Synechococcus Strains Research Articles

Enhancing the Biodiesel Production Potential of Synechococcus elongatus and Anabaena Cyanobacterial Strain Isolated from Saline Water Using Different Media Composition and Organic Carbon Sources

In the present study, Synechococcus elongatus and Anabaena, two cyanobacterial species were cultured using different media conditions such as ASN III, modified ASN III, BG-11, and BBM for the enrichment of biomass and lipid productivity. The experimental result clearly shows that BG 11 was the efficient and cost-effective medium for both the isolated cyanobacterial species such as Synechococcus elongatus and Anabaena. The influence of organic carbon sources on biomass and lipid productivity of the selected cyanobacterial species were studied when cultivated in a BG-11 medium using different organic carbon sources such as sucrose, glucose, sodium acetate and glycerol under mixotrophic conditions. Based on the experimental results, the isolated cyanobacterial strain Synechococcus elongatus and Anabaena showed an enriching effect on lipid production under mixotrophic conditions, but whereas Synechococcus elongatus showed a significant effect three times greater lipid productivity compared with Anabaena cyanobacterial strain, by the addition of glycerol as a supplement to the culture media.

Investigations on Isolation and Characterisation of Unicellular Cyanobacteria in Uzungöl and Cernek Lakes, Samsun, Türkiye

Isolation of unicellular cyanobacteria as pure cultures were carried out from the water samples collected from Uzungöl and Cernek lakes in Kızılırmak delta, Bafra, Samsun. Diluted water samples were spread onto a solid BG11 medium which was positively selective for cyanobacteria and isolated single colonies were observed after incubation under continuous light. Twenty colonies composed of unicellular cells were selected as unicellular isolates. These single colonies from each isolates were transferred into a liquid BG11 medium to grow as liquid cultures under continuous light. Characteristics of the cells in liquid cultures of each isolate were used to ıdentify subsection and form-genus of Cyanobacteria they might be included. The results showed that all the isolates were settled in Subsection 1 and form-genus Synechococcus. To support this identification, genomic DNAs were isolated from each of the isolates, 16S rRNA gene regions were amplified, and each amplicon was digested with EcoRI. Restriction fragment lengths were found to be consistent with those of the standard cyanobacterial strain Synechococcus sp. PCC7942. Since restriction fragment lengths and cultural characteristics were consistent, all the isolates were, therefore, evaluated to be included into Subsection 1 and form-genus Synechococcus.

Global Phylogeography of Marine Synechococcus in Coastal Areas Reveals Strong Community Shifts.

Marine Synechococcus comprise a numerically and ecologically prominent phytoplankton group, playing a major role in both carbon cycling and trophic networks in all oceanic regions except in the polar oceans. Despite their high abundance in coastal areas, our knowledge of Synechococcus communities in these environments is based on only a few local studies. Here, we use the global metagenome data set of the Ocean Sampling Day (June 21st, 2014) to get a snapshot of the taxonomic composition of coastal Synechococcus communities worldwide, by recruitment on a reference database of 141 picocyanobacterial genomes, representative of the whole Prochlorococcus, Synechococcus, and Cyanobium diversity. This allowed us to unravel drastic community shifts over small to medium scale gradients of environmental factors, in particular along European coasts. The combined analysis of the phylogeography of natural populations and the thermophysiological characterization of eight strains, representative of the four major Synechococcus lineages (clades I to IV), also brought novel insights about the differential niche partitioning of clades I and IV, which most often co-dominate the Synechococcus community in cold and temperate coastal areas. Altogether, this study reveals several important characteristics and specificities of the coastal communities of Synechococcus worldwide. IMPORTANCE Synechococcus is the second most abundant phytoplanktonic organism on Earth, and its wide genetic diversity allowed it to colonize all the oceans except for polar waters, with different clades colonizing distinct oceanic niches. In recent years, the use of global metagenomics data sets has greatly improved our knowledge of "who is where" by describing the distribution of Synechococcus clades or ecotypes in the open ocean. However, little is known about the global distribution of Synechococcus ecotypes in coastal areas, where Synechococcus is often the dominant phytoplanktonic organism. Here, we leverage the global Ocean Sampling Day metagenomics data set to describe Synechococcus community composition in coastal areas worldwide, revealing striking community shifts, in particular along the coasts of Europe. As temperature appears as an important driver of the community composition, we also characterize the thermal preferenda of 8 Synechococcus strains, bringing new insights into the adaptation to temperature of the dominant Synechococcus clades.

Diversity and Evolution of Iron Uptake Pathways in Marine Cyanobacteria from the Perspective of the Coastal Strain Synechococcus sp. Strain PCC 7002.

Marine cyanobacteria contribute to approximately half of the ocean primary production, and their biomass is limited by low iron (Fe) bioavailability in many regions of the open seas. The mechanisms by which marine cyanobacteria overcome Fe limitation remain unclear. In this study, multiple Fe uptake pathways have been identified in a coastal strain of Synechococcus sp. strain PCC 7002. A total of 49 mutants were obtained by gene knockout methods, and 10 mutants were found to have significantly decreased growth rates compared to the wild type (WT). The genes related to active Fe transport pathways such as TonB-dependent transporters and the synthesis and secretion of siderophores are found to be essential for the adaptation of Fe limitation in Synechococcus sp. PCC 7002. By comparing the Fe uptake pathways of this coastal strain with other open-ocean cyanobacterial strains, it can be concluded that the Fe uptake strategies from different cyanobacteria have a strong relationship with the Fe bioavailability in their habitats. The evolution and adaptation of cyanobacterial iron acquisition strategies with the change of iron environments from ancient oceans to modern oceans are discussed. This study provides new insights into the diversified strategies of marine cyanobacteria in different habitats from temporal and spatial scales. IMPORTANCE Iron (Fe) is an important limiting factor of marine primary productivity. Cyanobacteria, the oldest photosynthetic oxygen-evolving organisms on the earth, play crucial roles in marine primary productivity, especially in the oligotrophic ocean. How they overcome Fe limitation during the long-term evolution process has not been fully revealed. Fe uptake mechanisms of cyanobacteria have been partially studied in freshwater cyanobacteria but are largely unknown in marine cyanobacterial species. In this paper, the characteristics of Fe uptake mechanisms in a coastal model cyanobacterium, Synechococcus sp. PCC 7002, were studied. Furthermore, the relationship between Fe uptake strategies and Fe environments of cyanobacterial habitats has been revealed from temporal and spatial scales, which provides a good case for marine microorganisms adapting to changes in the marine environment.

New Strain of Cyphellophora olivacea Exhibits Striking Tolerance to Sodium Bicarbonate

The cyanobacterium strain Synechococcus cedrorum SAG 88.79 stock culture has fungal contamination stated by the Sammlung von Algenkulturen der Universität Göttingen itself. In this recent work, this particular fungal strain was isolated, identified, and morphologically characterised. The fungal strain AGSC12 belongs to the species Cyphellophora olivacea, with respect to the sequence similarity, phylogeny, and morphology of the strain. Colony morphology and growth capability were examined on SMA, EMMA, PDA, MEA, YEA, and YPA plates. Growth of the colonies was the most successful on YPA plates, followed by PDA and MEA containing plates. Surprisingly, the AGSC12 strain showed extreme tolerance to NaHCO3, albeit it, is is considered a general fungistatic compound. Moreover, positive association between the AGSC12 and SAG 88.79 strains was revealed, as the SAG 88.79 strain always attained higher cell density in co-cultures with the fungus than in mono-cultures. Besides, a taxonomic note on the SAG 88.79 strain itself was also stated.

Community context and pCO2 impact the transcriptome of the “helper” bacterium Alteromonas in co-culture with picocyanobacteria

Many microbial photoautotrophs depend on heterotrophic bacteria for accomplishing essential functions. Environmental changes, however, could alter or eliminate such interactions. We investigated the effects of changing pCO2 on gene transcription in co-cultures of 3 strains of picocyanobacteria (Synechococcus strains CC9311 and WH8102 and Prochlorococcus strain MIT9312) paired with the ‘helper’ bacterium Alteromonas macleodii EZ55. Co-culture with cyanobacteria resulted in a much higher number of up- and down-regulated genes in EZ55 than pCO2 by itself. Pathway analysis revealed significantly different transcription of genes involved in carbohydrate metabolism, stress response, and chemotaxis, with different patterns of up- or down-regulation in co-culture with different cyanobacterial strains. Gene transcription patterns of organic and inorganic nutrient transporter and catabolism genes in EZ55 suggested resources available in the culture media were altered under elevated (800 ppm) pCO2 conditions. Altogether, changing transcription patterns were consistent with the possibility that the composition of cyanobacterial excretions changed under the two pCO2 regimes, causing extensive ecophysiological changes in both members of the co-cultures. Additionally, significant downregulation of oxidative stress genes in MIT9312/EZ55 cocultures at 800 ppm pCO2 were consistent with a link between the predicted reduced availability of photorespiratory byproducts (i.e., glycolate/2PG) under this condition and observed reductions in internal oxidative stress loads for EZ55, providing a possible explanation for the previously observed lack of “help” provided by EZ55 to MIT9312 under elevated pCO2. If similar broad alterations in microbial ecophysiology occur in the ocean as atmospheric pCO2 increases, they could lead to substantially altered ecosystem functioning and community composition.

The phycoerythrobilin isomerization activity of MpeV in Synechococcus sp. WH8020 is prevented by the presence of a histidine at position 141 within its phycoerythrin-I β-subunit substrate.

Marine Synechococcus efficiently harvest available light for photosynthesis using complex antenna systems, called phycobilisomes, composed of an allophycocyanin core surrounded by rods, which in the open ocean are always constituted of phycocyanin and two phycoerythrin (PE) types: PEI and PEII. These cyanobacteria display a wide pigment diversity primarily resulting from differences in the ratio of the two chromophores bound to PEs, the green-light absorbing phycoerythrobilin and the blue-light absorbing phycourobilin. Prior to phycobiliprotein assembly, bilin lyases post-translationally catalyze the ligation of phycoerythrobilin to conserved cysteine residues on α- or β-subunits, whereas the closely related lyase-isomerases isomerize phycoerythrobilin to phycourobilin during the attachment reaction. MpeV was recently shown in Synechococcus sp. RS9916 to be a lyase-isomerase which doubly links phycourobilin to two cysteine residues (C50 and C61; hereafter C50, 61) on the β-subunit of both PEI and PEII. Here we show that Synechococcus sp. WH8020, which belongs to the same pigment type as RS9916, contains MpeV that demonstrates lyase-isomerase activity on the PEII β-subunit but only lyase activity on the PEI β-subunit. We also demonstrate that occurrence of a histidine at position 141 of the PEI β-subunit from WH8020, instead of a leucine in its counterpart from RS9916, prevents the isomerization activity by WH8020 MpeV, showing for the first time that both the substrate and the enzyme play a role in the isomerization reaction. We propose a structural-based mechanism for the role of H141 in blocking isomerization. More generally, the knowledge of the amino acid present at position 141 of the β-subunits may be used to predict which phycobilin is bound at C50, 61 of both PEI and PEII from marine Synechococcus strains.

Genomic analysis and biochemical profiling of an unaxenic strain of Synechococcus sp. isolated from the Peruvian Amazon Basin region.

Cyanobacteria are diverse photosynthetic microorganisms able to produce a myriad of bioactive chemicals. To make possible the rational exploitation of these microorganisms, it is fundamental to know their metabolic capabilities and to have genomic resources. In this context, the main objective of this research was to determine the genome features and the biochemical profile of Synechococcus sp. UCP002. The cyanobacterium was isolated from the Peruvian Amazon Basin region and cultured in BG-11 medium. Growth parameters, genome features, and the biochemical profile of the cyanobacterium were determined using standardized methods. Synechococcus sp. UCP002 had a specific growth rate of 0.086 ± 0.008μ and a doubling time of 8.08 ± 0.78h. The complete genome of Synechococcus sp. UCP002 had a size of ∼3.53Mb with a high coverage (∼200x), and its quality parameters were acceptable (completeness = 99.29%, complete and single-copy genes = 97.5%, and contamination = 0.35%). Additionally, the cyanobacterium had six plasmids ranging from 24 to 200kbp. The annotated genome revealed ∼3,422 genes, ∼ 3,374 protein-coding genes (with ∼41.31% hypothetical protein-coding genes), two CRISPR Cas systems, and 61 non-coding RNAs. Both the genome and plasmids had the genes for prokaryotic defense systems. Additionally, the genome had genes coding the transcription factors of the metalloregulator ArsR/SmtB family, involved in sensing heavy metal pollution. The biochemical profile showed primary nutrients, essential amino acids, some essential fatty acids, pigments (e.g., all-trans-β-carotene, chlorophyll a, and phycocyanin), and phenolic compounds. In conclusion, Synechococcus sp. UCP002 shows biotechnological potential to produce human and animal nutrients and raw materials for biofuels and could be a new source of genes for synthetic biological applications.

A Unique Set of Auxiliary Metabolic Genes Found in an Isolated Cyanophage Sheds New Light on Marine Phage-Host Interactions.

ABSTRACTCyanophages, viruses that infect cyanobacteria, are abundant and widely distributed in aquatic ecosystems, playing important roles in regulating the abundance, activity, diversity, and evolution of cyanobacteria. A T4-like cyanophage, S-SCSM1, infecting Synechococcus and Prochlorococcus strains of different ecotypes, was isolated from the South China Sea in this study. For the first time, a mannose-6-phosphate isomerase (MPI) gene was identified in the cultured cyanophage. At least 11 phylogenetic clusters of cyanophage MPIs were retrieved and identified from the marine metagenomic data sets, indicating that cyanophage MPIs in the marine environment are extremely diverse. The existence of 24 genes encoding 2-oxoglutarate (2OG)-Fe(II) oxygenase superfamily proteins in the S-SCSM1 genome emphasizes their potential importance and diverse functions in reprogramming host metabolism during phage infection. Novel cell wall synthesis and modification genes found in the S-SCSM1 genome indicate that diverse phenotypic modifications imposed by phages on cyanobacterial hosts remain to be discovered. Two noncoding RNAs of cis-regulatory elements in the S-SCSM1 genome were predicted to be associated with host exopolysaccharide metabolism and photosynthesis. The isolation and genomic characterization of cyanophage S-SCSM1 provide more information on the genetic diversity of cyanophages and phage-host interactions in the marine environment.IMPORTANCE Cyanophages play important ecological roles in aquatic ecosystems. Genomic and proteomic characterizations of the T4-like cyanophage S-SCSM1 indicate that novel and diverse viral genes and phage-host interactions in the marine environment remain unexplored. The first identified mannose-6-phosphate isomerase (MPI) gene from a cultured cyanophage was found in the S-SCSM1 genome, although MPIs were previously found in viral metagenomes at high frequencies similar to those of the cyanophage photosynthetic gene psbA. The presence of 24 genes encoding 2-oxoglutarate (2OG)-Fe(II) oxygenase superfamily proteins, novel cell wall synthesis and modification genes, a nonbleaching protein A gene, and 2 noncoding RNAs of cis-regulatory elements in the S-SCSM1 genome as well as the presence of a virion-associated regulatory protein indicate the diverse functions that cyanophages have in reprogramming the metabolism and modifying the phenotypes of hosts during infection.

Functional flexibility of cyanobacterial light harvesting phycobilisomes enable acclimation to the complex light regime of mixing marine water columns.

The light environment in a mixing water column is arguably the most erratic condition under which photosynthesis functions. Shifts in light intensity, by an order of magnitude, can occur over the time scale of hours. In marine Synechococcus, light is harvested by massive, membrane attached, phycobilisome chromophore-protein complexes (PBS). We examined the ability of a phycobilisome-containing marine Synechococcus strain (WH8102) to acclimate to illumination perturbations on this scale. Although changes in pigment composition occurred gradually over the course of days, we did observe significant and reversible changes in the pigment's fluorescence emission spectra on a time scale of hours. Upon transition to ten-fold higher intensities, we observed a decrease in the energy transferred to Photosystem II. At the same time, the spectral composition of PBS fluorescence emission shifted. Unlike fluorescence quenching mechanisms, this phenomenon resulted in increased fluorescence intensities. These data suggest a mechanism by which marine Synechococcus WH8102 detaches hexamers from the phycobilisome structure. The fluorescence yield of these uncoupled hexamers is high. The detachment process does not require protein synthesis as opposed to reattachment. Hence, the most likely process would be the degradation and resynthesis of labile PBS linker proteins. Experiments with additional species yielded similar results, suggesting that this novel mechanism might be broadly used among PBS-containing organisms.

Effect of Rising Temperature and Carbon Dioxide on the Growth, Photophysiology, and Elemental Ratios of Marine Synechococcus: A Multistressor Approach

Marine picocyanobacteria belonging to the genus Synechococcus are one of the most abundant photosynthetic organisms on Earth. They are often exposed to large fluctuations in temperature and CO2 concentrations in the ocean, which are expected to further change in the coming decades due to ocean acidification and warming resulting from rising atmospheric CO2 levels. To decipher the effect of changing temperature and CO2 levels on Synechococcus, six Synechococcus strains previously isolated from various coastal and open ocean sites were exposed to a matrix of three different temperatures (22 °C, 24 °C and 26 °C) and CO2 levels (400 ppm, 600 ppm and 800 ppm). Thereafter, the specific growth rates, photophysiological parameters (σPSII and Fv/Fm), C/N (mol/mol) ratios and the nitrogen stable isotopic composition (δ15N (‰)) of the strains were measured. Temperature was found to be a stronger driver of the changes in specific growth rates and photophysiology in the Synechococcus strains. Carbon-concentrating mechanisms (CCM) operational in these strains that shield the photosynthetic machinery from directly sensing ambient changes in CO2 possibly played a major role in causing minimal changes in the specific growth rates under the varying CO2 levels.

Bioavailable iron titrations reveal oceanic Synechococcus ecotypes optimized for different iron availabilities

The trace metal iron (Fe) controls the diversity and activity of phytoplankton across the surface oceans, a paradigm established through decades of in situ and mesocosm experimental studies. Despite widespread Fe-limitation within high-nutrient, low chlorophyll (HNLC) waters, significant contributions of the cyanobacterium Synechococcus to the phytoplankton stock can be found. Correlations among differing strains of Synechococcus across different Fe-regimes have suggested the existence of Fe-adapted ecotypes. However, experimental evidence of high- versus low-Fe adapted strains of Synechococcus is lacking, and so we investigated the transcriptional responses of microbial communities inhabiting the HNLC, sub-Antarctic region of the Southern Ocean during the Spring of 2018. Analysis of metatranscriptomes generated from on-deck incubation experiments reflecting a gradient of Fe-availabilities reveal transcriptomic signatures indicative of co-occurring Synechococcus ecotypes adapted to differing Fe-regimes. Functional analyses comparing low-Fe and high-Fe conditions point to various Fe-acquisition mechanisms that may allow persistence of low-Fe adapted Synechococcus under Fe-limitation. Comparison of in situ surface conditions to the Fe-titrations indicate ecological relevance of these mechanisms as well as persistence of both putative ecotypes within this region. This Fe-titration approach, combined with transcriptomics, highlights the short-term responses of the in situ phytoplankton community to Fe-availability that are often overlooked by examining genomic content or bulk physiological responses alone. These findings expand our knowledge about how phytoplankton in HNLC Southern Ocean waters adapt and respond to changing Fe supply.

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

This study examines how a large saltwater barrage [Thannermukkom barrage (TB)] in the Kochi backwaters (KBW), India's largest estuarine system on the west coast, affects the distribution of autotrophic small plankton communities (Synechococcus, picoeukaryotes, and nanoplankton). The KBW has two distinct regions when the shutters of TB are closed during non-monsoon periods: a mesohaline to euhaline northern region downstream and a limnetic southern region upstream. The current seasonal salinity distribution in the KBW revealed a significant decrease compared to that before the establishment of TB. Considering this, we hypothesised that the human-induced hydrographical transformation associated with the functioning of TB has implications for the ecology of the smaller plankton components in the system, and to test this, smaller autotrophic plankton and hydrographical variables were collected from the KBW during three different seasons, including the Pre-Monsoon [(PRM) March–May], the Southwest Monsoon (SWM) from June to September, and the Northeast Monsoon (NEM) from November to February. Seasonally, 13 locations along the KBW were sampled, with locations 1–4 in the downstream, 5–9 in the midstream, and 10–13 in the upstream. The findings revealed that, regardless of season, a mesohaline condition in the KBW is the most conducive one that sustains an increased abundance of autotrophic pico-and nano-plankton communities. Two strains of Synechococcus (SYN) [phycoerythrin rich SYN I and phycocyanin rich SYN II] contribute nearly equally in the mesohaline region, whereas a predominance of SYN II (>90%) was found in oligohaline/limnohaline conditions. We show that TB reduces the geographic extent of the mesohaline zone in the KBW that is most conducive to maintaining a high abundance of smaller plankton communities, reducing the total abundance and biomass of the smaller plankton community in the system.

A mechanistic study of the influence of nitrogen and energy availability on the NH4+ sensitivity of nitrogen assimilation in Synechococcus.

In most algae, NO3- assimilation is tightly controlled and is often inhibited by the presence of NH4+. In the marine, non-colonial, non-diazotrophic cyanobacterium Synechococcus UTEX 2380, NO3- assimilation is sensitive to NH4+ only when N does not limit growth. We sequenced the genome of Synechococcus UTEX 2380, studied the genetic organization of the nitrate assimilation related (NAR) genes, and investigated expression and kinetics of the main NAR enzymes, under N or light limitation. We found that Synechococcus UTEX 2380 is a β-cyanobacterium with a full complement of N uptake and assimilation genes and NAR regulatory elements. The nitrate reductase of our strain showed biphasic kinetics, previously observed only in freshwater or soil diazotrophic Synechococcus strains. Nitrite reductase and glutamine synthetase showed little response to our growth treatments, and their activity was usually much higher than that of nitrate reductase. NH4+ insensitivity of NAR genes may be associated with the stimulation of the binding of the regulator NtcA to NAR gene promoters by the high 2-oxoglutarate concentrations produced under N limitation. NH4+ sensitivity in energy-limited cells fits with the fact that, under these conditions, the use of NH4+ rather than NO3- decreases N-assimilation cost, whereas it would exacerbate N shortage under N limitation.

Effect of Culture pH on Properties of Exopolymeric Substances from Synechococcus PCC7942: Implications for Carbonate Precipitation

The role of culture conditions on the production of exopolymeric substances (EPS) by Synechococcus strain PCC7942 was investigated. Carbonate mineral precipitation in these EPS was assessed in forced precipitation experiments. Cultures were grown in HEPES-buffered medium and non-buffered medium. The pH of buffered medium remained constant at 7.5, but in non-buffered medium it increased to 9.5 within a day and leveled off at 10.5. The cell yield at harvest was twice as high in non-buffered medium than in buffered medium. High molecular weight (>10 kDa) and low molecular weight (3–10 kDa) fractions of EPS were obtained from both cultures. The cell-specific EPS production in buffered medium was twice as high as in non-buffered medium. EPS from non-buffered cultures contained more negatively charged macromolecules and more proteins than EPS from buffered cultures. The higher protein content at elevated pH may be due to the induction of carbon-concentrating mechanisms, necessary to perform photosynthetic carbon fixation in these conditions. Forced precipitation showed smaller calcite carbonate crystals in EPS from non-buffered medium and larger minerals in polymers from buffered medium. Vaterite formed only at low EPS concentrations. Experimental results are used to conceptually model the impact of pH on the potential of cyanobacterial blooms to produce minerals. We hypothesize that in freshwater systems, small crystal production may benefit the picoplankton by minimizing the mineral ballast, and thus prolonging the residence time in the photic zone, which might result in slow sinking rates.

Using Droplet Digital PCR to Detect Cyanobacteria in Human Lung Tissue

Objective and HypothesisCyanobacteria produce a variety of secondary metabolites, including toxins, that are harmful to human and animal health. The objective of this study is to develop a quantitative method that can reliably detect cyanobacteria in human lung tissue. We propose that with cyanobacteria‐specific 16S rDNA primers, droplet digital PCR (ddPCR) can provide a reliable and sensitive method to detect Cyanobacteria in human tissue.MethodsTwo cyanobacteria strains were used in this validation: a toxin‐producing strain, Microcystis aeruginosa (LB 2385), and a non‐toxin‐producing, strain Synechococcus sp. (LB 2390). Escherichia coli served as a negative bacterial control. DNA was extracted from cultures and frozen, human lung tissue with DNeasy Blood & Tissue kit (QIAGEN) and quantified using the dsDNA High‐Sensitivity Assay (Invitrogen) on a Qubit 3.0 fluorometer. The ddPCR reactions consisted of FAM labelled cyanobacterial 16S rDNA target primer/probe and HEX labelled RPP30 reference primer/probe (Bio‐RAD). Oil droplets were generated using the Bio‐Rad Automated Droplet Generator and the emulsified PCRs were run in a 96‐well plate on a C1000 Touch™ Thermal Cycler. The plates were read on a Bio‐Rad QX200 droplet reader and the data analyzed using the QuantaSoft v1.7.4 software. The Limit of Detection (LOD) was found by initially diluting LB 2385 and LB 2390 to 4 ng/mcL in nuclease‐free water, followed by serial 2x dilutions until it was below the detection limit by ddPCR. This was repeated using normal human lung tissue instead of nuclease‐free water.ResultsThe ddPCR assay developed in this study showed high specificity and sensitivity in detecting both strains of cyanobacteria. Under both conditions tested (spiking in water vs 10 ng human DNA (hDNA)), LB 2385 DNA could be reliably detected at a concentration of 61 fg/mcL, whereas LB 2390 DNA could be reliably detected at a concentration of 31 fg/mcL. This corresponds to 1‐2 copies/mcL of cyanobacterial DNA in the original samples. In the presence of hDNA, the sensitivity for cyanobacterial DNA decreased as the concentration of hDNA increased (2‐30 ng/mcL), possibly due to a physical barrier or competition between the primer/probes. In the hDNA condition, RPP30 could be reliably detected at 2‐3 copies/mcL with a concentration of ~60 pg/mcL. RPP30 is valuable for normalizing sample input, reducing biases caused by DNA quantification variations and pipetting errors. A strong linear correlation between sample input and the number of positive droplets (i.e., the concentration detected by ddPCR) was observed for both cyanobacteria 16S rDNA and RPP30 DNA targets. This assay also demonstrated high intra‐ and inter‐assay reproducibility.ConclusionsA highly specific and sensitive ddPCR assay was developed to detect the presence of cyanobacteria in human lung tissue. The ability to detect cyanobacteria in human samples will allow further research into the role cyanobacteria plays in human health and disease.

Novel functional insights into a modified sugar-binding protein from Synechococcus MITS9220

Paradigms of metabolic strategies employed by photoautotrophic marine picocyanobacteria have been challenged in recent years. Based on genomic annotations, picocyanobacteria are predicted to assimilate organic nutrients via ATP-binding cassette importers, a process mediated by substrate-binding proteins. We report the functional characterisation of a modified sugar-binding protein, MsBP, from a marine Synechococcus strain, MITS9220. Ligand screening of MsBP shows a specific affinity for zinc (KD ~ 1.3 μM) and a preference for phosphate-modified sugars, such as fructose-1,6-biphosphate, in the presence of zinc (KD ~ 5.8 μM). Our crystal structures of apo MsBP (no zinc or substrate-bound) and Zn-MsBP (with zinc-bound) show that the presence of zinc induces structural differences, leading to a partially-closed substrate-binding cavity. The Zn-MsBP structure also sequesters several sulphate ions from the crystallisation condition, including two in the binding cleft, appropriately placed to mimic the orientation of adducts of a biphosphate hexose. Combined with a previously unseen positively charged binding cleft in our two structures and our binding affinity data, these observations highlight novel molecular variations on the sugar-binding SBP scaffold. Our findings lend further evidence to a proposed sugar acquisition mechanism in picocyanobacteria alluding to a mixotrophic strategy within these ubiquitous photosynthetic bacteria.

The "Dark Side" of Picocyanobacteria: Life as We Do Not Know It (Yet).

Picocyanobacteria of the genus Synechococcus (together with Cyanobium and Prochlorococcus) have captured the attention of microbial ecologists since their description in the 1970s. These pico-sized microorganisms are ubiquitous in aquatic environments and are known to be some of the most ancient and adaptable primary producers. Yet, it was only recently, and thanks to developments in molecular biology and in the understanding of gene sequences and genomes, that we could shed light on the depth of the connection between their evolution and the history of life on the planet. Here, we briefly review the current understanding of these small prokaryotic cells, from their physiological features to their role and dynamics in different aquatic environments, focussing particularly on the still poorly understood ability of picocyanobacteria to adapt to dark conditions. While the recent discovery of Synechococcus strains able to survive in the deep Black Sea highlights how adaptable picocyanobacteria can be, it also raises more questions—showing how much we still do not know about microbial life. Using available information from brackish Black Sea strains able to perform and survive in dark (anoxic) conditions, we illustrate how adaptation to narrow ecological niches interacts with gene evolution and metabolic capacity.

Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal.

Isolates of the marine picocyanobacteria, Prochlorococcus and Synechococcus, are often accompanied by diverse heterotrophic “contaminating” bacteria, which can act as confounding variables in otherwise controlled experiments. Traditional microbiological methods for eliminating contaminants, such as direct streak-plating, are often unsuccessful with this particular group of microorganisms. While they will grow in pour plates, colonies often remain contaminated with heterotrophic bacteria that can migrate through the soft agar. Additionally, axenic clones of picocyanobacteria can be recovered via dilution-to-extinction in liquid medium, but the efficiency of recovery is low, often requiring large numbers of 96-well plates. Here, we detail a simple and effective protocol for rendering cultures of Synechococcus and Prochlorococcus strains free of bacterial contaminants while at the same time yielding clonal isolates. We build on the fact that co-culture with specific heterotrophs—“helper heterotrophs”—is often necessary to grow colonies of picocyanobacteria from single cells in agar. Suspecting that direct physical contact between the helper and the picocyanobacterial cells was not necessary for the “helper effect,” we developed a protocol in which the helper cells are embedded in soft agar pour plates, a filter overlaid on the surface, and a picocyanobacterial culture is diluted and then spotted on top of the filter. With this approach, motile contaminants cannot swim to the colonies, and it is possible to obtain the expected number of colonies from a given input (i.e., a Poisson distribution of colonies with an expected value equal to the input number of cells), thus ensuring clonal colonies. Using this protocol, we rendered three strains of Synechococcus, two strains of Prochlorococcus, and 19 new strains of Synechococcus from coastal seawater clonal and free of heterotrophic bacteria. The simplicity of this approach should expand the repertoire of axenic picocyanobacterial strains available for controlled physiological experiments. It will also enable the study of microdiversity in populations of picocyanobacteria by facilitating large-scale isolation of picocyanobacterial clones from a single source, including direct isolation from natural seawater.

Observation of the feeding behaviour of reared Japanese eel Anguilla japonica leptocephali fed picocyanobacteria Synechococcus spp.

The authors observed the feeding behaviour of artificially reared Japanese eel Anguilla japonica leptocephali, 7.5-19 mm total length (10-61 days post-hatch), fed Synechococcus sp., which is considered a potential food source of anguilliform larvae. Three strains of Synechococcus sp. (NIES-972, 976 and 979) were tested as the food material. Larvae across the entire length range could effectively ingest a suspension of pico-sized cyanobacteria (1-3μm in diameter). Video observations of the mid-hindgut of larvae under an epifluorescence microscope confirmed that the movement of microvilli of the intestinal epithelium allowed the cell particles to circulate in the mid-hindgut, before becoming solidified in the anal region. Significant differences in food intake were observed between larvae fed two strains of Synechococcus (NIES-972 and 976), and among different cell densities, which suggests feeding selectivity and density dependence. Comparisons of feeding behaviour under the light group (9L:15D) and the dark group (24D) showed significantly higher food intake (measured as an index of intestinal fullness) in the light group, although substantial and continuous ingestion was observed in the dark group, indicating continuous feeding by swallowing sea water. The authors hypothesise that the feeding ecology of anguilliform leptocephali is based on a survival strategy whereby the larvae do not compete with various higher-trophic-level fishes for food in an oligotrophic environment.