Synechococcus Research Articles

Photosystem I (PSI) is a photosynthetic protein–pigment complex that, upon photoexcitation, transfers electrons to ferredoxin, facilitating the production of NADPH. Isolated PSI reaction centers (RCs) have also been used in hybrid systems to reduce protons and produce ‘biohydrogen’. This review article examines how various cyanobacteria with similar photosynthetic machinery utilize different wavelengths of light to execute photosynthetic electron transport through PSI. Key factors, such as, the structure of the electron transfer cofactors, the protein environment surrounding the primary donor pigments and hydrogen-bonding interactions with the surrounding protein matrix are analyzed to understand their roles in maintaining efficient electron transfer when it is driven using photons of different energies. We compare PSI complexes with known atomic structures from four species of cyanobacteria, Thermosynechococcus elongatus, Acaryochloris marina, Halomicronema hongdechloris, and Fischerella thermalis. T. elongatus is typical of most oxygenic photosynthetic organisms in that it requires visible light and uses only chlorophyll a (Chl a) in PSI. In contrast, H. hongdechloris and F. thermalis are photoacclimating species capable of producing Chl f and Chl d that use red light when little visible light is available. A. marina, on the other hand, is adapted to red light conditions and consistently utilizes Chl d as its primary photosynthetic pigment, maintaining a stable pigment composition. Here, we explore the structural and functional differences between the PSI RCs of these organisms and the impact of these differences on electron transport. The structural differences in the cofactors influence both the absorption wavelengths of the cofactors and the energy levels of the intermediate states of electron transfer. An analysis of the surrounding protein shows how it has been adapted and underscores the interplay between the pigment structure, protein environment, and hydrogen bonding networks in tuning the efficiency and adaptability of photosynthetic mechanisms across different species of cyanobacteria.

Differential effects of the D1/S264V mutation in photosystem II with either PsbA1 or PsbA3 on QB, non-heme Iron, and the associated hydrogen-bond network.

Phycocyanin is a biliprotein that has been used as a natural food colorant due to its brilliant color. However, their application is limited by their poor stability. In this study, biosynthesis pathways of two phycocyanin holo-β subunits, CpcBA from mesophilic Arthrospira platensis and CpcBT from thermophilic Thermosynechococcus elongatus BP-1, were constructed in Escherichia coli. Coexpression of ferredoxin (Fd), Fd-NADP+ reductase (FNR), and NADP-specific glutamate dehydrogenase (gdhA) enabled full chromophorylation of these recombinant CpcBs in recombinant E. coli. These fully chromophorylated CpcBs were visually redder and had higher hydroxyl radical and peroxyl radical scavenging activities than the partially chromophorylated CpcBs. Comparative study on thermostability showed that at high temperature the CpcBT had lower denature rate constants and longer half-life values than the CpcBA. Both proteins were stable at acidic pH (3.0-6.6), except for the CpcBA at pH 3.0. Under a combinational treatment of acid pH and heat, CpcBA showed remarkable losses (93.6-98.4%) while CpcBT showed much less losses (20.0-49.6%). All the results indicated that CpcBT was a stable phycocyanin and could potentially be developed as an excellent colorant in the food industry.

We establish a general kinetic scheme for the energy transfer and radical-pair dynamics in photosystem I (PSI) of Chlamydomonas reinhardtii, Synechocystis PCC6803, Thermosynechococcus elongatus and Spirulina platensis grown under white-light conditions. With the help of simultaneous target analysis of transient-absorption data sets measured with two selective excitations, we resolved the spectral and kinetic properties of the different species present in PSI. WL-PSI can be described as a Bulk Chl a in equilibrium with a higher-energy Chl a, one or two Red Chl a and a reaction-center compartment (WL-RC). Three radical pairs (RPs) have been resolved with very similar properties in the four model organisms. The charge separation is virtually irreversible with a rate of ≈900 ns-1. The second rate, of RP1 → RP2, ranges from 70-90 ns-1 and the third rate, of RP2 → RP3, is ≈30 ns-1. Since RP1 and the Red Chl a are simultaneously present, resolving the RP1 properties is challenging. In Chlamydomonas reinhardtii, the excited WL-RC and Bulk Chl a compartments equilibrate with a lifetime of ≈0.28 ps, whereas the Red and the Bulk Chl a compartments equilibrate with a lifetime of ≈2.65 ps. We present a description of the thermodynamic properties of the model organisms at room temperature.

Picophytoplankton populations [Prochlorococcus, Synechococcus (SYN), and picoeukaryotes] are dominant primary producers in the open ocean and projected to become more important with climate change. Their fates can vary, however, with microbial food web complexities. In the California Current Ecosystem, picophytoplankton biomass and abundance peak in waters of intermediate productivity and decrease at higher production. Using experimental data from eight cruises crossing the pronounced CCE trophic gradient, we tested the hypothesis that these declines are driven by intensified grazing on heterotrophic bacteria (HBAC) passed to similarly sized picophytoplankton via shared predators. Results confirm previously observed distributions as well as significant increases in bacterial abundance, cell growth, and grazing mortality with primary production. Mortalities of picophytoplankton, however, diverge from the bacterial mortality trend such that relative grazing rates on SYN compared to HBAC decline by 12-fold between low and high productivity waters. The large shifts in mortality rate ratios for coexisting populations are not explained by size variability but rather suggest high selectivity of grazer assemblages or tightly coupled tradeoffs in microbial growth advantages and grazing vulnerabilities. These findings challenge the long-held view that protistan grazing mainly determines overall biomass of microbial communities while viruses uniquely regulate diversity by "killing the winners".

Malaysia is home to a number of hot springs that are rich in microbial diversity including the photosynthetic cyanobacteria. Although this microbial community has been characterised based on metagenomics approach, the culturable thermophilic isolates have not been isolated and characterised extensively. Compared to the mesophiles, information on plant growth promoting (PGP) properties of these thermophiles remain largely untapped. As the amount of arable land for microbial bioprospecting is decreasing due to extensive human activities, the search for alternative source for microbial strains with PGP properties is important for the development of potential biofertilisers. This study sought to isolate and characterise culturable cyanobacteria strains from two local hot springs – Sungai Klah (SK) and Lubuk Timah (LT) located in Perak using morphological and molecular methods. The IAA production from the axenic cultures were measured. The PGP properties were also measured by priming the rice seeds with cyanobacterial water extracts. A total of six strains were isolated from both hot springs. Strains LTM and LTW from LT were identified as Leptolyngbya sp. whereas strains SEM, SEH, STH and STM were identified as Thermosynechococcus elongatus. All six strains produced IAA ranged from 670.10 pg/μL to 2010 pg/μL. The water extracts were found to increase the seed amylase activity of the rice seeds from 5th day of germination (DAG) to 10th DAG. In general, the IAA production and increased seed amylase activity might have contributed in enhancing the longest root length, shoot length and root-to-shoot (RS) ratio. To conclude, the thermophilic cyanobacteria from hot springs can be further exploited as a novel source of PGP microbes for the development of biofertilsers.

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution

The Sembawang Hot Spring in Singapore lies at the foot of a major regional geological feature called the Bentong-Raub Suture Zone. Amid an extensively managed surface geothermal park, an undisturbed hot spring emerges with source water at 61°C, pH 6.8, and 1 mg/L dissolved sulfide. A small main pool at the source supported orange-green benthic flocs, whereas the outflow channel with gradually less extreme environmental stress supported extensive vivid green microbial mats. Microscopy revealed that cyanobacterial morphotypes were distinct in flocs and mats at several intervals along the environmental gradient, and we describe a spiraling pattern in the oscillatorian cyanobacteria that may reflect response to poly-extreme stress. Estimation of diversity using 16S rRNA gene sequencing revealed assemblages that were dominated by phototrophic bacteria. The most abundant taxa in flocs at 61°C/1 mg/L sulfide were Roseiflexus sp. and Thermosynechococcus elongatus, whilst the mats at 45.7–55.3°C/0–0.5 mg/L sulfide were dominated by Oscillatoriales cyanobacterium MTP1 and Chloroflexus sp. Occurrence of diverse chemoautotrophs and heterotrophs reflected known thermal ranges for taxa, and of note was the high abundance of thermophilic cellulolytic bacteria that likely reflected the large allochthonous leaf input. A clear shift in ASV-defined putative ecotypes occurred along the environmental stress gradient of the hot spring and overall diversity was inversely correlated to environmental stress. Significant correlations for abiotic variables with observed biotic diversity were identified for temperature, sulfide, and carbonate. A network analysis revealed three putative modules of biotic interactions that also reflected the taxonomic composition at intervals along the environmental gradient. Overall, the data indicated that three distinct microbial communities were supported within a small spatial scale along the poly-extreme environmental gradient. The findings add to the growing inventory of hot spring microbiomes and address an important biogeographic knowledge gap for the region.

Energetics and proton release in photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA2 or psbA3 gene

Carboxysomes are bacterial microcompartments, whose structural features enable the encapsulated Rubisco holoenzyme to operate in a high-CO2 environment. Consequently, Rubiscos housed within these compartments possess higher catalytic turnover rates relative to their plant counterparts. This particular enzymatic property has made the carboxysome, along with associated transporters, an attractive prospect to incorporate into plant chloroplasts to increase future crop yields. To date, two carboxysome types have been characterized, the α-type that has fewer shell components and the β-type that houses a faster Rubisco. While research is underway to construct a native carboxysome in planta, work investigating the internal arrangement of carboxysomes has identified conserved Rubisco amino acid residues between the two carboxysome types which could be engineered to produce a new, hybrid carboxysome. In theory, this hybrid carboxysome would benefit from the simpler α-carboxysome shell architecture while simultaneously exploiting the higher Rubisco turnover rates in β-carboxysomes. Here, we demonstrate in an Escherichia coli expression system, that the Thermosynechococcus elongatus Form IB Rubisco can be imperfectly incorporated into simplified Cyanobium α-carboxysome-like structures. While encapsulation of non-native cargo can be achieved, T. elongatus Form IB Rubisco does not interact with the Cyanobium carbonic anhydrase, a core requirement for proper carboxysome functionality. Together, these results suggest a way forward to hybrid carboxysome formation.

Numerous technologies are currently in development for use in next-generation protein sequencing platforms. A notable published approach employs fluorescently-tagged binding proteins to identity the N-terminus of immobilized peptides, in-between rounds of digestion. This approach makes use of N-terminal amino acid binder (NAAB) proteins, which would identify amino acids by chemical and shape complementarity. One source of NAABs is the ClpS protein family, which serve to recruit proteins to bacterial proteosomes based on the identity of the N-terminal amino acid. In this study, a Thermosynechococcus vestitus (also known as Thermosynechococcus elongatus) ClpS2 protein was used as the starting point for direct evolution of an NAAB with affinity and specificity for N-terminal leucine. Enriched variants were analyzed and shown to improve the interaction between the ClpS surface and the peptide chain, without increasing promiscuity. Interestingly, interactions were found that were unanticipated which favor different charged residues located at position 5 from the N-terminus of a target peptide.

Three psbA genes (psbA1, psbA2, and psbA3) encoding the D1 subunit of photosystem II (PSII) are present in the thermophilic cyanobacterium Thermosynechococcus elongatus and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, leading to the narrowing of the channel, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, hence an enhancement of oxygen evolution in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.

Ethylene and isoprene are essential platform chemicals necessary to produce polymers and materials. However, their current production methods based on fossil fuels are not very efficient and result in significant environmental pollution. For a successful transition more sustainable economic model, producing these key polymeric building blocks from renewable and sustainable resources such as biomass or CO2 is essential. Here, inspired by the symbiotic relationship of natural microbial communities, artificial consortia composed of E. coli strains producing volatile platform chemicals: ethylene and isoprene and two strains of cyanobacteria phototrophically synthesizing and exporting sucrose to feed these heterotrophs were developed. Disaccharide produced by transgenic cyanobacteria was used as a carbon and electron shuttle between the two community components. The E. coli cscB gene responsible for sucrose transport was inserted into two cyanobacterial strains, Thermosynechococcus elongatus PKUAC-SCTE542 and Synechococcus elongatus PCC7942, resulting in a maximal sucrose yield of 0.14 and 0.07 g/L, respectively. These organisms were co-cultured with E. coli BL21 expressing ethylene-forming enzyme or isoprene synthase and successfully synthesized volatile hydrocarbons. Productivity parameters of these co-cultures were higher than respective transgenic cultures of E. coli grown individually at similar sucrose concentrations, highlighting the positive impact of the artificial consortia on the production of these platform chemicals.

The detergent-free extraction of integral membrane proteins using styrene-maleic acid copolymers (SMAs) has shown promise as a potentially effective technique to isolate proteins in a more native-like conformation. As the field continues to develop, the protein selectivity and extraction efficiency of many analogues of traditional SMAs are being investigated. Recently, we discovered that the monoesterification of SMAs with alkoxy ethoxylate sidechains drastically affects the bioactivity of these copolymers in the extraction of photosystem I from the cyanobacterium Thermosynechococcus elongatus. However, subsequent investigations also revealed that the conditions under which these esterified SMA polymer analogues are prepared, purified, and stored can alter the structure of the alkoxy ethoxylate-functionalized SMA and perturb the protein extraction process. Herein, we demonstrate that the basic conditions required to solubilize SMA analogues may lead to deleterious saponification side reactions, cleaving the sidechains of an esterified SMA and dramatically decreasing its efficacy for protein extraction. We found that this process is highly dependent on temperature, with polymer samples being prepared and stored at lower temperatures exhibiting significantly fewer saponification side reactions. Furthermore, the effects of small-molecule impurities and exposure to light were also investigated, both of which are shown to have significant effects on the polymer structure and/or protein extraction process.

How to correct relative voxel scale factors for calculations of vector-difference Fourier maps in cryo-EM

Thermosynechococcus is a genus of thermophilic unicellular cyanobacteria that dominates microbial mats in Asian non-acidic hot springs. These cyanobacteria are the major primary producers in their ecological niches and are promising sources of thermostable enzymes for biotechnology applications. To improve our understanding of these organisms, we conducted whole-genome sequencing of a novel strain for comparative analysis with other representatives in the same genus. This newly characterized strain, Thermosynechococcus sp. TA-1, was isolated from the Taian hot springs in Taiwan. Analyses based on average nucleotide identity (ANI) and genome-scale phylogeny suggested that TA-1 and another Taiwanese strain CL-1 belong to a novel species-level taxon. Two metagenome-assembled genomes (MAGs) originated from India represent the sister group, and Thermosynechococcus elongatus PKUAC-SCTE542 from China is the next closest lineage. All cultivated strains and MAGs from Japan form a separate monophyletic clade and could be classified into two species-level taxa. Intriguingly, although TA-1 and CL-1 share 97.0% ANI, the genome alignment identified at least 16 synteny breakpoints that are mostly associated with transposase genes, which illustrates the dynamic nature of their chromosomal evolution. Gene content comparisons identified multiple features distinct at species- or strain-level among these Thermosynechococcus representatives. Examples include genes involved in bicarbonate transportation, nitric oxide protection, urea utilization, kanamycin resistance, restriction-modification system, and chemotaxis. Moreover, we observed the insertion of type II inteins in multiple genes of the two Taiwanese strains and inferred putative horizontal transfer of an asparagine synthase gene (asnB) associated with exopolysaccharides gene cluster. Taken together, while previous work suggested that strains in this genus share a highly conserved genomic core and no clear genetic differentiation could be linked to environmental factors, we found that the overall pattern of gene content divergence is largely congruent with core genome phylogeny. However, it is difficult to distinguish between the roles of phylogenetic relatedness and geographic proximity in shaping the genetic differentiation. In conclusion, knowledge of the genomic differentiation among these strains provides valuable resources for future functional characterization.

Progress in structural membrane biology has been significantly accelerated by the ongoing ‘Resolution Revolution’ in cryo-electron microscopy (cryo-EM). In particular, structure determination by single-particle analysis has evolved into the most powerful method for atomic model building of multisubunit membrane protein complexes. This has created an ever-increasing demand in cryo-EM machine time, which to satisfy is in need of new and affordable cryo-electron microscopes. Here, we review our experience in using the JEOL CRYO ARM 200 prototype for the structure determination by single-particle analysis of three different multisubunit membrane complexes: the Thermus thermophilus V-type ATPase VO complex, the Thermosynechococcus elongatus photosystem I monomer and the flagellar motor lipopolysaccharide peptidoglycan ring (LP ring) from Salmonella enterica.

Small angle neutron scattering and lipidomic analysis of a native, trimeric PSI-SMALP from a thermophilic cyanobacteria

Response of smaller plankton to the saltwater barrage-altered hydrography of a tropical estuarine system on India's southwest coast

Glucosylsucroses are potentially useful as additives in cosmetic and pharmaceutical formulations. Although enzymatic synthesis of glucosylsucroses is the most efficient method for their production, the key enzyme that produces them has remained unknown. Here, we report that glucosylsucrose synthase from (TeGSS) catalyzes the synthesis of glucosylsucrose using sucrose and UDP-glucose as substrates. These saccharides are homologous to glucosylsucroses produced by sp. PCC 7120 (referred to as protein alr1000). When the ratio of UDP-glucose to sucrose is relatively high, TeGSS from cyanobacteria can hydrolyze excess UDP-glucose to UDP and glucose, indicating that sucrose provides a feedback mechanism for the control of glucosylsucrose synthesis. In the present study, we solved the crystal structure of TeGSS bound to UDP and sucrose. Our structure shows that the catalytic site contains a circular region that may allow glucosylsucroses with a right-hand helical structure to enter the catalytic site. Because active site residues Tyr18 and Arg179 are proximal to UDP and sucrose, we mutate these residues (., Y18F and R179A) and show that they exhibit very low activity, supporting their role as catalytic groups. Overall, our study provides insight into the catalytic mechanism of TeGSS.