Synechococcus elongatus Argonaute reduces natural transformation efficiency and provides immunity against exogenous plasmids.

IMPORTANCE: S. elongatus is an important cyanobacterial model organism for the study of its prokaryotic circadian clock, photosynthesis, and other biological processes. It is also widely used for genetic engineering to produce renewable biochemicals. Our findings reveal an SeAgo-based defense mechanism in S. elongatus against the horizontal transfer of genetic material. We demonstrate that deletion of the ago gene facilitates genetic studies and genetic engineering of S. elongatus.

Effects of RuBisCO and CO2 concentration on cyanobacterial growth and carbon isotope fractionation.

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 specific range of genetic and environmental factors that may impact ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO across varying atmospheric CO2 concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO2 fixation versus CO2 supply, and thus whole-cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO2 concentrations on both carbon isotope biosignatures and cyanobacterial physiology, including cell growth and oxygen evolution rates. We found that an increased pool of active RuBisCO does not significantly affect the 13 C/12 C isotopic discrimination (εp ) at all tested CO2 concentrations, yielding εp of ≈ 23‰ for both wild-type and mutant strains at elevated CO2 . We therefore suggest that expected variation in cyanobacterial RuBisCO expression patterns should not confound carbon isotope biosignature interpretation. A deeper understanding of environmental, evolutionary, and intracellular factors that impact cyanobacterial physiology and isotope discrimination is crucial for reconciling microbially driven carbon biosignatures with those preserved in the geologic record.

An unexpected role for leucyl aminopeptidase in UV tolerance revealed by a genome-wide fitness assessment in a model cyanobacterium.

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.

Heterologous Expression in Anabaena of the Columbamide Pathway from the Cyanobacterium Moorena bouillonii and Production of New Analogs.

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 Moorena bouillonii PNG5-198 collected from the coastal waters of Papua New Guinea. The columbamide biosynthetic gene cluster (BGC) had been identified using bioinformatics, but not confirmed by experimental evidence. Here, we report the heterologous expression in Anabaena (Nostoc) PCC 7120 of the 28.5 kb BGC that encodes for columbamide biosynthesis. The production of columbamides in Anabaena is investigated under several different culture conditions, and several new columbamide analogs are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition to previously characterized columbamides A, B, and C, new columbamides I-M are produced in these experiments, and the structure of the most abundant monochlorinated analog, columbamide K (11), is fully characterized. The other new columbamide analogs are produced in only small quantities, and structures are proposed based on high-resolution-MS, MS/MS, and 1H NMR data. Overexpression of the pathway's predicted halogenases resulted in increased productions of di- and trichlorinated compounds. The most significant change in production of columbamides in Anabaena is correlated with the concentration of NaCl in the medium.

Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time.

The earliest geochemical indicators of microbes-and the enzymes that powered them-extend back ∼3.8 Ga on Earth. Paleobiologists often attempt to understand these indicators by assuming that the behaviors of extant microbes and enzymes are uniform with those of their predecessors. This consistency in behavior seems at odds with our understanding of the inherent variability of living systems. Here, we examine whether a uniformitarian assumption for an enzyme thought to generate carbon isotope indicators of biological activity, RuBisCO, can be corroborated by independently studying the history of changes recorded within RuBisCO's genetic sequences. We resurrected a Precambrian-age RuBisCO by engineering its ancient DNA inside a cyanobacterium genome and measured the engineered organism's fitness and carbon-isotope-discrimination profile. Results indicate that Precambrian uniformitarian assumptions may be warranted but with important caveats. Experimental studies illuminating early innovations are crucial to explore the molecular foundations of life's earliest traces.

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug 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.

The circadian clock and darkness control natural competence in cyanobacteria.

The cyanobacterium Synechococcus elongatus is a model organism for the study of circadian rhythms. It is naturally competent for transformation-that is, it takes up DNA from the environment, but the underlying mechanisms are unclear. Here, we use a genome-wide screen to identify genes required for natural transformation in S. elongatus, including genes encoding a conserved Type IV pilus, genes known to be associated with competence in other bacteria, and others. Pilus biogenesis occurs daily in the morning, while natural transformation is maximal when the onset of darkness coincides with the dusk circadian peak. Thus, the competence state in cyanobacteria is regulated by the circadian clock and can adapt to seasonal changes of day length.

Expression of Heterologous OsDHAR Gene Improves Glutathione (GSH)-Dependent Antioxidant System and Maintenance of Cellular Redox Status in Synechococcus elongatus PCC 7942.

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 ß-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.

Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting.

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.

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus.

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, PixJSe (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 PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

Development of a cyanobacterial heterologous polyketide production platform.

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.

High-throughput interaction screens illuminate the role of c-di-AMP in cyanobacterial nighttime survival.

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.

Expression of OsTPX Gene Improves Cellular Redox Homeostasis and Photosynthesis Efficiency in Synechococcus elongatus PCC 7942.

Cyanobacterial 2-Cys peroxiredoxin (thioredoxin peroxidase, TPX) comprises a family of thiol antioxidant enzymes critically involved in cell survival under oxidative stress. In our previous study, a putative TPX was identified using a proteomics analysis of rice (Oryza sativa L. japonica, OsTPX) seedlings exposed to oxidative stress. This OsTPX gene is structurally similar to the Synechococcus elongatus TPX gene in the highly conserved redox-active disulfide bridge (Cys114, Cys236) and other highly conserved regions. In the present study, the OsTPX gene was cloned into rice plants and S. elongatus PCC 7942 strain to study hydrogen peroxide (H2O2) stress responses. The OsTPX gene expression was confirmed using semi-quantitative RT-PCR and western blot analysis. The OsTPX gene expression increased growth under oxidative stress by decreasing reactive oxygen species and malondialdehyde level. Additionally, the OsTPX gene expression in S. elongatus PCC 7942 (OT) strain exhibited a reduced loss of chlorophyll and enhanced photosynthesis efficiency under H2O2 stress, thereby increasing biomass yields twofold compared with that of the control wild type (WT) strain. Furthermore, redox balance, ion homeostasis, molecular chaperone, and photosynthetic systems showed upregulation of some genes in the OT strain than in the WT strain by RNA-Seq analysis. Thus, OsTPX gene expression enhances oxidative stress tolerance by increasing cell defense regulatory networks through the cellular redox homeostasis in the rice plants and S. elongatus PCC 7942.

NOT Gate Genetic Circuits to Control Gene Expression in Cyanobacteria.

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.

Enhanced biomass and oxidative stress tolerance of Synechococcus elongatus PCC 7942 overexpressing the DHAR gene from Brassica juncea.

OBJECTIVES: 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). RESULTS: 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. CONCLUSION: 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.

Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges.

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.

Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942.

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 co-existed 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.

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria.

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 broad-host-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.

Detection of horizontal transfer of individual genes by anomalous oligomer frequencies.

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.

Gene transfer in Leptolyngbya sp. strain BL0902, a cyanobacterium suitable for production of biomass and bioproducts.

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.