Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Cyanobacterial Harmful Algal Mats (CyanoHAMs) in tropical rivers of central Mexico and their potential risks through toxin production.

Cyanobacteria inhabiting lotic environments have been poorly studied and characterized in Mexico, despite their potential risks from cyanotoxin production. This article aims to fill this knowledge gap by assessing the importance of benthic cyanobacteria as potential cyanotoxin producers in central Mexican rivers through: (i) the taxonomic identification of cyanobacteria found in these rivers, (ii) the environmental characterization of their habitats, and (iii) testing for the presence of toxin producing genes in the encountered taxa. Additionally, we introduce and discuss the use of the term "CyanoHAMs" for lotic water environments. Populations of cyanobacteria were collected from ten mountain rivers and identified using molecular techniques. Subsequently, these taxa were evaluated for genes producing anatoxins and microcystins via PCR. Through RDA analyses, the collected cyanobacteria were grouped into one of three categories based on their environmental preferences for the following: (1) waters with high ionic concentrations, (2) cold-temperate waters, or (3) waters with high nutrient enrichment. Populations from six locations were identified to genus level: Ancylothrix sp., Cyanoplacoma sp., and Oxynema sp. The latter was found to contain the gene that produces anatoxins and microcystins in siliceous rivers, while Oxynema tested positive for the gene that produces microcystins in calcareous rivers. Our results suggest that eutrophic environments are not necessarily required for toxin-producing cyanobacteria. Our records of Compactonostoc, Oxynema, and Ancylothrix represent the first for Mexico. Four taxa were identified to species level: Wilmottia aff. murrayi, Nostoc tlalocii, Nostoc montejanii, and Dichothrix aff. willei, with only the first testing positive using PCR for anatoxin and microcystin-producing genes in siliceous rivers. Due to the differences between benthic growths with respect to planktonic ones, we propose the adoption of the term Cyanobacterial Harmful Algal Mats (CyanoHAMs) as a more precise descriptor for future studies.

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle.

Gansulinema gen. nov. and Komarkovaeasiopsis gen. nov.: Novel Oculatellacean genera (Cyanobacteria) isolated from desert soils and hot spring.

To increase the understanding of simple thin filamentous cyanobacteria in harsh environmental areas, we previously isolated and identified four strains (XN101, XN102, GS121, NX122) from desert soils and hot spring in China. As a result, two new Oculatellacean genera of these four strains, Gansulinema gen. nov. and Komarkovaeasiopsis gen. nov., are described based on a polyphasic approach. The ultrastructure of these strains showed a similar arrangement of peripheral thylakoids with three to four parallel layers, indicating that they belonged to the orders Nodosilineales, Oculatellales, or Leptolyngbyales. In the 16S rRNA gene phylogeny, two sequences of the Gansulinema strains and the two sequences of the Komarkovaeasiopsis strains formed two independent and robust clusters, within the order Oculatellales. The 16S rRNA gene sequences of strains of Komarkovaeasiopsis and Gansulinema showed low identity to each other (≤93.2%) and to other sequences of the Oculatellacean genera (≤94.5% and ≤93.3%, respectively). Furthermore, the 16S-23S internal transcribed spacer rRNA region secondary structures of strains of Komarkovaeasiopsis and Gansulinema were not consistent with all existing descriptions of Oculatellacean taxa. These data suggest that cyanobacterial communities are rich sources of new taxa in under-exploited areas, such as desert soils and hot spring in China.

Description of six new cyanobacterial species from soil biocrusts on San Nicolas Island, California, in three genera previously restricted to Brazil.

As the taxonomic knowledge of cyanobacteria from terrestrial environments increases, it remains important to analyze biodiversity in areas that have been understudied to fully understand global and endemic diversity. This study was completed as part of a larger algal biodiversity study of the soil biocrusts of San Nicholas Island, California, USA. Among the taxa isolated were several new species in three genera (Atlanticothrix, Pycnacronema, and Konicacronema) which were described from, and previously restricted to, Brazil. New taxa are described herein using a polyphasic approach to cyanobacterial taxonomy that considers morphological, molecular, ecological, and biogeographical factors. Morphological data corroborated by molecular analysis including sequencing of the 16S rRNA gene, and the associated 16S-23S ITS rRNA region was used to delineate three new species of Atlanticothrix, two species of Pycnacronema, and one species of Konicacronema. The overlap of genera from San Nicolas Island and Brazil suggests that cyanobacterial genera may be widely distributed across global hemispheres, whereas the presence of distinct lineages may indicate that this is not true at the species level. Our data suggest that based upon global wind patterns, cyanobacteria in both Northern and Southern hemispheres of the Americas may have a more recent common ancestor in Northern Africa, but this common ancestry is distant enough that speciation has occurred since transatlantic dispersal.

Antagonistic actions of Paucibacter aquatile B51 and its lasso peptide paucinodin toward cyanobacterial bloom-forming Microcystis aeruginosa PCC7806.

Superior antagonistic activity against axenic Microcystis aeruginosa PCC7806 was observed with Paucibacter sp. B51 isolated from cyanobacterial bloom samples among 43 tested freshwater bacterial species. Complete genome sequencing, analyzing average nucleotide identity and digital DNA-DNA hybridization, designated the B51 strain as Paucibacter aquatile. Electron and fluorescence microscopic image analyses revealed the presence of the B51 strain in the vicinity of M. aeruginosa cells, which might provoke direct inhibition of the photosynthetic activity of the PCC7806 cells, leading to perturbation of cellular metabolisms and consequent cell death. Our speculation was supported by the findings that growth failure of the PCC7806 cells led to low pH conditions with fewer chlorophylls and down-regulation of photosystem genes (e.g., psbD and psaB) during their 48-h co-culture condition. Interestingly, the concentrated ethyl acetate extracts obtained from B51-grown supernatant exhibited a growth-inhibitory effect on PCC7806. The physical separation of both strains by a filter system led to no inhibitory activity of the B51 cells, suggesting that contact-mediated anti-cyanobacterial compounds might also be responsible for hampering the growth of the PCC7806 cells. Bioinformatic tools identified 12 gene clusters that possibly produce secondary metabolites, including a class II lasso peptide in the B51 genome. Further chemical analysis demonstrated anti-cyanobacterial activity from fractionated samples having a rubrivinodin-like lasso peptide, named paucinodin. Taken together, both contact-mediated inhibition of photosynthesis and the lasso peptide secretion of the B51 strain are responsible for the anti-cyanobacterial activity of P. aquatile B51.

Use of 16S rRNA gene sequences to identify cyanobacteria that can grow in far-red light.

Although most cyanobacteria use visible light (VL; λ = 400-700 nm) for photosynthesis, some have evolved strategies to use far-red light (FRL; λ = 700-800 nm). These cyanobacteria are defined as far-red light-utilizing cyanobacteria (FRLCyano), including two groups: (1) chlorophyll d-producing Acaryochloris spp. and (2) polyphyletic cyanobacteria that produce chlorophylls d and f in response to FRL. Numerous ecological studies examine pigments, such as chlorophylls d and f, to investigate the presence of FRLCyano in the environment. This method is not ideal because it can only detect FRLCyano that have made chlorophylls d or f. Here we develop a new method, far-red cyanobacteria identification (FRCI), to identify FRLCyano based on 16S rRNA gene sequences. From public databases and published articles, 62 16S rRNA gene sequences of FRLCyano were extracted. Comparing with related lineages, we determined that 97% sequence identity is the optimal cut-off for distinguishing FRLCyano from other cyanobacteria. To test the method experimentally, we collected samples from 17 sites in Taipei, Taiwan, and conducted VL and FRL enrichments. Our results demonstrate that FRCI can detect FRLCyano during FRL enrichments more sensitively than pigment analysis. FRCI can also resolve the composition of FRLCyano at the genus level, which pigment analysis cannot do. In addition, we applied FRCI to published datasets and discovered putative FRLCyano in diverse environments, including soils, hot springs and deserts. Overall, our results indicate that FRCI is a sensitive and high-resolution method using 16S rRNA gene sequences to identify FRLCyano.

Genome-wide analysis of lectins in cyanobacteria: from evolutionary mode to motif patterns.

Lectins are glycoproteins that can bind to specific carbohydrates, and different lectin families exhibit different biological activities. They are also present in the cyanobacteria and many of them have shown excellent therapeutic effect, which deserve for bioprospecting. However, in comparison to those from terrestrial plants, the current knowledge on cyanobacterial lectins is very limited. To this end, genome-wide analyses were performed to find out their evolutionary mode and motif patterns in 316 genomes of representative taxa. In results, 196 putative cyanobacterial lectins were dig out and 105 of them were classified into known families. Seven lectins were found to be belonged to distinct two lectin families, and they may have the potential activities of both lectin families. Whereas no MFP-2, Chitin, and Nictaba family lectins were found. What's more, the Legume lectin-like lectin family was found to be the richest and most complex in cyanobacteria, which could be a main research direction for future cyanobacterial lectin bioprospecting and development. Our classification and prediction of cyanobacteria lectins is expected to provide assistance in the development of lectin-based medicine and provide solutions to the current thorny viral and tumor diseases in humans.

Stringent Response of Cyanobacteria and Other Bacterioplankton during Different Stages of a Harmful Cyanobacterial Bloom.

We conducted a field study to investigate the role of stringent response in cyanobacteria and coexisting bacterioplankton during nutrient-deprived periods at various stages of bloom in a freshwater lake (Utah Lake) for the first time. Using metagenomics and metatranscriptomics analyses, we examined the cyanobacterial ecology and expression of important functional genes related to stringent response, N and P metabolism, and regulation. Our findings mark a significant advancement in understanding the mechanisms by which toxic cyanobacteria survive and proliferate during nitrogen (N) and phosphorus (P) limitations. We successfully identified and analyzed the metagenome-assembled genomes (MAGs) of the dominant bloom-forming cyanobacteria, namely, Dolichospermum circinale, Aphanizomenon flos-aquae UKL13-PB, Planktothrix agardhii, and Microcystis aeruginosa. By mapping RNA-seq data to the coding sequences of the MAGs, we observed that these four prevalent cyanobacteria species activated multiple functions to adapt to the depletion of inorganic nutrients. During and after the blooms, the four dominant cyanobacteria species expressed high levels of transcripts related to toxin production, such as microcystins (mcy), anatoxins (ana), and cylindrospermopsins (cyr). Additionally, genes associated with polyphosphate (poly-P) storage and the stringent response alarmone (p)ppGpp synthesis/hydrolysis, including ppk, relA, and spoT, were highly activated in both cyanobacteria and bacterioplankton. Under N deficiency, the main N pathways shifted from denitrification and dissimilatory nitrate reduction in bacterioplankton toward N2-fixing and assimilatory nitrate reduction in certain cyanobacteria with a corresponding shift in the community composition. P deprivation triggered a stringent response mediated by spoT-dependent (p)ppGpp accumulation and activation of the Pho regulon in both cyanobacteria and bacterioplankton, facilitating inorganic and organic P uptake. The dominant cyanobacterial MAGs exhibited the presence of multiple alkaline phosphatase (APase) transcripts (e.g., phoA in Dolichospermum, phoX in Planktothrix, and Microcystis), suggesting their ability to synthesize and release APase enzymes to convert ambient organic P into bioavailable forms. Conversely, transcripts associated with bacterioplankton-dominated pathways like denitrification were low and did not align with the occurrence of intense cyanoHABs. The strong correlations observed among N, P, stringent response metabolisms and the succession of blooms caused by dominant cyanobacterial species provide evidence that the stringent response, induced by nutrient limitation, may activate unique N and P functions in toxin-producing cyanobacteria, thereby sustaining cyanoHABs.

Description of four new filamentous cyanobacterial taxa from freshwater habitats in the Azores Archipelago.

Simple filamentous cyanobacteria comprise a diverse and polyphyletic group of species, primarily in the orders Leptolyngbyales and Oscillatoriales, that need more sampling to improve their taxonomy. Oceanic islands, such as the Azores archipelago, present unique habitats and biogeographic conditions that harbor an unknown range of diversity of microorganisms. Filamentous cyanobacteria isolated from aquatic habitats in the Azores and maintained in the BACA culture collection were described using morphology, both light and transmission electron microscopy, ecology, and genetic data of the 16S rRNA gene sequences and 16S-23S Internal Transcribed Spacer (ITS) rRNA region secondary structure. Our analyses revealed two new monophyletic genera: Tumidithrix elongata gen. sp. nov. (Pseudanabaenaceae) and Radiculonema aquaticum gen. sp. nov. (Leptolyngbyaceae). In addition, two new species Leptodesmis lacustris sp. nov. (Leptolyngbyaceae) and Pycnacronema lacustrum sp. nov. (Wilmottiaceae) are reported as the first aquatic species for these genera. The description of these new taxa and the genetic study of an isolate of Leptodesmis alaskaensis from the Azores followed the polyphasic approach, identifying diacritical features. Our results reinforce the need for taxonomic studies on cyanobacteria from less-studied habits and geographic regions, which have a potential for new taxa description.

Albertania and Egbenema gen. nov. from Nigeria and the United States, expanding biodiversity in the Oculatellaceae (cyanobacteria).

Knowledge of the tropical terrestrial cyanobacterial flora from the African continent is still limited. Of 31 strains isolated from soil and subaerial samples collected in Lagos State, Nigeria, three were found to be in the Oculatellaceae, including two species in a new genus. Subsequently, isolates from microbial mats in White Sands National Park in New Mexico, United States, and from a rock near the ocean in Puerto Rico, United States, were found to belong to the new genus as well. Cyanobacterial isolates were characterized microscopically, sequenced for the 16S rRNA gene and associated ITS region, and phylogenetically analyzed. Egbenema gen. nov., with three new species, as well as two new species of Albertania were differentiated from all other Oculatellaceae. Both genera belong to a supported clade within the Oculatellaceae that includes Trichotorquatus and Komarkovaea. The two new species of Albertania, A. egbensis and A. latericola, were from the same sample, but were evolutionarily separate based on 16S rRNA gene phylogenies, percent identity below the 98.7% threshold, and ITS rRNA percent dissimilarity >7.0%. Egbenema aeruginosum gen. et sp. nov. was phylogenetically separated from Trichotorquatus and Albertania but was in a clade with other strains belonging to Egbenema. The two Egbenema strains from the United States are here named Egbenema epilithicum sp. nov. and Egbenema gypsiphilum sp. nov. Our results support the hypothesis that further species discoveries of novel cyanobacteria will likely be made in soils and subaerial habitats, as these habitats continue to be studied, both in tropical and temperate biomes.

Tolyporphins-Exotic Tetrapyrrole Pigments in a Cyanobacterium-A Review.

Tolyporphins were discovered some 30 years ago as part of a global search for antineoplastic compounds from cyanobacteria. To date, the culture HT-58-2, comprised of a cyanobacterium-microbial consortium, is the sole known producer of tolyporphins. Eighteen tolyporphins are now known-each is a free base tetrapyrrole macrocycle with a dioxobacteriochlorin (14), oxochlorin (3), or porphyrin (1) chromophore. Each compound displays two, three, or four open ß-pyrrole positions and two, one, or zero appended C-glycoside (or -OH or -OAc) groups, respectively; the appended groups form part of a geminal disubstitution motif flanking the oxo moiety in the pyrroline ring. The distinct structures and repertoire of tolyporphins stand alone in the large pigments-of-life family. Efforts to understand the cyanobacterial origin, biosynthetic pathways, structural diversity, physiological roles, and potential pharmacological properties of tolyporphins have attracted a broad spectrum of researchers from diverse scientific areas. The identification of putative biosynthetic gene clusters in the HT-58-2 cyanobacterial genome and accompanying studies suggest a new biosynthetic paradigm in the tetrapyrrole arena. The present review provides a comprehensive treatment of the rich science concerning tolyporphins.

Comparative Genome analysis of the Genus Curvibacter and the Description of Curvibacter microcysteis sp. nov. and Curvibacter cyanobacteriorum sp. nov., Isolated from Fresh Water during the Cyanobacterial Bloom Period.

The three Gram-negative, catalase- and oxidase-positive bacterial strains RS43T, HBC28, and HBC61T, were isolated from fresh water and subjected to a polyphasic study. Comparison of 16S rRNA gene sequence initially indicated that strains RS43T, HBC28, and HBC61T were closely related to species of genus Curvibacter and shared the highest sequence similarity of 98.14%, 98.21%, and 98.76%, respectively, with Curvibacter gracilis 7-1T. Phylogenetic analysis based on genome sequences placed all strains within the genus Curvibacter. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the three strains and related type strains supported their recognition as two novel genospecies in the genus Curvibacter. Comparative genomic analysis revealed that the genus possessed an open pangenome. Based on KEGG BlastKOALA analyses, Curvibacter species have the potential to metabolize benzoate, phenylacetate, catechol, and salicylate, indicating their potential use in the elimination of these compounds from the water systems. The results of polyphasic characterization indicated that strain RS43T and HBC61T represent two novel species, for which the name Curvibacter microcysteis sp. nov. (type strain RS43T =KCTC 92793T=LMG 32714T) and Curvibacter cyanobacteriorum sp. nov. (type strain HBC61T =KCTC 92794T =LMG 32713T) are proposed.

Phylogenetic distribution, structural analysis and interaction of nucleotide excision repair proteins in cyanobacteria.

Cyanobacteria are photosynthetic Gram-negative, oxygen evolving prokaryotes with cosmopolitan distribution. Ultraviolet radiation (UVR) and other abiotic stresses result in DNA lesions in cyanobacteria. Nucleotide excision repair (NER) pathway removes the DNA lesions produced by UVR to normal DNA sequence. In cyanobacteria, detailed knowledge about NER proteins is poorly studied. Therefore, we have studied the NER proteins in cyanobacteria. Analyses of 289 amino acids sequence from 77 cyanobacterial species have revealed the presence of a minimum of one copy of NER protein in their genome. Phylogenetic analysis of NER protein shows that UvrD has maximal rate of amino acid substitutions which resulted in increased branch length. The motif analysis shows that UvrABC proteins is more conserved than UvrD, Further, UvrA with UvrB protein interacts with each other and form stable complex which have DNA binding domain on the surface of the complex. UvrB also have DNA binding domain. Positive electrostatic potential was found in the DNA binding region, which is followed by negative and neutral electrostatic potential. Additionally, the surface accessibility values at the DNA strands of T5-T6 dimer binding site were maximal. Protein nucleotide interaction shows the strong binding of T5-T6 dimer with NER proteins of Synechocystis sp. PCC 6803. This process repairs the UV-induced DNA lesions in dark when photoreactivation is inactive. Regulation of NER proteins protect cyanobacterial genome and maintain the fitness of organism under different abiotic stresses.

Cyanobacterial phycobilisomes as a platform for the stable production of heterologous enzymes and other proteins.

Efforts to stably over-express recombinant proteins in cyanobacteria are hindered due to cellular proteasome activity that efficiently degrades foreign proteins. Recent work from this lab showed that a variety of exogenous genes from plants, humans, and bacteria can be successfully and stably over-expressed in cyanobacteria, as fusion constructs with the abundant ß-subunit of phycocyanin (the cpcB gene product) in quantities up to 10-15% of the total cell protein. The CpcB*P fusion proteins did not simply accumulate in a soluble free-floating form in the cell but, rather, they assembled as functional (α,ß*P)3CpcG1 heterohexameric light-harvesting phycocyanin antenna discs, where α is the CpcA α-subunit of phycocyanin, ß*P is the CpcB*P fusion protein, the asterisk denoting fusion, and CpcG1 is the 28.9 kDa phycocyanin disc linker polypeptide (Hidalgo Martinez et al., 2022). The present work showed that the CpcA α-subunit of phycocyanin and the CpcG1 28.9 kDa phycocyanin disc linker polypeptide can also successfully serve as leading sequences in functional heterohexameric (α*P,ß)3CpcG1 and (α,ß)3CpcG1*P fusion constructs that permit stable recombinant protein over-expression and accumulation. These were shown to form a residual light-harvesting antenna and to contribute to photosystem-II photochemistry in the cyanobacterial cells. The work suggested that cyanobacterial cells need phycocyanin for light absorption, photosynthesis, and survival and, therefore, may tolerate the presence of heterologous recombinant proteins, when the latter are in a fusion construct configuration with essential cellular proteins, e.g., phycocyanin, thus allowing their substantial and stable accumulation.

How diverse a genus can be: An integrated multi-layered analysis into Desmonostoc (Nostocaceae, Cyanobacteriota).

Cyanobacteria (Phylum Cyanobacteriota) are Gram-negative bacteria capable of performing oxygenic photosynthesis. Although the taxonomic classification of cyanobacteria was for a long time based primarily on morphological characters, the application of other techniques (e.g. molecular phylogeny), especially in recent decades, has contributed to a better resolution of cyanobacteria systematics, leading to a revision of the phylum. Although Desmonostoc occurs as a new genus/cluster and some species have been described recently, relatively few studies have been carried out to elucidate its diversity, which encompasses strains from different ecological origins, or examine the application of new characterization tools. In this context, the present study investigated the diversity within Desmonostoc, based on morphological, molecular, metabolic, and physiological characteristics. Although the usage of physiological parameters is unusual for a polyphasic approach, they were efficient in the characterization performed here. The phylogenetic analysis based on 16S rRNA gene sequences put all studied strains (25) into the D1 cluster and indicated the emergence of novel sub-clusters. It was also possible to observe that nifD and nifH exhibited different evolutionary histories within the Desmonostoc strains. Collectively, metabolic and physiological data, coupled with the morphometric data, were in general, in good agreement with the separation based on the phylogeny of the 16S rRNA gene. Furthermore, the study provided important information on the diversity of Desmonostoc strains collected from different Brazilian biomes by revealing that they were cosmopolitan strains, acclimatized to low luminous intensities, with a large metabolic diversity and great biotechnological potential.

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.

Unique Initiation and Termination Mechanisms Involved in the Biosynthesis of a Hybrid Polyketide-Nonribosomal Peptide Lyngbyapeptin B Produced by the Marine Cyanobacterium Moorena bouillonii.

Lyngbyapeptin B is a hybrid polyketide-nonribosomal peptide isolated from particular marine cyanobacteria. In this report, we carried out genome sequence analysis of a producer cyanobacterium Moorena bouillonii to understand the biosynthetic mechanisms that generate the unique structural features of lyngbyapeptin B, including the (E)-3-methoxy-2-butenoyl starter unit and the C-terminal thiazole moiety. We identified a putative lyngbyapeptin B biosynthetic (lynB) gene cluster comprising nine open reading frames that include two polyketide synthases (PKSs: LynB1 and LynB2), four nonribosomal peptide synthetases (NRPSs: LynB3, LynB4, LynB5, and LynB6), a putative nonheme diiron oxygenase (LynB7), a type II thioesterase (LynB8), and a hypothetical protein (LynB9). In vitro enzymatic analysis of LynB2 with methyltransferase (MT) and acyl carrier protein (ACP) domains revealed that the LynB2 MT domain (LynB2-MT) catalyzes O-methylation of the acetoacetyl-LynB2 ACP domain (LynB2-ACP) to yield (E)-3-methoxy-2-butenoyl-LynB2-ACP. In addition, in vitro enzymatic analysis of LynB7 revealed that LynB7 catalyzes the oxidative decarboxylation of (4R)-2-methyl-2-thiazoline-4-carboxylic acid to yield 2-methylthiazole in the presence of Fe2+ and molecular oxygen. This result indicates that LynB7 is responsible for the last post-NRPS modification to give the C-terminal thiazole moiety in lyngbyapeptin B biosynthesis. Overall, we identified and characterized a new marine cyanobacterial hybrid PKS-NRPS biosynthetic gene cluster for lyngbyapeptin B production, revealing two unique enzymatic logics.

Ancient Rapid Radiation Explains Most Conflicts Among Gene Trees and Well-Supported Phylogenomic Trees of Nostocalean Cyanobacteria.

Prokaryotic genomes are often considered to be mosaics of genes that do not necessarily share the same evolutionary history due to widespread horizontal gene transfers (HGTs). Consequently, representing evolutionary relationships of prokaryotes as bifurcating trees has long been controversial. However, studies reporting conflicts among gene trees derived from phylogenomic data sets have shown that these conflicts can be the result of artifacts or evolutionary processes other than HGT, such as incomplete lineage sorting, low phylogenetic signal, and systematic errors due to substitution model misspecification. Here, we present the results of an extensive exploration of phylogenetic conflicts in the cyanobacterial order Nostocales, for which previous studies have inferred strongly supported conflicting relationships when using different concatenated phylogenomic data sets. We found that most of these conflicts are concentrated in deep clusters of short internodes of the Nostocales phylogeny, where the great majority of individual genes have low resolving power. We then inferred phylogenetic networks to detect HGT events while also accounting for incomplete lineage sorting. Our results indicate that most conflicts among gene trees are likely due to incomplete lineage sorting linked to an ancient rapid radiation, rather than to HGTs. Moreover, the short internodes of this radiation fit the expectations of the anomaly zone, i.e., a region of the tree parameter space where a species tree is discordant with its most likely gene tree. We demonstrated that concatenation of different sets of loci can recover up to 17 distinct and well-supported relationships within the putative anomaly zone of Nostocales, corresponding to the observed conflicts among well-supported trees based on concatenated data sets from previous studies. Our findings highlight the important role of rapid radiations as a potential cause of strongly conflicting phylogenetic relationships when using phylogenomic data sets of bacteria. We propose that polytomies may be the most appropriate phylogenetic representation of these rapid radiations that are part of anomaly zones, especially when all possible genomic markers have been considered to infer these phylogenies. [Anomaly zone; bacteria; horizontal gene transfer; incomplete lineage sorting; Nostocales; phylogenomic conflict; rapid radiation; Rhizonema.].