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Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO

UV radiation (UVR) has significant physiological effects on organisms living at or near the Earth’s surface, yet the full suite of genes required for fitness of a photosynthetic organism in a UVR-rich environment remains unknown. This study reports a genome-wide fitness assessment of the genes that affect UVR tolerance under environmentally relevant UVR dosages in the model cyanobacterium Synechococcus elongatus PCC 7942. Our results highlight the importance of specific genes that encode proteins involved in DNA repair, glutathione synthesis, and the assembly and maintenance of photosystem II, as well as genes that encode hypothetical proteins and others without an obvious connection to canonical methods of UVR tolerance. Disruption of a gene that encodes a leucyl aminopeptidase (LAP) conferred the greatest UVR-specific decrease in fitness. Enzymatic assays demonstrated a strong pH-dependent affinity of the LAP for the dipeptide cysteinyl-glycine, suggesting an involvement in glutathione catabolism as a function of night-time cytosolic pH level. A low differential expression of the LAP gene under acute UVR exposure suggests that its relative importance would be overlooked in transcript-dependent screens. Subsequent experiments revealed a similar UVR-sensitivity phenotype in LAP knockouts of other organisms, indicating conservation of the functional role of LAPs in UVR tolerance.

Columbamides are chlorinated acyl amide natural products, several of which exhibit cannabinomimetic activity. These compounds were originally discovered from a culture of the filamentous marine cyanobacterium

Uniformitarian assumptions underlie the oldest evidence for living organisms on Earth, the distinct isotope fractionation between inorganic and organic carbon. Aside from a handful of compelling deviations, the 13C/12C isotopic mean of preserved organic carbon ({delta}13Corg) has remained remarkably unchanged through time. RuBisCO is the principal carboxylase/oxygenase biomolecular component that is thought to primarily account for the generation of these distinct carbon isotopic signals. However, it is difficult to reconcile a mostly unchanging mean {delta}13Corg with several known factors that can affect the isotope fractionation of RuBisCO, such as atmospheric composition and the amino acid composition of the enzyme itself, which have each changed markedly over Earth history. Here we report the resurrection and genetic incorporation of a Precambrian-age, Form IB RuBisCO in a modern cyanobacterial host. The isotopic composition of biomass relative to CO2 ({varepsilon}p) in ancestral and control strains were much greater when grown under Precambrian CO2 concentrations compared to modern ambient levels, but displaying values within a nominal envelope of modern-day RuBisCO IB enzyme variants. We infer that these isotopic differences derive indirectly from the decreased fitness of the AncIB strain, which includes diminished growth capacity and total cell RuBisCO activity. We argue that to answer the greatest questions of deep-time paleobiology, ancient biogeochemical signals should be reproduced in the laboratory through the synthesis of the geologic record with experimentally-derived constraints on underlying ancient molecular biology. Significance StatementThe earliest geochemical indicators of microbes, and the enzymes that powered them, extend back almost 3.8 billion years on our planet. Paleobiologists often attempt to understand these indicators by assuming that the behaviors of modern microbes and enzymes are consistent (uniform) with those of their predecessors. This assumption seems uncomfortably at odds with the great variability of Earths environment and its highly adaptive microbes. Here we examine whether a uniformitarian assumption for an enzyme thought to generate these indicators, RuBisCO, can be corroborated by independently studying the history of changes recorded within RuBisCOs genetic sequences. We outline a new approach to paleobiology that informatively links molecule-level evolutionary changes with planet-level geochemical conditions in Earths deep past.

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations), molecular and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a range of genetic and environmental factors that may have impacted ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO and characterized the resultant physiological and isotope fractionation effects. We specifically investigated how both increased atmospheric CO2 concentrations and RuBisCO regulation influence cell growth, oxygen evolution rate, and carbon isotope fractionation in cyanobacteria. We found that >2% CO2 increases the growth rate of wild-type and mutant strains, and that the pool of active RuBisCO enzyme increases with increased expression. At elevated CO2, carbon isotope discrimination (ep) is increased by ~8‰, whereas RuBisCO overexpression does not significantly affect isotopic discrimination at all tested CO2 concentrations. Our results show that understanding the environmental factors that impact RuBisCO regulation, physiology, and evolution is crucial for reconciling microbially driven carbon isotope fractionation with the geologic record of organic and inorganic carbon isotope signatures. IMPORTANCE Carbon isotope biosignatures preserved in the geologic record are interpreted to reflect the long-term evolution of microbial carbon fixation and provide the earliest evidence of life on Earth. RuBisCO enzymes, distinctive and early-evolved catalysts that fix atmospheric CO2, have likely been responsible for the bulk of primary productivity through Earth history. Thus, a comprehensive understanding of the molecular, physiological, environmental, and evolutionary factors that influence the isotopic discrimination of cyanobacteria that utilize RuBisCO is essential for the interpretation of ancient isotopic biosignatures. For example, the vastly different atmospheric CO2 levels that characterized the Precambrian may have influenced the expression and regulation of the ancient RuBisCO protein complex. These observations underscore the need to consider how a broader range of environmental conditions and subcellular processes may have shaped isotopic discrimination over geologic time. In this study, we establish a cyanobacterial metabolic-engineering strategy that can test such hypotheses and offer insights into the biogeochemical record of life.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, non-ribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drugs leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide, however high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.Abstract Figure

In the heterocyst-forming cyanobacterium Anabaena (also Nostoc) sp. strain PCC 7120, the nitrogen-fixation (nif) genes are expressed specifically in heterocysts during the late stages of development. We used gfp-reporter fusions to examine the regulation of the nifH and nifD genes. Plasmid-borne reporter fusions containing up to 500-bp of the nifH upstream region, which extended into the nifU gene, did not show developmentally regulated GFP fluorescence after removal of a nitrogen source from the growth medium, which indicated that sequences essential for transcriptional regulation are outside of the tested regions. Therefore, a gfp-reporter fusion was engineered into the chromosome at the 5′ end of nifD to produce strain AMC1774. This reporter construct at the native locus showed strong developmentally regulated GFP expression in differentiating heterocysts by 18 h after removal of combined nitrogen. We then screened for UV-induced mutants of AMC1774 that differentiated heterocysts but failed to ...

To downregulate gene expression in cyanobacteria, we constructed NOT gate genetic circuits using orthogonal promoters and their cognate repressors regulated translationally by synthetic riboswitches. Four NOT gates were tested and characterized in five cyanobacterial strains using fluorescent reporter-gene assays. In comparison to alternative systems used to downregulate gene expression in cyanobacteria, these NOT gates performed well, reducing YFP reporter expression by 4 to 50-fold. We further evaluated these NOT gates by controlling the expression of the ftsZ gene, which encodes a prokaryotic tubulin homologue that is required for cell division and is essential for Synechococcus elongatus PCC 7942. These NOT gates would facilitate cyanobacterial genetic engineering or the study of essential cellular processes.

An excess of reactive oxygen species (ROS) can cause severe oxidative damage to cellular components in photosynthetic cells. Antioxidant systems, such as the glutathione (GSH) pools, regulate redox status in cells to guard against such damage. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of oxidized ascorbate (dehydroascorbate) and contains a redox active site and glutathione binding-site. The DHAR gene is important in biological and abiotic stress responses involving reduction of the oxidative damage caused by ROS. In this study, transgenic Synechococcus elongatus PCC 7942 (TA) was constructed by cloning the Oryza sativa L. japonica DHAR (OsDHAR) gene controlled by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter (Ptrc) into the cyanobacterium to study the functional activities of OsDHAR under oxidative stress caused by hydrogen peroxide exposure. OsDHAR expression increased the growth of S. elongatus PCC 7942 under oxidative stress by reducing the levels of hydroperoxides and malondialdehyde (MDA) and mitigating the loss of chlorophyll. DHAR and glutathione S-transferase activity were higher than in the wild-type S. elongatus PCC 7942 (WT). Additionally, overexpression of OsDHAR in S. elongatus PCC 7942 greatly increased the glutathione (GSH)/glutathione disulfide (GSSG) ratio in the presence or absence of hydrogen peroxide. These results strongly suggest that DHAR attenuates deleterious oxidative effects via the glutathione (GSH)-dependent antioxidant system in cyanobacterial cells. The expression of heterologous OsDHAR in S. elongatus PCC 7942 protected cells from oxidative damage through a GSH-dependent antioxidant system via GSH-dependent reactions at the redox active site and GSH binding site residues during oxidative stress.

To facilitate the genetic engineering of diverse cyanobacterial strains, we have modified broad host-range RSF1010-based plasmids to improve transmissibility, increase copy number, and facilitate cloning. RSF1010-based plasmids replicate in diverse bacterial strains but produce low amounts of useable DNA for cloning. We previously engineered a mobAY25F mutation in RSF1010-based plasmids that improved cloning but decreased conjugation efficiency. Here, we engineered RSF1010-based plasmids to restore conjugation efficiency, which was demonstrated in three diverse laboratory strains of cyanobacteria. We then used an improved RSF1010-based plasmid in mating experiments with cultured samples of wild cyanobacteria. This plasmid, which confers antibiotic resistance and carries a yfp reporter gene, allowed selection of exconjugant cyanobacteria and facilitated the isolation of genetically tractable strains from mixed wild cultures. Improved RSF1010 vectors can be used for bioprospecting genetically tractable strains and are compatible with the CYANO-VECTOR cloning system, a versatile toolbox for constructing plasmids for cyanobacterial genetic engineering.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJ(Se) (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis. Plate-based phototaxis assays indicate that UTEX 3055 uses PixJ(Se) to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJ(Se) controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

The development of new heterologous hosts for polyketides production represents an excellent opportunity to expand the genomic, physiological, and biochemical backgrounds that better fit the sustainable production of these valuable molecules. Cyanobacteria are particularly attractive for the production of natural compounds because they have minimal nutritional demands and several strains have well established genetic tools. Using the model strain Synechococcus elongatus, a generic platform was developed for the heterologous production of polyketide synthase (PKS)-derived compounds. The versatility of this system is based on interchangeable modules harboring promiscuous enzymes for PKS activation and the production of PKS extender units, as well as inducible circuits for a regulated expression of the PKS biosynthetic gene cluster. To assess the capability of this platform, we expressed the mycobacterial PKS-based mycocerosic biosynthetic pathway to produce multimethyl-branched esters (MBE). This work is a foundational step forward for the production of high value polyketides in a photosynthetic microorganism. Fil: Roulet, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Taton, Arnaud. University of California at San Diego; Estados Unidos Fil: Golden, James W.. University of California at San Diego; Estados Unidos Fil: Arabolaza, Ana Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Burkart, Michael D.. University of California at San Diego; Estados Unidos Fil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

The broadly conserved signaling nucleotide cyclic di-adenosine monophosphate (c-di-AMP) is essential for viability in most bacteria where it has been studied. However, characterization of the cellular functions and metabolism of c-di-AMP has largely been confined to the class Bacilli, limiting our functional understanding of the molecule among diverse phyla. We identified the cyclase responsible for c-di-AMP synthesis and characterized the molecule’s role in survival of darkness in the model photosynthetic cyanobacterium Synechococcus elongatus PCC 7942. In addition to the use of traditional genetic, biochemical, and proteomic approaches, we developed a high-throughput genetic interaction screen (IRB-Seq) to determine pathways where the signaling nucleotide is active. We found that in S. elongatus c-di-AMP is produced by an enzyme of the diadenylate cyclase family, CdaA, which was previously unexplored experimentally. A cdaA-null mutant experiences increased oxidative stress and death during the nighttime portion of day-night cycles, in which potassium transport is implicated. These findings suggest that c-di-AMP is biologically active in cyanobacteria and has non-canonical roles in the phylum including oxidative stress management and day-night survival. The pipeline and analysis tools for IRB-Seq developed for this study constitute a quantitative high-throughput approach for studying genetic interactions., Author summary Cyclic di-adenosine monophosphate (c-di-AMP) is a molecule that has significant roles in many microorganisms. This work shows the existence of c-di-AMP for the first time in photosynthetic microorganisms, cyanobacteria, and demonstrates its role in survival during the light-to-dark shifts that occur in day-night cycles. Despite the obvious importance of adaptation to these daily cycles for organisms that are fundamentally reliant on light, such as cyanobacteria, understanding of diurnal physiology is lacking because most cyanobacterial research is conducted during growth in constant light. To identify other players in c-di-AMP’s function we developed a low-cost and efficient method for finding interactions between genes. The technique combines one mutation, in this case for the gene that encodes the enzyme for synthesis of c-di-AMP, with thousands of other individual mutations to find pairwise interactions that affect fitness of the resulting mutants. Mutants are tagged with DNA barcodes to allow their survival to be easily tracked in a population of cells. The method enables us to place the function of c-di-AMP within the context of pathways previously known to be involved in day-night survival. Taken together, this work expands the known roles of c-di-AMP, improves our understanding of cyanobacterial survival in day-night cycles, and presents an improved approach for determining genetic interactions.

To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H2O2, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR). Under H2O2 stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H2O2 level decreased. In addition, under H2O2 stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains. By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H2O2 stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

© 2016 The Authors. To facilitate development of synthetic biology tools for genetic engineering of cyanobacterial strains, we constructed pANS-derived self-replicating shuttle vectors that are based on the minimal replication element of the Synechococcus elongatus strain PCC 7942 plasmid pANS. To remove the possibility of homologous recombination events between the shuttle plasmids and the native pANS plasmid, the endogenous pANS was cured through plasmid incompatibility-mediated spontaneous loss. A heterologous toxin-antitoxin cassette was incorporated into the shuttle vectors for stable plasmid maintenance in the absence of antibiotic selection. The pANS-based shuttle vectors were shown to be able to carry a large 20 kb DNA fragment containing a gene cluster for biosynthesis of the omega-3 fatty acid eicosapentaenoic acid. Based on quantitative PCR analysis, there are about 10 copies of pANS and 3 copies of the large native plasmid pANL per chromosome in S. elongatus. Fluorescence levels of GFP reporter genes in a pANS-based vector were about 2.5-fold higher than when in pANL or integrated into the chromosome. In addition to its native host, pANS-based shuttle vectors were also found to replicate stably in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. There were about 27 copies of a pANS-based shuttle vector, 9 copies of a pDU1-based shuttle vector and 3 copies of an RSF1010-based shuttle vector per genome when these three plasmids coexisted in Anabaena cells. The endogenous pANS from our S. elongatus laboratory strain was cloned in Escherichia coli, re-sequenced and re-annotated to update previously published sequencing data.

© The Author(s) 2017. Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Both ground-based and satellite data show that parts of Antarctica have entered a period of rapid climate change, which already affects the functioning and productivity of limnetic ecosystems. To predict the consequences of future climate anomalies for lacustrine microbial communities, we not only need better baseline information on their biodiversity but also on the climate-related environmental factors structuring these communities. Here we applied denaturing gradient gel electrophoresis (DGGE) of the small subunit ribosomal DNA (SSU rDNA) to assess the genetic composition and distribution of Cyanobacteria and eukaryotes in 37 benthic microbial mat: samples from east Antarctic lakes. The lakes were selected to span a wide range of environmental gradients governed by differences in lake morphology and chemical limnology across 5 ice-free oases. Sequence analysis of selected DGGE bands revealed a high degree of potential endemism among the Cyanobacteria (mainly represented by Oscillatoriales and Nostocales), and the presence of a variety of protists (alveolates, stramenopiles and green algae), fungi, tardigrades and nematodes, which corroborates previous microscopy-based observations. Variation partitioning analyses revealed that the microbial mat community structure is largely regulated by both geographical and local environmental factors of which salinity (and related variables), lake water depth and nutrient concentrations are of major importance. These 3 groups of environmental variables have previously been shown to change drastically in Antarctica in response to climate change. Together, these results have obvious consequences for predicting the trajectory of biodiversity under changing climate conditions and call for the continued assessment of the biodiversity of these unique ecosystems.

We isolated 59 strains of cyanobacteria from the benthic microbial mats of 23 Antarctic lakes, from five locations in two regions, in order to characterize their morphological and genotypic diversity. On the basis of their morphology, the cyanobacteria were assigned to 12 species that included four Antarctic endemic taxa. Sequences of the ribosomal RNA gene were determined for 56 strains. In general, the strains closely related at the 16S rRNA gene level belonged to the same morphospecies. Nevertheless, divergences were observed concerning the diversity in terms of species richness, novelty, and geographical distribution. For the 56 strains, 21 operational taxonomic units (OTUs, defined as groups of partial 16S rRNA gene sequences with more than 97.5% similarity) were found, including nine novel and three exclusively Antarctic OTUs. Sequences of Petalonema cf. involvens and Chondrocystis sp. were determined for the first time. The internally transcribed spacer (ITS) between the 16S and the 23S rRNA genes was sequenced for 33 strains, and similar groupings were observed with the 16S rRNA gene and the ITS, even when the strains were derived from different lakes and regions. In addition, 48 strains were screened for antimicrobial and cytotoxic activities, and 17 strains were bioactive against the gram-positive Staphylococcus aureus, or the fungi Aspergillus fumigatus and Cryptococcus neoformans. The bioactivities were not in coincidence with the phylogenetic relationships, but rather were specific to certain strains.

For the first time, the cyanobacterial diversity from microbial mats in lakes of Eastern Antarctica was investigated using microscopic and molecular approaches. The present study assessed the biogeographical distribution of cyanobacteria in Antarctica. Five samples were taken from four lakes spanning a range of different ecological environments in Larsemann Hills, Vestfold Hills and Rauer Islands to evaluate the influence of lake characteristics on the cyanobacterial diversity. Seventeen morphospecies and 28 16S rRNA gene-based operational taxonomic units belonging to the Oscillatoriales, Nostocales and Chroococcales were identified. The internal transcribed spacer was evaluated to complement the 16S rRNA gene data and showed similar but more clear-cut tendencies. The molecular approach suggested that potential Antarctic endemic species, including a previously undiscovered diversity, are more abundant than has been estimated by morphological methods. Moreover, operational taxonomic units, also found outside Antarctica, were more widespread over the continent than potential endemics. The cyanobacterial diversity of the most saline lakes was found to differ from the others, and correlations between the sampling depth and the cyanobacterial communities can also be drawn. Comparison with database sequences illustrated the ubiquity of several cyanobacterial operational taxonomic units and their remarkable range of tolerance to harsh environmental conditions.

1. Lakes and ponds in the Larsemann Hills and Bølingen Islands (East-Antarctica) were characterised by cyanobacteria-dominated, benthic microbial mats. A 56-lake dataset representing the limnological diversity among the more than 150 lakes and ponds in the region was developed to identify and quantify the abiotic conditions associated with cyanobacterial and diatom communities. 2. Limnological diversity in the lakes of the Larsemann Hills and Bølingen Islands was associated primarily with conductivity and conductivity-related variables (concentrations of major ions and alkalinity), and variation in lake morphometry (depth, catchment and lake area). Low concentrations of pigments, phosphate, nitrogen, DOC and TOC in the water column of most lakes suggest extremely low water column productivity and hence high water clarity, and may thus contribute to the ecological success of benthic microbial mats in this region. 3. Benthic communities consisted of prostrate and sometimes finely laminated mats, flake mats, epilithic and interstitial microbial mats. Mat physiognomy and carotenoid/chlorophyll ratios were strongly related to lake depth, but not to conductivity. 4. Morphological-taxonomic analyses revealed the presence of 26 diatom morphospecies and 33 cyanobacterial morphotypes. Mats of shallow lakes (interstitial and flake mats) and those of deeper lakes (prostrate mats) were characterised by different dominant cyanobacterial morphotypes. No relationship was found between the distribution of these morphotypes and conductivity. In contrast, variation in diatom species composition was strongly related to both lake depth and conductivity. Shallow ponds were mainly characterised by aerial diatoms (e.g. Diadesmis cf. perpusilla and Hantzschia spp.). In deep lakes, communities were dominated by Psammothidium abundans and Stauroforma inermis. Lakes with conductivities higher than ±1.5 mS cm-1 became susceptible to freezing out of salts and hence pronounced conductivity fluctuations. In these lakes P. abundans and S. inermis were replaced by Amphora veneta. Stomatocysts were important only in shallow freshwater lakes. 5. Ice cover influenced microbial mat structure and composition both directly by physical disturbance in shallow lakes and by influencing light availability in deeper lakes, as well as indirectly by generating conductivity increases and promoting the development of seasonal anoxia. 6. The relationships between diatom species composition and conductivity, and diatom species composition and depth, were statistically significant. Transfer functions based on these data can therefore be used in paleolimnological reconstruction to infer changes in the precipitation-evaporation balance in continental Antarctic lakes.

To compile reference data for palaeolimnological studies using fossil pigments, we examined the extent to which environmental variables, gross morphology and species composition influence the modern pigment content of in situ microbial communities in 62 east Antarctic lakes. Pigment contents, measured using HPLC, were compared with 32 environmental variables, gross microbial mat morphology and cyanobacterial species composition in each lake. Results showed low concentrations or an absence of pigments in the water columns of most lakes. For benthic microbial communities, multivariate statistical analyses identified lake depth as the most important factor explaining pigment composition. In deeper lakes the pigment composition was dominated by chloro- phylls, in intermediate depth lakes by chlorophylls and carotenoids, and in shallow lakes by scytone- mins, ultraviolet-screening pigments found in cyanobacteria. In addition to lake depth, conductivity, turbidity, dissolved oxygen, sulphate and geographical location were all significant (p ≤ 0.05) in ex- plaining variance in the pigment content. Significant differences in microbial mat gross morphologies occurred at different lake depths (p ≤ 0.01), and were characterised by significant differences in their pigment content (p ≤ 0.004). Despite the high abundance of scytonemin in shallow lakes, there were only limited changes in the absolute concentrations of chlorophylls and carotenoids. We conclude that lake depth is the most significant factor influencing both gross mat morphology and pigment content, presumably as a result of its influence on the light climate. In general, the ability of the cyanobacteria to regulate their pigment content, morphology, community composition and motility to best exploit the light environment at different lake depths may explain their dominance in these systems.

Currently, there is no consensus concerning the geographic distribution and extent of endemism in Antarctic cyanobacteria. In this paper we describe the phenotypic and genotypic diversity of cyanobacteria in a field microbial mat sample from Lake Fryxell and in an artificial cold-adapted sample cultured in a benthic gradient chamber (BGC) by using an inoculum from the same mat. Light microscopy and molecular tools, including 16S rRNA gene clone libraries, denaturing gradient gel electrophoresis, and sequencing, were used. For the first time in the study of cyanobacterial diversity of environmental samples, internal transcribed spacer (ITS) sequences were retrieved and analyzed to complement the information obtained from the 16S rRNA gene. Microscopy allowed eight morphotypes to be identified, only one of which is likely to be an Antarctic endemic morphotype. Molecular analysis, however, revealed an entirely different pattern. A much higher number of phylotypes (15 phylotypes) was found, but no sequences from Nodularia and Hydrocoryne , as observed by microscopy, were retrieved. The 16S rRNA gene sequences determined in this study were distributed in 11 phylogenetic lineages, 3 of which were exclusively Antarctic and 2 of which were novel. Collectively, these Antarctic sequences together with all the other polar sequences were distributed in 22 lineages, 9 of which were exclusively Antarctic, including the 2 novel lineages observed in this study. The cultured BGC mat had lower diversity than the field mat. However, the two samples shared three morphotypes and three phylotypes. Moreover, the BGC mat allowed enrichment of one additional phylotype. ITS sequence analysis revealed a complex signal that was difficult to interpret. Finally, this study provided evidence of molecular diversity of cyanobacteria in Antarctica that is much greater than the diversity currently known based on traditional microscopic analysis. Furthermore, Antarctic endemic species were more abundant than was estimated on the basis of morphological features. Decisive arguments concerning the global geographic distribution of cyanobacteria should therefore incorporate data obtained with the molecular tools described here.

During several expeditions to Papua New Guinea (1986-1997), fragments of carbonate substrates (especially coral skeletons) were sampled in the intertidal as well as in the infralittoral zones. Their endolithic blue-green algal flora was studied by classical light microscopical methods. A total of seventeen endolithic blue-green algal taxa belonging to the genera Hapalosiphon, Herpyzonema, Hornwthonema, Hyella, Kyrtuthrix, Leptolyngbya, Lithonema, Mastigocoleus, and Solentia could be distinguished. In the intertidal zone, exposed during low tides to drying and rain, Herpyzonema intermedium, Hormathonema violaceonigrum, H. luteabrimneum, Hyella spp., Kyrtuthrix dalmatica, Mastigocoleus testarum, and Solentia spp. were frequently found. The true-branching Lithonema adriaticum was frequently observed in the intertidal zone, but always in low abundance. In the upper intertidal zone, besides Herpyzonema intermedium, a second stigonematalean endolithic blue-green alga was occasionally observed; it probably represents a new species tentatively placed in the genus Hapalosiphon. Although not quantitatively of great importance, the greatest diversity occurred in the genus Hyella with six distinct taxa, two of which may represent new species. In the infralittoral zone, endolithic blue-green algae were less diverse (mainly Leptolyngbya terebrans and Mastigocoleus testarum) and they were often accompanied by endolithic green algae (especially Ostreobium sp.), especially inside living corals.

© The Author(s) 2014. Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broadhost-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

Current cyanobacterial model organisms were not selected for their growth traits or potential for the production of renewable biomass, biofuels, or other products. The cyanobacterium strain BL0902 emerged from a search for strains with superior growth traits. Morphology and 16S rRNA sequence placed strain BL0902 in the genus Leptolyngbya. Leptolyngbya sp. strain BL0902 (hereafter Leptolyngbya BL0902) showed robust growth at temperatures from 22°C to 40°C and tolerated up to 0.5 M NaCl, 32 mM urea, high pH, and high solar irradiance. Its growth rate under outdoor conditions rivaled Arthrospira ("pirulina" strains. Leptolyngbya BL0902 accumulated higher lipid content and a higher proportion of monounsaturated fatty acids than Arthrospira strains. In addition to these desirable qualities, Leptolyngbya BL0902 is amenable to genetic engineering that is reliable, efficient, and stable. We demonstrated conjugal transfer from Escherichia coli of a plasmid based on RSF1010 and expression of spectinomycin/streptomycin resistance and yemGFP reporter transgenes. Conjugation efficiency was investigated in biparental and triparental matings with and without a "elper"plasmid that carries DNA methyltransferase genes, and with two different conjugal plasmids. We also showed that Leptolyngbya BL0902 is amenable to transposon mutagenesis with a Tn5 derivative. To facilitate genetic manipulation of Leptolyngbya BL0902, a conjugal plasmid vector was engineered to carry a trc promoter upstream of a Gateway recombination cassette. These growth properties and genetic tools position Leptolyngbya BL0902 as a model cyanobacterial production strain. © 2012 Taton et al.

Many genes related to the circadian clock have been discovered and studied in Synechococcus elongatus PCC 7942, the model organism for cyanobacterial circadian rhythms. However, the partners of some known clock components are still unidentified, and undiscovered pathways are predicted to exist that connect the central clock to other cellular functions. Identification of all clock components in S. elongatus is necessary for fully elucidating molecular mechanisms of the cyanobacterial circadian clock, as well as the relationship of the circadian clock to metabolism and other essential cellular activities. We adopted a transposon-mediated in vitro mutagenesis and sequencing strategy to disrupt essentially every locus in the genome and screen each insertional mutant for altered circadian phenotypes in S. elongatus. The completion of the genome sequence by the Department of Energy Joint Genome Institute greatly facilitated our functional genomics project, which is very close to the finish line with 88% of the genome mutagenized and more than 75% of loci screened for circadian function. Among the first 700 genes surveyed, 70 new clock loci were discovered that represent an array of functional categories.

BioBIKE (biobike.csbc.vcu.edu) is a web-based environment enabling biologists with little programming expertise to combine tools, data, and knowledge in novel and possibly complex ways, as demanded by the biological problem at hand. BioBIKE is composed of three integrated components: a biological knowledge base, a graphical programming interface and an extensible set of tools. Each of the five current BioBIKE instances provides all available information (genomic, metabolic, experimental) appropriate to a given research community. The BioBIKE programming language and graphical programming interface employ familiar operations to help users combine functions and information to conduct biologically meaningful analyses. Many commonly used tools, such as Blast and PHYLIP, are built-in, allowing users to access them within the same interface and to pass results from one to another. Users may also invent their own tools, packaging complex expressions under a single name, which is immediately made accessible through the graphical interface. BioBIKE represents a partial solution to the difficult question of how to enable those with no background in computer programming to work directly and creatively with mass biological information. BioBIKE is distributed under the MIT Open Source license. A description of the underlying language and other technical matters is available at www.Biobike.org.

The filamentous cyanobacterium Nostoc sp. strain PCC 7120 is capable of fixing atmospheric nitrogen. The labile nature of the core process requires the terminal differentiation of vegetative cells to form heterocysts, specialized cells with altered cellular and metabolic infrastructure to mediate the N2-fixing process. We present an investigation targeting the cellular proteomic expression of the heterocysts compared to vegetative cells of a population cultured under N2-fixing conditions. New 8-plex iTRAQ reagents were used on enriched replicate heterocyst and vegetative cells, and replicate N2-fixing and non-N2-fixing filaments to achieve accurate measurements. With this approach, we successfully identified 506 proteins, where 402 had confident quantifications. Observations provided by purified heterocyst analysis enabled the elucidation of the dominant metabolic processes between the respective cell types, while emphasis on the filaments enabled an overall comparison. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production.

Aims: To exploit the cyanobacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes for the discovery of novel antibiotic and antitumour activities. Methods and results: In all, 51 Antarctic cyanobacteria isolated from benthic mats were cultivated in the laboratory by optimizing temperature, irradiance and mixing. Productivity was generally very low (

Perennially cold environments in which temperatures remain below 5°C are common throughout the biosphere (Margesin and Haggblom 2007). In these habitats, the persistent cold temperatures are often accompanied by freeze–thaw cycles, extreme fluctuations in irradiance (including ultraviolet radiation), and large variations in nutrient supply and salinity. As a result of these constraints, polar and alpine environments

For the first time, the cyanobacterial diversity from microbial mats in lakes of Eastern Antarctica was investigated using microscopic and molecular approaches. The present study assessed the biogeographical distribution of cyanobacteria in Antarctica. Five samples were taken from four lakes spanning a range of different ecological environments in Larsemann Hills, Vestfold Hills and Rauer Islands to evaluate the influence of lake characteristics on the cyanobacterial diversity. Seventeen morphospecies and 28 16S rRNA gene-based operational taxonomic units belonging to the Oscillatoriales, Nostocales and Chroococcales were identified. The internal transcribed spacer was evaluated to complement the 16S rRNA gene data and showed similar but more clear-cut tendencies. The molecular approach suggested that potential Antarctic endemic species, including a previously undiscovered diversity, are more abundant than has been estimated by morphological methods. Moreover, operational taxonomic units, also found outside Antarctica, were more widespread over the continent than potential endemics. The cyanobacterial diversity of the most saline lakes was found to differ from the others, and correlations between the sampling depth and the cyanobacterial communities can also be drawn. Comparison with database sequences illustrated the ubiquity of several cyanobacterial operational taxonomic units and their remarkable range of tolerance to harsh environmental conditions.

Background Understanding the history of life requires that we understand the transfer of genetic material across phylogenetic boundaries. Detecting genes that were acquired by means other than vertical descent is a basic step in that process. Detection by discordant phylogenies is computationally expensive and not always definitive. Many have used easily computed compositional features as an alternative procedure. However, different compositional methods produce different predictions, and the effectiveness of any method is not well established. Results The ability of octamer frequency comparisons to detect genes artificially seeded in cyanobacterial genomes was markedly increased by using as a training set those genes that are highly conserved over all bacteria. Using a subset of octamer frequencies in such tests also increased effectiveness, but this depended on the specific target genome and the source of the contaminating genes. The presence of high frequency octamers and the GC content of the contaminating genes were important considerations. A method comprising best practices from these tests was devised, the Core Gene Similarity (CGS) method, and it performed better than simple octamer frequency analysis, codon bias, or GC contrasts in detecting seeded genes or naturally occurring transposons. From a comparison of predictions with phylogenetic trees, it appears that the effectiveness of the method is confined to horizontal transfer events that have occurred recently in evolutionary time. Conclusions The CGS method may be an improvement over existing surrogate methods to detect genes of foreign origin.