Molds in Simulated Silica Sinter: Implications for the Identification of Microfossils

Siliceous sinters that precipitate around modern hot spring systems are able to fossilize the indigenous microbial communities, forming molds that accurately outline the shape of the microorganisms. Over time, the biomass decays, and only silica molds or their infill may remain as evidence of the former living cells. However, little is known regarding the fidelity of such silica molds in terms of size and morphology, and the preservation of critical parameters for the identification of ancient silicified microorganisms by silica molds remains untested. Here we report experiments examining the formation of microbial molds of the cyanobacterium Synechococcus elongatus in silica gel. We demonstrate that post-depositional processes, primarily desiccation, are crucial for obtaining accurate and robust molds, and that initial desiccation acts to strengthen cell molds against further alteration. However, all silica gel treatments systematically created preservational biases (changes in size, additional structures) that may be misleading and may complicate the identification of fossil microorganisms.     

Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus

Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635–2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion. Received: 28 July 1997 / Accepted: 26 November 1997     

Biotransformation of 5‐Hydroxymethylfurfural to 2,5‐Furandicarboxylic Acid by a Syntrophic Consortium of Engineered Synechococcus elongatus and Pseudomonas putida

2,5‐furandicarboxylic acid (FDCA) is one of the top platform chemicals that can be produced from biomass feedstock. To make the cost of industrial FDCA production compatible with plastics made from fossils, the price of substrates and process complexity should be reduced. The aim of this research is to create a CO₂‐driven syntrophic consortium for the catalytic conversion of renewable biomass‐derived 5‐hydroxymethylfurfural (HMF) to FDCA. Sucrose produced from carbon fixation by the engineered Synechococcus elongatus serves as the sole carbon source for the engineered Pseudomonas putida to catalyze the reaction of HMF to FDCA. The yield of FDCA by the consortium reaches around 70% while the conversion of HMF is close to 100%. With further surface engineering to clump the two strains, the FDCA yield is elevated to almost 100% via the specific association between an Src homology 3 (SH3) domain and its ligand. The syntrophic consortium successfully demonstrates its green and cost‐effective characteristics for the conversion of CO₂ and biomass into platform chemicals.     

聚球藻Synechococcus sp.PCC7942高CO_2需求突变株的筛选及其超微结构观察(英文)

以单细胞蓝藻聚球藻Synechococcussp.PCC7942为材料,利用甲基磺酸乙酯(EMS)进行化学诱变获得了一个高CO2 需求突变株。它能在 4%CO2 下生长而不能在空气中生长。对突变株的初检表明:其回复突变率约为 10 -7。该突变株从高CO2 条件下转到空气中后,细胞在 2～ 3d内逐渐趋于死亡;其光合作用对外源无机碳的依赖性高于野生型细胞,碳酸酐酶活性也低于野生型细胞。在超微结构水平,突变株细胞内出现了不同类型的异常羧体:有的为棒状;有的为不规则状;有的为 空羧体",而且,类囊体周围糖原颗粒增多。进一步说明该突变株在CO2 吸收和利用功能上有缺陷。此外,对低碳条件对羧体的诱导及羧体的生物发生也作了一些探讨     

Metabolic rewiring of synthetic pyruvate dehydrogenase bypasses for acetone production in cyanobacteria

Designing synthetic pathways for efficient CO₂ fixation and conversion is essential for sustainable chemical production. Here we have designed a synthetic acetate‐acetyl‐CoA/malonyl‐CoA (AAM) bypass to overcome an enzymatic activity of pyruvate dehydrogenase complex. This synthetic pathway utilizes acetate assimilation and carbon rearrangements using a methyl malonyl‐CoA carboxyltransferase. We demonstrated direct conversion of CO₂ into acetyl‐CoA‐derived acetone as an example in photosynthetic Synechococcus elongatus PCC 7942 by increasing the acetyl‐CoA pools. The engineered cyanobacterial strain with the AAM‐bypass produced 0.41 g/L of acetone at 0.71 m/day of molar productivity. This work clearly shows that the synthetic pyruvate dehydrogenase bypass (AAM‐bypass) is a key factor for the high‐level production of an acetyl‐CoA‐derived chemical in photosynthetic organisms.     

Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.     

High-Throughput Functional Analysis of the Synechococcus elongatus PCC 7942 Genome

Synechococcus elongatus PCC 7942 was the first cyanobacterialstrain to be reliably transformed by exogenously added DNA andhas become the model organism for cyanobacterial circadian rhythms.With a small genome (2.7 Mb) and well-developed genetic tools,PCC 7942 provides an exceptional opportunity to elucidate thecircadian mechanism through genetics. We describe a projectto create mutations in every locus of the genome, both to assayeach locus for its potential contribution to the circadian clockand to archive data for the cyanobacterial community. Cosmidclones that carry inserts of PCC 7942 DNA are saturated withtransposon insertions in vitro to provide sequencing templatesand substrates for mutagenesis of the PCC 7942 genome via homologousrecombination. We have mutagenized 53% of the chromosome from50 chromosome-bearing cosmids and identified the positions ofinsertions in 31 of those cosmids and the 46 kb plasmid, pANL.PCC 7942 mutants defective for 490 different genes have beenscreened for circadian phenotypes. Mutagenesis of three apparentlyessential loci, including clpPIIclpX, resulted in circadianphenotypes. We developed an effective antisense suppressionmethod to further the analysis of essential genes. When completed,the set of comprehensive mutations will provide the communitywith a unique resource whose impact will extend beyond circadianresearch.     

Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.     

Oxidative Processes in Photosystem I of Thermophilic Cyanobacteria at High Temperatures

We studied the mechanisms of the relationships between the generation of millisecond delayed fluorescence in photosystem I (DF) and the oxidative destruction of chlorophyll in the membranes of a thermophilic cyanobacteria Synechococcus elongatusin the temperature range 60–80°C at various irradiation levels and in the presence of substances affecting the intensity of DF. Light and temperature dependencies of the chlorophyll oxidation rates were similar to those of the DF of PSI. Anions Cl^–, Br^–, and NO^– ₃, which quench the triplet states of chlorophyll, almost completely inhibited the chlorophyll oxidation and reduced the intensity of the DF maximum by 70%. Under anaerobic conditions and in the presence of sodium ascorbate, the rate of chlorophyll oxidation also markedly decreased. We found that the long-wavelength chlorophyll forms were the most susceptible to oxidation and related the temperature-dependent changes in the DF of PSI and in the oxidative processes in the membranes of thermophilic cyanobacteria to an increase in the concentration of the triplet states of P₇₀₀and other chlorophyll forms. The latter result from the temperature-dependent inactivation of carotenoids and the inhibition of electron transfer to ferredoxin in PSI.     

Physiological changes in marine picocyanobacterial Synechococcus strains exposed to elevated CO2 partial pressure

Unicellular marine cyanobacteria are abundant in both coastal and oligotrophic environments, where they contribute substantially to primary production. The physiological effect of future increases in atmospheric CO₂ concentrations on the marine picocyanobacteria is still poorly known. We studied the physiological changes in marine phycocyanin (PC)-rich and phycoerythrin (PE)-rich Synechococcus strains under different CO₂ partial pressures (350, 600 and 800 ppm). The PE strain showed no significant change in growth rate over the experimental CO₂ range. A significant increase (25.4%) in carbohydrate was observed at 800 ppm CO₂, but no significant change in protein and RNA/DNA ratio was observed in any CO₂ treatment. The PC strain showed a significant increase (36.7%) in growth rate at 800 ppm CO₂, but no significant change in carbohydrate or protein content was observed over the entire CO₂ range. The RNA/DNA ratio increased with increasing CO₂ concentration and was positively correlated with growth rate. Cellular red fluorescence and orange fluorescence of the PE strain tended to decline in all CO₂ treatments. However, no such decline was observed at higher CO₂ treatments in the PC strain. Our results suggest that the PC strain would probably benefit more than the PE strain from future increases in atmospheric CO₂ concentrations.     

The Physiological Response of Synechococcus elongatus to Salinity: A Potential Biomarker for Ancient Salinity in Evaporative Environments

The purpose of this study was to characterize the physiological response of Synechococcus elongatus, a brackish-water cyanobacterium, to salt stress. S. elongatus was grown in artificial sea water medium with different salinities. The response was measured by analysis of extracellular polymeric substances (EPS) and membrane lipids. The EPS yields were positively correlated (r² = 0.99) with the salinity. The ratio of unsaturated to saturated fatty acids (U/S) increased with salinity in the range of 2.1‰ to 31.5‰ and decreased at 52.5‰. A positive linear correlation (r² = 0.92) was observed between the average chain length (ACL) of fatty acids and the salinity. These data indicate that S. elongatus adapted to salt stress by the secretion of EPS and by adjusting the membrane fluidity through the changes in ACL or desaturation of fatty acids. These variations in EPS and fatty acids may be used as geochemical biomarkers in sediments to unravel changes in the salinity of ancient evaporative environments.     

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 g^–1 microcystin-RR. The results showed that the growth of Synechococcus elongatus (expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Significant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.     

聚球藻7942光自养培养的碳代谢和能量代谢

代谢通量分析是研究微藻光自养培养过程中CO2和光能利用的一个非常有用的工具。本研究建立了聚球藻7942光自养培养代谢网络,并通过代谢通量方法分析了不同入射光强下的碳代谢流分布和能量代谢。研究结果表明,CO2固定是代谢能量和还原力消耗的主要途径,分别约占总消耗能量的85%和70%。研究还发现在一定光强范围,基于ATP生成的细胞得率和最大细胞得率基本维持不变,分别约为2.80g/molATP和2.95g/molATP,但基于总吸收光能的细胞得率和对应的光能转换效率则随着光强的增加而降低。     

Nitrite accumulation by Synechococcus 6301 as a consequence of carbon- or sulfur-deficiency

Abstract Air grown cultures of the cyanobacterium Synechococcus 6301, when incubated under continuous illumination with nitrate as the sole nitrogen source, started to liberate nitrite from the second day of inoculation. Nitrite accumulation depended on culture density and was caused by CO₂ deficiency since it could be prevented by addition of 5% CO₂ to the gas stream. Nitrite excreted during growth with air (0.035% CO₂) was taken up after an increase in CO₂ concentration to 5%.
In sulfur depleted cultures, nitrite excretion took place also with saturating CO₂ concentration. In this case nitrite accumulation could be reversed by addition of a suitable sulfur source.
Under both conditions for nitrite accumulation, carbon and sulfur deficiency, a significant decrease in nitrite reductase activity was observed which might account for nitrite liberation.