Genome-scale identification and comparative analysis of transcription factors in thermophilic cyanobacteria.

BACKGROUND: The transcription factors (TFs) in thermophilic cyanobacteria might represent a uniquely evolved gene repertoire in light of the strong selective pressure caused by hostile habitats. Understanding the molecular composition of the TF genes in thermophilic cyanobacteria will facilitate further studies regarding verifying their exact biochemical functions and genetic engineering. However, limited information is available on the TFs of thermophilic cyanobacteria. Herein, a thorough investigation and comparative analysis were performed to gain insights into the molecular composition of the TFs in 22 thermophilic cyanobacteria. RESULTS: The results suggested a fascinating diversity of the TFs among these thermophiles. The abundance and type of TF genes were diversified in these genomes. The identified TFs are speculated to play various roles in biological regulations. Further comparative and evolutionary genomic analyses revealed that HGT may be associated with the genomic plasticity of TF genes in Thermostichus and Thermosynechococcus strains. Comparative analyses also indicated different pattern of TF composition between thermophiles and corresponding mesophilic reference cyanobacteria. Moreover, the identified unique TFs of thermophiles are putatively involved in various biological regulations, mainly as responses to ambient changes, may facilitating the thermophiles to survive in hot springs. CONCLUSION: The findings herein shed light on the TFs of thermophilic cyanobacteria and fundamental knowledge for further research regarding thermophilic cyanobacteria with a broad potential for transcription regulations in responses to environmental fluctuations.

Genomic and phenotypic characterization of Thermosynechococcus-like strains reveals eight species within the genus Thermosynechococcus and a novel genus Parathermosynechococcus gen. nov.

Thermophilic unicellular cyanobacteria of the family Thermosynechococcaceae are essential primary producers and integral components of many microbial mats found in hot springs of Asia and North America. Historically, based on their simple morphology, these organisms, along with members of taxonomically unrelated thermophilic Thermostichaceae have been described with a generic term, "Synechococcus", used for elongated unicellular cyanobacteria. This has created significant misperception in the scientific literature regarding the taxonomic status of these essential thermophilic primary producers and their relationship with Synechococcus sensu stricto. In this manuscript, we attempted a genome-driven taxonomic reevaluation of the family Thermosynechococcaceae. Application of genomic analyses such as GTDB classification, ANI/AAI and phylogenomics support the delineation of eight species within genus Thermosynechococcus. Two subspecies were further identified within T. taiwanensis by dDDH and phylogenomics. Moreover, the results also suggest the presence of two putative new genera phylogenetically alongside genus Thermosynechococcus, a thermophilic genus Parathermosynechococcus represented by PCC 6715 and a non-thermophilic genus represented by PCC 6312. The proposed genospecies and new genera were further integrated with morphological and/or ecological information. Interestingly, the phylogeny of 16S-23S ITS achieved a better taxonomic relationship than that of 16S rRNA and supported the genome-based classification of Thermosynechococcus spp. Finally, the pan-genome analysis indicated a conserved pattern of genomic core among known members of Thermosynechococcus.

Thermophilic cyanobacteria-exciting, yet challenging biotechnological chassis.

Thermophilic cyanobacteria are prokaryotic photoautotrophic microorganisms capable of growth between 45 and 73 °C. They are typically found in hot springs where they serve as essential primary producers. Several key features make these robust photosynthetic microbes biotechnologically relevant. These are highly stable proteins and their complexes, the ability to actively transport and concentrate inorganic carbon and other nutrients, to serve as gene donors, microbial cell factories, and sources of bioactive metabolites. A thorough investigation of the recent progress in thermophilic cyanobacteria reveals a significant increase in the number of newly isolated and delineated organisms and wide application of thermophilic light-harvesting components in biohybrid devices. Yet despite these achievements, there are still deficiencies at the high-end of the biotechnological learning curve, notably in genetic engineering and gene editing. Thermostable proteins could be more widely employed, and an extensive pool of newly available genetic data could be better utilised. In this manuscript, we attempt to showcase the most important recent advances in thermophilic cyanobacterial biotechnology and provide an overview of the future direction of the field and challenges that need to be overcome before thermophilic cyanobacterial biotechnology can bridge the gap with highly advanced biotechnology of their mesophilic counterparts. KEY POINTS: • Increased interest in all aspects of thermophilic cyanobacteria in recent years • Light harvesting components remain the most biotechnologically relevant • Lack of reliable molecular biology tools hinders further development of the chassis.

Microalgae are not an umbrella solution for power industry waste abatement but could play a role in their valorization.

Microalgae have long been regarded as a promising solution for biological carbon abatement from the power industry, offering renewable biomass without competing for land or water resources used for food crops. In this study, we extensively examined the application of photosynthetic microorganisms for closing carbon, nitrogen, and micronutrient loops in the power industry. Subsequently, we explored the bottom-up integration of algal biorefineries into power industry waste streams for increased economic benefits and reduced environmental impacts. Analysis of the available data indicated that microalgae integration with the power industry is primarily performed using flue-gas-assisted cultivation. This approach allows for carbon sequestration typically below one gram per liter per day, too low to significantly impact carbon abatement at achievable scales of microalgae cultivation. Alternative approaches are also being explored. For example, soluble bicarbonate platforms allow for higher biomass productivity and temporary carbon storage. Meanwhile, the use of ashes and waste heat and thermophilic strains can result in lower cultivation costs and better control of cultivation conditions. These approaches offer further incremental improvement to microalgae-based carbon abatement systems in the power industry but are unlikely to be an umbrella solution for carbon reduction. Consequently, in the near term, microalgae-based carbon valorization systems are likely to be limited to niche applications involving the synthesis of high-value products. For microalgae to truly transform carbon abatement processes radical improvements in both biology and engineering approaches are urgently needed.

Characterization of Molecular Diversity and Organization of Phycobilisomes in Thermophilic Cyanobacteria.

Thermophilic cyanobacteria are cosmopolitan and abundant in the thermal environment. Their light-harvesting complexes, phycobilisomes (PBS), are highly important in photosynthesis. To date, there is limited information on the PBS composition of thermophilic cyanobacteria whose habitats are challenging for survival. Herein, genome-based methods were used to investigate the molecular components of PBS in 19 well-described thermophilic cyanobacteria. These cyanobacteria are from the genera Leptolyngbya, Leptothermofonsia, Ocullathermofonsia, Thermoleptolyngbya, Trichothermofonsia, Synechococcus, Thermostichus, and Thermosynechococcus. According to the phycobiliprotein (PBP) composition of the rods, two pigment types are observed in these thermophiles. The amino acid sequence analysis of different PBP subunits suggests several highly conserved cysteine residues in these thermophiles. Certain amino acid contents in the PBP of thermophiles are significantly higher than their mesophilic counterparts, highlighting the potential roles of specific substitutions of amino acid in the adaptive thermostability of light-harvesting complexes in thermophilic cyanobacteria. Genes encoding PBS linker polypeptides vary among the thermophiles. Intriguingly, motifs in linker apcE indicate a photoacclimation of a far-red light by Leptolyngbya JSC-1, Leptothermofonsia E412, and Ocullathermofonsia A174. The composition pattern of phycobilin lyases is consistent among the thermophiles, except for Thermostichus strains that have extra homologs of cpcE, cpcF, and cpcT. In addition, phylogenetic analyses of genes coding for PBPs, linkers, and lyases suggest extensive genetic diversity among these thermophiles, which is further discussed with the domain analyses. Moreover, comparative genomic analysis suggests different genomic distributions of PBS-related genes among the thermophiles, indicating probably various regulations of expression. In summary, the comparative analysis elucidates distinct molecular components and organization of PBS in thermophilic cyanobacteria. These results provide insights into the PBS components of thermophilic cyanobacteria and fundamental knowledge for future research regarding structures, functions, and photosynthetic improvement.

Discovery of Five New Ethylene-Forming Enzymes for Clean Production of Ethylene in E. coli.

Ethylene is an essential platform chemical with a conjugated double bond, which can produce many secondary chemical products through copolymerisation. At present, ethylene production is mainly from petroleum fractionation and cracking, which are unsustainable in the long term, and harmful to our environment. Therefore, a hot research field is seeking a cleaner method for ethylene production. Based on the model ethylene-forming enzyme (Efe) AAD16440.1 (6vp4.1.A) from Pseudomonas syringae pv. phaseolicol, we evaluated five putative Efe protein sequences using the data derived from phylogenetic analyses and the conservation of their catalytic structures. Then, pBAD expression frameworks were constructed, and relevant enzymes were expressed in E. coli BL21. Finally, enzymatic activity in vitro and in vivo was detected to demonstrate their catalytic activity. Our results show that the activity in vitro measured by the conversion of α-ketoglutarate was from 0.21-0.72 µmol ethylene/mg/min, which varied across the temperatures. In cells, the activity of the new Efes was 12.28-147.43 µmol/gDCW/h (DCW, dry cellular weight). Both results prove that all the five putative Efes could produce ethylene.

The Genome Copy Number of the Thermophilic Cyanobacterium Thermosynechococcus elongatus E542 Is Controlled by Growth Phase and Nutrient Availability.

The recently isolated thermophilic cyanobacterium Thermosynechococcus elongatus PKUAC-SCTE542 (here Thermosynechococcus E542) is a promising strain for fundamental and applied research. Here, we used several improved ploidy estimation approaches, which include quantitative PCR (qPCR), spectrofluorometry, and flow cytometry, to precisely determine the ploidy level in Thermosynechococcus E542 across different growth stages and nutritional and stress conditions. The distribution of genome copies per cell among the populations of Thermosynechococcus E542 was also analyzed. The strain tends to maintain 3 or 4 genome copies per cell in lag phase, early growth phase, or stationary phase under standard conditions. Increased ploidy (5.5 ± 0.3) was observed in exponential phase; hence, the ploidy level is growth phase regulated. Nearly no monoploid cells were detected in all growth phases, and prolonged stationary phase could not yield ploidy levels lower than 3 under standard conditions. During the late growth phase, a significantly higher ploidy level was observed in the presence of bicarbonate (7.6 ± 0.7) and high phosphate (6.9 ± 0.2) at the expense of reduced percentages of di- and triploid cells. Meanwhile, the reduction in phosphates decreased the average ploidy level by increasing the percentages of mono- and diploid cells. In contrast, temperature and antibiotic stresses reduced the percentages of mono-, di-, and triploid cells yet maintained average ploidy. The results indicate a possible causality between growth rate, stress, and genome copy number across the conditions tested, but the exact mechanism is yet to be elucidated. Furthermore, the spectrofluorometric approach presented here is a quick and straightforward ploidy estimation method with reasonable accuracy.IMPORTANCE The present study revealed that the genome copy number (ploidy) status in the thermophilic cyanobacterium Thermosynechococcus E542 is regulated by growth phase and various environmental parameters to give us a window into understanding the role of polyploidy. An increased ploidy level is found to be associated with higher metabolic activity and increased vigor by acting as backup genetic information to compensate for damage to the other chromosomal copies. Several improved ploidy estimation approaches that may upgrade the ploidy estimation procedure for cyanobacteria in the future are presented in this work. Furthermore, the new spectrofluorometric method presented here is a rapid and straightforward method of ploidy estimation with reasonable accuracy compared to other laborious methods.

Modifying the Cyanobacterial Metabolism as a Key to Efficient Biopolymer Production in Photosynthetic Microorganisms.

Cyanobacteria are photoautotrophic bacteria commonly found in the natural environment. Due to the ecological benefits associated with the assimilation of carbon dioxide from the atmosphere and utilization of light energy, they are attractive hosts in a growing number of biotechnological processes. Biopolymer production is arguably one of the most critical areas where the transition from fossil-derived chemistry to renewable chemistry is needed. Cyanobacteria can produce several polymeric compounds with high applicability such as glycogen, polyhydroxyalkanoates, or extracellular polymeric substances. These important biopolymers are synthesized using precursors derived from central carbon metabolism, including the tricarboxylic acid cycle. Due to their unique metabolic properties, i.e., light harvesting and carbon fixation, the molecular and genetic aspects of polymer biosynthesis and their relationship with central carbon metabolism are somehow different from those found in heterotrophic microorganisms. A greater understanding of the processes involved in cyanobacterial metabolism is still required to produce these molecules more efficiently. This review presents the current state of the art in the engineering of cyanobacterial metabolism for the efficient production of these biopolymers.

Complete Genome Sequence and Comparative Analysis of Synechococcus sp. CS-601 (SynAce01), a Cold-Adapted Cyanobacterium from an Oligotrophic Antarctic Habitat.

Marine picocyanobacteria belonging to Synechococcus are major contributors to the global carbon cycle, however the genomic information of its cold-adapted members has been lacking to date. To fill this void the genome of a cold-adapted planktonic cyanobacterium Synechococcus sp. CS-601 (SynAce01) has been sequenced. The genome of the strain contains a single chromosome of approximately 2.75 MBp and GC content of 63.92%. Gene prediction yielded 2984 protein coding sequences and 44 tRNA genes. The genome contained evidence of horizontal gene transfer events during its evolution. CS-601 appears as a transport generalist with some specific adaptation to an oligotrophic marine environment. It has a broad repertoire of transporters of both inorganic and organic nutrients to survive in inhospitable environments. The cold adaptation of the strain exhibited characteristics of a psychrotroph rather than psychrophile. Its salt adaptation strategy is likely to rely on the uptake and synthesis of osmolytes, like glycerol or glycine betaine. Overall, the genome reveals two distinct patterns of adaptation to the inhospitable environment of Antarctica. Adaptation to an oligotrophic marine environment is likely due to an abundance of genes, probably acquired horizontally, that are associated with increased transport of nutrients, osmolytes, and light harvesting. On the other hand, adaptations to low temperatures are likely due to prolonged evolutionary changes.

Accelerated CO2 Hydration with Thermostable Sulfurihydrogenibium azorense Carbonic Anhydrase-Chitin Binding Domain Fusion Protein Immobilised on Chitin Support.

Carbonic anhydrases (CAs) represent a group of enzymes that catalyse important reactions of carbon dioxide hydration and dehydration, a reaction crucial to many biological processes and environmental biotechnology. In this study we successfully constructed a thermostable fusion enzyme composed of the Sulfurihydrogenibium azorense carbonic anhydrase (Saz_CA), the fastest CA discovered to date, and the chitin binding domain (ChBD) of chitinase from Bacillus circulans. Introduction of ChBD to the Saz_CA had no major impact on the effect of ions or inhibitors on the enzymatic activity. The fusion protein exhibited no negative effects up to 60 °C, whilst the fusion partner appears to protect the enzyme from negative effects of magnesium. The prepared biocatalyst appears to be thermally activated at 60 °C and could be partially purified with heat treatment. Immobilisation attempts on different kinds of chitin-based support results have shown that the fusion enzyme preferentially binds to a cheap, untreated chitin with a large crystallinity index over more processed forms of chitin. It suggests significant potential economic benefits for large-scale deployment of immobilised CA technologies such as CO2 utilisation or mineralisation.

Temperature-controlled thermophilic bacterial communities in hot springs of western Sichuan, China.

BACKGROUND: Ganzi Prefecture in Western China is situated geographically at the transition regions between Tibetan Plateau and Sichuan Basin in a highly tectonically active boundary area between the India and Eurasia plates. The region hosts various hot springs that span a wide range of temperature from 30 to 98 °C and are located at high altitude (up to 4200 m above sea level) in the region of large geothermal anomalies and active Xianshuihe slip-fault that has been active since Holocene. The site represents a biodiversity reservoir for thermophiles, yet their diversity and relationship to geochemical parameters are largely unknown. In the present work, bacterial diversity and community structure in 14 hot springs of Ganzi were investigated using Illumina MiSeq sequencing. RESULTS: Bacterial community compositions were evidently distinct among the 14 hot springs, and the bacterial communities in hot springs were majorly abundant in phyla Aquificae, Cyanobacteria and Proteobacteria. Both clustering and PCoA analysis suggested the existence of four bacterial community patterns in these hot springs. Temperature contributed to shaping bacterial community structure of hot springs as revealed by correlation analysis. Abundant unassigned-genus sequences detected in this study strongly implied the presence of novel genera or genetic resources in these hot springs. CONCLUSION: The diversity of hot springs of Ganzi prefecture in Western Sichuan, China is evidently shaped by temperature. Interestingly disproportionally abundant unassigned-genus sequences detected in this study show indicate potential of novel genera or phylotypes. We hypothesize that frequent earthquakes and rapidly changing environment might have contributed to evolution of these potentially new lineages. Overall, this study provided first insight into the bacterial diversity of hot springs located in Western Sichuan, China and its comparison with other similar communities worldwide.

Transcriptomic Profiling and Gene Disruption Revealed that Two Genes Related to PUFAs/DHA Biosynthesis May be Essential for Cell Growth of Aurantiochytrium sp.

Aurantiochytrium sp. PKU#SW7 is a thraustochytrid strain that was found to exhibit high potential for docosahexaenoic acid (DHA, C22:6n-3) production. In this work, the transcriptome of Aurantiochytrium sp. PKU#SW7 was analyzed for the study of genes involved in basic metabolic functions and especially in the mechanisms of DHA biosynthesis. Sequence annotation and functional analysis revealed that the strain contains components of fatty acid synthesis (FAS) and polyketide synthase (PKS) pathways. Fatty acid desaturases and elongases were identified as components of FAS pathway, whilst key components of PKS pathway were also found in the cDNA library. The relative contribution of the two pathways to the synthesis of DHA was unknown, as both pathways appeared to be lacking full complement of genes for standalone synthesis of DHA. Further analysis of two putative genes encoding the very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase and dehydrase/isomerase involved in FAS and PKS pathways, respectively, revealed that under various salinity conditions, their relative expression levels changed corresponding to the variation of DHA content in Aurantiochytrium sp. Independent knock outs of these genes in Aurantiochytrium sp. resulted in poor cell growth, probably due to little or no intracellular DHA accumulation. Hence, it can be speculated that both genes are engaged in DHA biosynthesis and DHA in Aurantiochytrium sp. could be produced by jointed actions of both FAS and PKS systems.

DArT-based characterisation of genetic diversity in a Miscanthus collection from Poland.

MAIN CONCLUSION: Analysis of 180 accessions of Miscanthus using a DArT platform revealed high diversity. The phylogenetic analysis revealed that M. × giganteus accessions fall into two genetically distinct groups. Miscanthus is a genus of perennial rhizomatous grasses that has emerged in last 20 years as a feedstock for bioenergy and biofuel production. Currently, the most widely used accession for bioenergy purposes is Miscanthus × giganteus, a sterile triploid hybrid between Miscanthus sinensis and Miscanthus sacchariflorus. However, previous reports have shown that genetic diversity of Miscanthus × giganteus is limited. Here, we report development of Diversity Arrays Technology platform for the analysis of genetic structure of a Miscanthus collection of 180 accessions. A total of 906 markers were obtained of which around 25.5% exhibited polymorphism information content value in the range of 0.40 and 0.50 and are considered particularly informative. Newly developed marker system will serve as an additional resource to assist crop improvement, germplasm preservation and genetic studies. Three types of analysis indicated that 180 accessions from the collection were well differentiated and presented high diversity. Interestingly, the analysis revealed that there are two separate groups of plants, significantly differing in genetic diversity, that are commercially available as M. × giganteus. We suggest that one of these groups is most likely mutants or somaclonal variants of original M. × giganteus. The other group is recent hybrids of Miscanthus of higher genetic diversity. This study indicates that the diversity of commercially available M. × giganteus is higher than commonly assumed. Development of the new marker system can significantly assist breeding of new commercial cultivars of Miscanthus for bioenergy use.

Efficient production of triacylglycerols rich in docosahexaenoic acid (DHA) by osmo-heterotrophic marine protists.

Thraustochytrids have recently emerged as a promising source for docosahexaenoic acid (DHA) production due to their high growth rate and oil content. In this study, two thraustochytrid isolates, Aurantiochytrium sp. PKU#SW7 and Thraustochytriidae sp. PKU#Mn16 were used for DHA production. Following growth parameters were optimized to maximize DHA production: temperature, pH, salinity, and glucose concentration. Both isolates achieved the highest DHA yield at the cultivation temperature of 28 °C, pH 6, 100 % seawater, and 2 % glucose. A DHA yield of 1.395 g/l and 1.426 g/l was achieved under the optimized culture conditions. Further investigation revealed that both isolates possess simple fatty acids profiles with palmitic acid and DHA as their dominant constituents, accounting for ∼79 % of total fatty acids. To date, very few studies have focused on the DHA distribution in various lipid fractions which is an important factor for identifying strains with a potential for industrial DHA production. In the present study, the lipids profiles of each strain both revealed that the majority of DHA was distributed in neutral lipids (NLs), and the DHA distribution in NLs of PKU#SW7 was exclusively in the form of triacylglycerols (TAGs) which suggest that PKU#SW7 could be utilized as an alternative source of DHA for dietary supplements. The fermentation process established for both strains also indicating that Aurantiochytrium sp. PKU#SW7 was more suitable for cultivation in fermenter. In addition, the high percentage of saturated fatty acids produced by the two thraustochytrids indicates their potential application in biodiesel production. Overall, our findings suggest that two thraustochytrid isolates are suitable candidates for biotechnological applications.

Complete genome sequence and transcriptome response to vitamin C supplementation of Novacetimonas hansenii SI1 - producer of highly-stretchable cellulose.

Novacetimonas hansenii SI1, previously known as Komagataeibacter hansenii, produces bacterial nanocellulose (BNC) with unique ability to stretch. The addition of vitamin C in the culture medium increases the porosity of the membranes and their stretchability making them highly moldable. To better understand the genetic background of this strain, we obtained its complete genome sequence using a hybrid sequencing and assembly strategy. We described the functional regions in the genome which are important for the synthesis of BNC and acetan-like II polymer. We next investigated the effect of 1% vitamin C supplementation on the global gene expression profile using RNA sequencing. Our transcriptomic readouts imply that vitamin C functions mainly as a reducing agent. We found that the changes in cellular redox status are balanced by strong repression of the sulfur assimilation pathway. Moreover, in the reduced conditions, glucose oxidation is decreased and alternative pathways for energy generation, such as acetate accumulation, are activated. The presence of vitamin C negatively influences acetan-like II polymer biosynthesis, which may explain the lowered yield and changed mechanical properties of BNC. The results of this study enrich the functional characteristics of the genomes of the efficient producers of the N. hansenii species. Improved understanding of the adaptation to the presence of vitamin C at the molecular level has important guiding significance for influencing the biosynthesis of BNC and its morphology.

Polyphasic characterization of a novel hot-spring cyanobacterium Thermocoleostomius sinensis gen et sp. nov. and genomic insights into its carbon concentration mechanism.

Thermophilic cyanobacteria play a crucial role as primary producers in hot spring ecosystems, yet their microbiological, taxonomic, and ecological characteristics are not extensively studied. This study aimed to characterize a novel strain of thermophilic cyanobacteria, PKUAC-SCTA174 (A174), using a combination of traditional polyphasic methods and modern genomic-based approaches. The study included 16S rRNA-based phylogeny, ITS secondary structure prediction, morphological and habitat analyses, as well as high-quality genome sequencing with corresponding phylogenomic analyses. The results of the 16S rRNA, 16S-23S ITS secondary structure, morphology, and habitat analyses supported the classification of the strain as a member of a novel genus within the family Oculatellaceae, closely related to Albertania and Trichotorquatus. Genomic analysis revealed the presence of a sophisticated carbon-concentrating mechanism (CCM) in the strain, involving two CO2 uptake systems NDH-I3, and NDH-I4, three types of bicarbonate transporters (BCT1, bicA, sbtA,) and two distinct putative carboxysomal carbonic anhydrases (ccaA1 and ccaA2). The expression of CCM genes was investigated with a CO2 shift experiment, indicating varying transcript abundance among different carbon uptake systems. Based on the comprehensive characterization, the strain was delineated as Thermocoleostomius sinensis, based on the botanical code. The study of the complete genome of strain A174 contributes valuable insights into the genetic characteristics of the genus Thermocoleostomius and related organisms and provides a systematic understanding of thermophilic cyanobacteria. The findings presented here offer valuable data that can be utilized for future research in taxogenomics, ecogenomics, and geogenomics.

Distinct Molecular Patterns of Two-Component Signal Transduction Systems in Thermophilic Cyanobacteria as Revealed by Genomic Identification.

Two-component systems (TCSs) play crucial roles in sensing and responding to environmental signals, facilitating the acclimation of cyanobacteria to hostile niches. To date, there is limited information on the TCSs of thermophilic cyanobacteria. Here, genome-based approaches were used to gain insights into the structure and architecture of the TCS in 17 well-described thermophilic cyanobacteria, namely strains from the genus Leptodesmis, Leptolyngbya, Leptothermofonsia, Thermoleptolyngbya, Thermostichus, and Thermosynechococcus. The results revealed a fascinating complexity and diversity of the TCSs. A distinct composition of TCS genes existed among these thermophilic cyanobacteria. A majority of TCS genes were classified as orphan, followed by the paired and complex cluster. A high proportion of histidine kinases (HKs) were predicted to be cytosolic subcellular localizations. Further analyses suggested diversified domain architectures of HK and response regulators (RRs), putatively in association with various functions. Comparative and evolutionary genomic analyses indicated that the horizontal gene transfer, as well as duplications events, might be involved in the evolutionary history of TCS genes in Thermostichus and Thermosynechococcus strains. A comparative analysis between thermophilic and mesophilic cyanobacteria indicated that one HK cluster and one RR cluster were uniquely shared by all the thermophilic cyanobacteria studied, while two HK clusters and one RR cluster were common to all the filamentous thermophilic cyanobacteria. These results suggested that these thermophile-unique clusters may be related to thermal characters and morphology. Collectively, this study shed light on the TCSs of thermophilic cyanobacteria, which may confer the necessary regulatory flexibility; these findings highlight that the genomes of thermophilic cyanobacteria have a broad potential for acclimations to environmental fluctuations.

Characterization of a novel thermophilic cyanobacterium within Trichocoleusaceae, Trichothermofontia sichuanensis gen. et sp. nov., and its CO2-concentrating mechanism.

Thermophiles from extreme thermal environments have shown tremendous potential regarding ecological and biotechnological applications. Nevertheless, thermophilic cyanobacteria remain largely untapped and are rarely characterized. Herein, a polyphasic approach was used to characterize a thermophilic strain, PKUAC-SCTB231 (hereafter B231), isolated from a hot spring (pH 6.62, 55.5°C) in Zhonggu village, China. The analyses of 16S rRNA phylogeny, secondary structures of 16S-23S ITS and morphology strongly supported strain B231 as a novel genus within Trichocoleusaceae. Phylogenomic inference and three genome-based indices further verified the genus delineation. Based on the botanical code, the isolate is herein delineated as Trichothermofontia sichuanensis gen. et sp. nov., a genus closely related to a validly described genus Trichocoleus. In addition, our results suggest that Pinocchia currently classified to belong to the family Leptolyngbyaceae may require revision and assignment to the family Trichocoleusaceae. Furthermore, the complete genome of Trichothermofontia B231 facilitated the elucidation of the genetic basis regarding genes related to its carbon-concentrating mechanism (CCM). The strain belongs to ß-cyanobacteria according to its ß-carboxysome shell protein and 1B form of Ribulose bisphosphate Carboxylase-Oxygenase (RubisCO). Compared to other thermophilic strains, strain B231contains a relatively low diversity of bicarbonate transporters (only BicA for HCO3- transport) but a higher abundance of different types of carbonic anhydrase (CA), ß-CA (ccaA) and γ-CA (ccmM). The BCT1 transporter consistently possessed by freshwater cyanobacteria was absent in strain B231. Similar situation was occasionally observed in freshwater thermal Thermoleptolyngbya and Thermosynechococcus strains. Moreover, strain B231 shows a similar composition of carboxysome shell proteins (ccmK1-4, ccmL, -M, -N, -O, and -P) to mesophilic cyanobacteria, the diversity of which was higher than many thermophilic strains lacking at least one of the four ccmK genes. The genomic distribution of CCM-related genes suggests that the expression of some components is regulated as an operon and others in an independently controlled satellite locus. The current study also offers fundamental information for future taxogenomics, ecogenomics and geogenomic studies on distribution and significance of thermophilic cyanobacteria in the global ecosystem.

Reevaluation of Parasynechococcus-like Strains and Genomic Analysis of Their Microsatellites and Compound Microsatellites.

Morphologically similar to Synechococcus, a large number of Parasynechococcus strains were misclassified, resulting in extreme underestimation of their genetic diversity. In this study, 80 Synechococcus-like strains were reevaluated using a combination of 16S rRNA phylogeny and genomic approach, identifying 54 strains as Parasynechococcus-like strains and showing considerably intragenus genetic divergence among the subclades identified. Further, bioinformatics analysis disclosed diversified patterns of distribution, abundance, density, and diversity of microsatellites (SSRs) and compound microsatellites (CSSRs) in genomes of these Parasynechococcus-like strains. Variations of SSRs and CSSRs were observed amongst phylotypes and subclades. Both SSRs and CSSRs were in particular unequally distributed among genomes. Dinucleotide SSRs were the most widespread, while the genomes showed two patterns in the second most abundant repeat type (mononucleotide or trinucleotide SSRs). Both SSRs and CSSRs were predominantly observed in coding regions. These two types of microsatellites showed positive correlation with genome size (p < 0.01) but negative correlation with GC content (p < 0.05). Additionally, the motif (A)n, (AG)n and (AGC)n was a major one in the corresponding category. Meanwhile, distinctive motifs of CSSRs were found in 39 genomes. This study characterizes SSRs and CSSRs in genomes of Parasynechococcus-like strains and will be useful as a prerequisite for future studies regarding their distribution, function, and evolution. Moreover, the identified SSRs may facilitate fast acclimation of Parasynechococcus-like strains to fluctuating environments and contribute to the extensive distribution of Parasynechococcus species in global marine environments.

Comparative Genomic Analysis Revealed Distinct Molecular Components and Organization of CO2-Concentrating Mechanism in Thermophilic Cyanobacteria.

Cyanobacteria evolved an inorganic carbon-concentrating mechanism (CCM) to perform effective oxygenic photosynthesis and prevent photorespiratory carbon losses. This process facilitates the acclimation of cyanobacteria to various habitats, particularly in CO2-limited environments. To date, there is limited information on the CCM of thermophilic cyanobacteria whose habitats limit the solubility of inorganic carbon. Here, genome-based approaches were used to identify the molecular components of CCM in 17 well-described thermophilic cyanobacteria. These cyanobacteria were from the genus Leptodesmis, Leptolyngbya, Leptothermofonsia, Thermoleptolyngbya, Thermostichus, and Thermosynechococcus. All the strains belong to ß-cyanobacteria based on their ß-carboxysome shell proteins with 1B form of Rubisco. The diversity in the Ci uptake systems and carboxysome composition of these thermophiles were analyzed based on their genomic information. For Ci uptake systems, two CO2 uptake systems (NDH-13 and NDH-14) and BicA for HCO3 - transport were present in all the thermophilic cyanobacteria, while most strains did not have the Na+/HCO3 - Sbt symporter and HCO3 - transporter BCT1 were absent in four strains. As for carboxysome, the ß-carboxysomal shell protein, ccmK2, was absent only in Thermoleptolyngbya strains, whereas ccmK3/K4 were absent in all Thermostichus and Thermosynechococcus strains. Besides, all Thermostichus and Thermosynechococcus strains lacked carboxysomal ß-CA, ccaA, the carbonic anhydrase activity of which may be replaced by ccmM proteins as indicated by comparative domain analysis. The genomic distribution of CCM-related genes was different among the thermophiles, suggesting probably distinct expression regulation. Overall, the comparative genomic analysis revealed distinct molecular components and organization of CCM in thermophilic cyanobacteria. These findings provided insights into the CCM components of thermophilic cyanobacteria and fundamental knowledge for further research regarding photosynthetic improvement and biomass yield of thermophilic cyanobacteria with biotechnological potentials.