Structural color in the bacterial domain: The ecogenomics of a 2-dimensional optical phenotype.

Structural color is an optical phenomenon resulting from light interacting with nanostructured materials. Although structural color (SC) is widespread in the tree of life, the underlying genetics and genomics are not well understood. Here, we collected and sequenced a set of 87 structurally colored bacterial isolates and 30 related strains lacking SC. Optical analysis of colonies indicated that diverse bacteria from at least two different phyla (Bacteroidetes and Proteobacteria) can create two-dimensional packing of cells capable of producing SC. A pan-genome-wide association approach was used to identify genes associated with SC. The biosynthesis of uroporphyrin and pterins, as well as carbohydrate utilization and metabolism, was found to be involved. Using this information, we constructed a classifier to predict SC directly from bacterial genome sequences and validated it by cultivating and scoring 100 strains that were not part of the training set. We predicted that SCr is widely distributed within gram-negative bacteria. Analysis of over 13,000 assembled metagenomes suggested that SC is nearly absent from most habitats associated with multicellular organisms except macroalgae and is abundant in marine waters and surface/air interfaces. This work provides a large-scale ecogenomics view of SC in bacteria and identifies microbial pathways and evolutionary relationships that underlie this optical phenomenon.

Cryopreservation and recovery of a complex hypersaline microbial mat community.

Cryopreservation of microorganisms is an essential tool in industrial- and food applications where conservation of microbial activity and critical beneficial traits need to be guaranteed to provide a consistent product or production process. This often refers to simple, single species or low diversity assemblages in liquid cultures that can easily be revived and regrown to perform the desired process. Cryopreservation is also of essence for scientific experimentation where many environmental samples are taken in remote sampling sites and at high costs. Biobanking, or the long term preservation and potential revival of complex, structured samples come with an additional challenge related to maintaining the structure upon revival. Here we look at cryopreserving and reviving a complex photosynthesis driven microbial mat from a hypersaline ecosystem. Amplicon sequencing of the 16S and 18S ribosomal RNA gene was used to determine the community composition of bacteria and eukaryotes respectively. The tests included the use of different cryopreservative agents and different times of cryopreservation at -150 °C. Upon revival, the cryopreservatives cannot be separated from the preserved samples without disturbing the community structure, while carryover of these compounds may influence reconstitution of the communities. Indeed, although both glycerol and Me2SO are good cryopreservatives of microbial assemblages, carryover of these compounds had a profound negative effect on the reestablishment of a functional microbial mat. Best cryopreservation and reconstitution results were obtained in the absence of a cryopreservative agent or when methanol was used.

Phototrophic marine benthic microbiomes: the ecophysiology of these biological entities.

Phototrophic biofilms are multispecies, self-sustaining and largely closed microbial ecosystems. They form macroscopic structures such as microbial mats and stromatolites. These sunlight-driven consortia consist of a number of functional groups of microorganisms that recycle the elements internally. Particularly, the sulfur cycle is discussed in more detail as this is fundamental to marine benthic microbial communities and because recently exciting new insights have been obtained. The cycling of elements demands a tight tuning of the various metabolic processes and require cooperation between the different groups of microorganisms. This is likely achieved through cell-to-cell communication and a biological clock. Biofilms may be considered as a macroscopic biological entity with its own physiology. We review the various components of some marine phototrophic biofilms and discuss their roles in the system. The importance of extracellular polymeric substances (EPS) as the matrix for biofilm metabolism and as substrate for biofilm microorganisms is discussed. We particularly assess the importance of extracellular DNA, horizontal gene transfer and viruses for the generation of genetic diversity and innovation, and for rendering resilience to external forcing to these biological entities.

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.

Bioremediation of chromium contaminated water by diatoms with concomitant lipid accumulation for biofuel production.

Hexavalent chromium compounds such as chromate and dichromate, commonly designated as Cr (VI) compounds, are widely used heavy metals in different industries and are considered highly toxic to most life forms. Unfortunately, they have become a major pollutant of groundwater and rivers around dichromate using industries. Bioremediation is widely used to decrease the amount of dichromate in wastewater but requires large amounts of precious fresh water. Here we tested two marine micro-algal species, Phaeodactylum tricornutum strain CCY0033 and Navicula pelliculosa strain CCMP543, for their ability of dichromate bioremediation and concomitantly producing lipids that can serve as biofuel. Dichromate tolerance of the strains was investigated under different growth conditions in order to obtain high biomass yields, high lipid accumulation and high dichromate removal from the medium. Both algal strains grew well and produced high biomass in media containing up to 1 mg of dichromate per liter. Variations in growth conditions revealed that dichromate removal from the medium correlated positively with biomass yield. Dichromate removal using living cells was in the same order of magnitude as with autoclaved dead cells or when using extracted extracellular polymeric substances (EPS). This suggests biosorption of dichromate to cell-associated polymeric substances as the major mechanism of the bioremediation process. For both strains, optimal dichromate removal and lipid production were achieved at a light intensity of 55 µmol m-2s-1 and at a sodium nitrate concentration of 3 mM. The optimal temperature for dichromate removal and lipid production was 23 °C for P. tricornutum and 27 °C for N. pelliculosa. Compared to P. tricornutum strain CCY0033, N. pelliculosa strain CCMP543 produced an overall higher lipid yield under these conditions.

Transcriptome analysis of Haloquadratum walsbyi: vanity is but the surface.

BACKGROUND: Haloquadratum walsbyi dominates saturated thalassic lakes worldwide where they can constitute up to 80-90% of the total prokaryotic community. Despite the abundance of the enigmatic square-flattened cells, only 7 isolates are currently known with 2 genomes fully sequenced and annotated due to difficulties to grow them under laboratory conditions. We have performed a transcriptomic analysis of one of these isolates, the Spanish strain HBSQ001 in order to investigate gene transcription under light and dark conditions. RESULTS: Despite a potential advantage for light as additional source of energy, no significant differences were found between light and dark expressed genes. Constitutive high gene expression was observed in genes encoding surface glycoproteins, light mediated proton pumping by bacteriorhodopsin, several nutrient uptake systems, buoyancy and storage of excess carbon. Two low expressed regions of the genome were characterized by a lower codon adaptation index, low GC content and high incidence of hypothetical genes. CONCLUSIONS: Under the extant cultivation conditions, the square hyperhalophile devoted most of its transcriptome towards processes maintaining cell integrity and exploiting solar energy. Surface glycoproteins are essential for maintaining the large surface to volume ratio that facilitates light and organic nutrient harvesting whereas constitutive expression of bacteriorhodopsin warrants an immediate source of energy when light becomes available.

A versatile method for simultaneous stable carbon isotope analysis of DNA and RNA nucleotides by liquid chromatography/isotope ratio mass spectrometry.

RATIONALE: Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is currently the most accurate and precise technique for the measurement of compound-specific stable carbon isotope ratios ((13)C/(12)C) in biological metabolites, at their natural abundance. However, until now this technique could not be applied for the analysis of nucleic acids, the building blocks of the carriers of genetic information in living cells and viruses, DNA and RNA. METHODS: Mixed-mode chromatography (MMC) was applied to obtain the complete separation of nine nucleotides (eight originating from DNA/RNA and one nucleotide (inosine monophosphate) that may serve as an internal standard) in a single run using LC/IRMS. We also developed and validated a method for DNA and RNA extraction and an enzymatic hydrolysis protocol for natural samples, which is compatible with LC/IRMS analysis as it minimizes the carbon blank. The method was used to measure the concentration and stable carbon isotope ratio of DNA and RNA nucleotides in marine sediment and in the common marine macro alga (Ulva sp.) at natural abundance levels as well as for (13)C-enriched samples. RESULTS: The detection limit of the LC/IRMS method varied between 1.0 nmol for most nucleotides and 2.0 nmol for late-eluting compounds. The intraday and interday reproducibility of nucleotide concentration measurements was better than, respectively, 4.1% and 8.9% and for δ(13)C measurements better than, respectively, 0.3‰ and 0.5‰. The obtained nucleic acid concentrations and nucleic acid synthesis rates were in good agreement with values reported in the literature. CONCLUSIONS: This new method gives reproducible results for the concentration and δ(13)C values of nine nucleotides. This solvent-free chromatographic method may also be used for other purposes, such as for instance to determine nucleotide concentrations using spectrophotometric detection. This sensitive method offers a new avenue for the study of DNA and RNA biosynthesis that can be applied in various fields of research.

Draft Genome Sequences of Synechococcus sp. strains CCAP1479/9, CCAP1479/10, CCAP1479/13, CCY0621, and CCY9618: Five Freshwater Syn/Pro Clade Picocyanobacteria.

Picocyanobacteria are essential primary producers in freshwaters yet little is known about their genomic diversity and ecological niches. We report here five draft genomes of freshwater picocyanobacteria: Synechococcus sp. CCAP1479/9, Synechococcus sp. CCAP1479/10, and Synechococcus sp. CCAP1479/13 isolated from Lake Windermere in the Lake District, UK; and Synechococcus sp. CCY0621 and Synechococcus sp. CCY9618 isolated from lakes in The Netherlands. Phylogenetic analysis reveals all five strains belonging to sub-cluster 5.2 of the Synechococcus and Prochlorococcus clade of Cyanobacteria. These five strains are divergent from Synechococcus elongatus, an often-used model for freshwater Synechococcus. Functional annotation revealed significant differences in the number of genes involved in the transport and metabolism of several macro-molecules between freshwater picocyanobacteria from sub-cluster 5.2 and Synechococcus elongatus, including amino acids, lipids, and carbohydrates. Comparative genomic analysis identified further differences in the presence of photosynthesis-associated proteins while gene neighbourhood comparisons revealed alternative structures of the nitrate assimilation operon nirA.

Diversity of the holopelagic Sargassum microbiome from the Great Atlantic Sargassum Belt to coastal stranding locations.

The holopelagic brown macroalgae Sargassum natans and Sargassum fluitans form essential habitats for attached and mobile fauna which contributes to a unique biodiversity in the Atlantic Ocean. However, holopelagic Sargassum natans (genotype I & VIII) and Sargassum fluitans (genotype III) have begun forming large accumulations with subsequent strandings on the western coast of Africa, the Caribbean and northern Brazil, threatening local biodiversity of coastal ecosystems and triggering economic losses. Moreover, stranded masses of holopelagic Sargassum may introduce or facilitate growth of bacteria that are not normally abundant in coastal regions where Sargassum is washing ashore. Hitherto, it is not clear how the holopelagic Sargassum microbiome varies across its growing biogeographic range and what factors drive the microbial composition. We determined the microbiome associated with holopelagic Sargassum from the Great Atlantic Sargassum Belt to coastal stranding sites in Mexico and Florida. We characterized the Sargassum microbiome via amplicon sequencing of the 16S V4 region hypervariable region of the rRNA gene. The microbial community of holopelagic Sargassum was mainly composed of photo(hetero)trophs, organic matter degraders and potentially pathogenic bacteria from the Pseudomonadaceae, Rhodobacteraceae and Vibrionaceae. Sargassum genotypes S. natans I, S. natans VIII and S. fluitans III contained similar microbial families, but relative abundances and diversity varied. LEfSE analyses further indicated biomarker genera that were indicative of Sargassum S. natans I/VIII and S. fluitans III. The holopelagic Sargassum microbiome showed biogeographic patterning with high relative abundances of Vibrio spp., but additional work is required to determine whether that represents health risks in coastal environments. Our study informs coastal management policy, where the adverse sanitary effects of stranded Sargassum might impact the health of coastal ecosystems.

Stratified prokaryote network in the oxic-anoxic transition of a deep-sea halocline.

The chemical composition of the Bannock basin has been studied in some detail. We recently showed that unusual microbial populations, including a new division of Archaea (MSBL1), inhabit the NaCl-rich hypersaline brine. High salinities tend to reduce biodiversity, but when brines come into contact with fresher water the natural haloclines formed frequently contain gradients of other chemicals, including permutations of electron donors and acceptors, that may enhance microbial diversity, activity and biogeochemical cycling. Here we report a 2.5-m-thick chemocline with a steep NaCl gradient at 3.3 km within the water column betweeen Bannock anoxic hypersaline brine and overlying sea water. The chemocline supports some of the most biomass-rich and active microbial communities in the deep sea, dominated by Bacteria rather than Archaea, and including four major new divisions of Bacteria. Significantly higher metabolic activities were measured in the chemocline than in the overlying sea water and underlying brine; functional analyses indicate that a range of biological processes is likely to occur in the chemocline. Many prokaryotic taxa, including the phylogenetically new groups, were confined to defined salinities, and collectively formed a diverse, sharply stratified, deep-sea ecosystem with sufficient biomass to potentially contribute to organic geological deposits.

Sulfur cycling and methanogenesis primarily drive microbial colonization of the highly sulfidic Urania deep hypersaline basin.

Urania basin in the deep Mediterranean Sea houses a lake that is >100 m deep, devoid of oxygen, 6 times more saline than seawater, and has very high levels of methane and particularly sulfide (up to 16 mM), making it among the most sulfidic water bodies on Earth. Along the depth profile there are 2 chemoclines, a steep one with the overlying oxic seawater, and another between anoxic brines of different density, where gradients of salinity, electron donors and acceptors occur. To identify and differentiate the microbes and processes contributing to the turnover of organic matter and sulfide along the water column, these chemoclines were sampled at a high resolution. Bacterial cell numbers increased up to a hundredfold in the chemoclines as a consequence of elevated nutrient availability, with higher numbers in the upper interface where redox gradient was steeper. Bacterial and archaeal communities, analyzed by DNA fingerprinting, 16S rRNA gene libraries, activity measurements, and cultivation, were highly stratified and metabolically more active along the chemoclines compared with seawater or the uniformly hypersaline brines. Detailed analysis of 16S rRNA gene sequences revealed that in both chemoclines delta- and epsilon-Proteobacteria, predominantly sulfate reducers and sulfur oxidizers, respectively, were the dominant bacteria. In the deepest layers of the basin MSBL1, putatively responsible for methanogenesis, dominated among archaea. The data suggest that the complex microbial community is adapted to the basin's extreme chemistry, and the elevated biomass is driven largely by sulfur cycling and methanogenesis.

Differences in gene expression patterns between cultured and natural Haloquadratum walsbyi ecotypes.

Solar crystallizer ponds are characterized by high population density with a relatively simple community structure in terms of species composition. The microbial community in the solar saltern of Santa Pola (Alicante, Spain), is largely dominated by the hyperhalophilic square archaeon Haloquadratum walsbyi. Here we studied metatranscriptomes retrieved from a crystallizer pond during the winter of 2012 and summer of 2014 and compared Hqr. walsbyi's transcription patterns with that of the cultured strain Hqr. walsbyi HBSQ001. Significant differences were found between natural and the cultured grown strain in the distribution of transcript levels per gene. This likely reflects the adaptation of the cultured strain to the relative homogeneous growth conditions while the natural species, which is represented by multiple ecotypes, is adapted to heterogeneous environmental conditions and challenges of nutrient competition, viral attack, and other stressors. An important consequence of this study is that expression patterns obtained under artificial cultivation conditions cannot be directly extrapolated to gene expression under natural conditions. Moreover, we found 195 significantly differential expressed genes between the seasons, with 140 genes being higher expressed in winter and mainly encode proteins involved in energy and carbon source acquiring processes, and in stress responses.

Draft genome sequences of three filamentous cyanobacteria isolated from brackish habitats.

Brackish cyanobacterial genome sequences are relatively rare. Here, we report the 5.5 Mbp, 5.8 Mbp and 6.1 Mbp draft genomes of Spirulina sp. CCY15215, Leptolyngbya sp. CCY15150 and Halomicronema sp. CCY15110 isolated from coastal microbial mats on the North Sea beach of the island of Schiermonnikoog in the Netherlands. Large scale phylogenomic analyses reveal that Spirulina sp. CCY15215 is a large cell diameter cyanobacterium, whereas Leptolyngbya sp. CCY15150 and Halomicronema sp. CCY15110 are the first reported brackish genomes belonging to the LPP clade consisting primarily of Leptolyngbya, Plectonema and Phormidium spp. Further genome mining divulges that all new draft genomes contain, ggpS and ggpP , the genes responsible for synthesising glucosylglycerol (GG), a compatible solute found in moderately salt-tolerant cyanobacteria.

Stromatolites as Biosignatures of Atmospheric Oxygenation: Carbonate Biomineralization and UV-C Resilience in a Geitlerinema sp. - Dominated Culture.

Modern stromatolites are key to the record of past microbial activity preserved in fossil carbonate deposits. Mono-phototrophic cultures dominated by the cyanobacterium Geitlerinema sp. were obtained from a laboratory-maintained, low magnesium-calcite stromatolite originating from Lagoa Vermelha, Brazil. This lagoonal system has been described as a Precambrian analog, illustrating a period of photosynthetically induced atmospheric oxygenation, which created a global sanctuary from shortwave solar radiation and enabled the evolution of modern life on Earth. The enrichment cultures precipitate carbonates in minimal media, suggesting that cyanobacterial photosynthesis and extracellular polymeric substance production may be crucial in the mineralization of the studied stromatolite. We further show that Geitlerinema sp. can build and maintain filamentous mats under long-term UV-C exposure. Our results suggest that present day stromatolites dominated by cyanobacteria may be interpreted as biosignatures of atmospheric oxygenation and have implications for the search for putative biological traces on Mars.

Circadian clock-controlled gene expression in co-cultured, mat-forming cyanobacteria.

Natural coastal microbial mat communities are multi-species assemblages that experience fluctuating environmental conditions and are shaped by resource competition as well as by cooperation. Laboratory studies rarely address the natural complexity of microbial communities but are usually limited to homogeneous mono-cultures of key species grown in liquid media. The mat-forming filamentous cyanobacteria Lyngbya aestuarii and Coleofasciculus chthonoplastes were cultured under different conditions to investigate the expression of circadian clock genes and genes that are under their control. The cyanobacteria were grown in liquid medium or on a solid substrate (glass beads) as mono- or as co-cultures under a light-dark regime and subsequently transferred to continuous light. TaqMan-probe based qPCR assays were used to quantify the expression of the circadian clock genes kaiA, kaiB, and kaiC, and of four genes that are under control of the circadian clock: psbA, nifH, ftsZ, and prx. Expression of kaiABC was influenced by co-culturing the cyanobacteria and whether grown in liquid media or on a solid substrate. Free-running (i.e. under continuous light) expression cycle of the circadian clock genes was observed in L. aestuarii but not in C. chthonoplastes. In the former organism, maximum expression of psbA and nifH occurred temporally separated and independent of the light regime, although the peak shifted in time when the culture was transferred to continuous illumination. Although functionally similar, both species of cyanobacteria displayed different 24-h transcriptional patterns in response to the experimental treatments, suggesting that their circadian clocks have adapted to different life strategies adopted by these mat-forming cyanobacteria.

Seasonal development of a coastal microbial mat.

Growth and activity of coastal microbial mats is strongly seasonal. The development of these mats starts in early spring and fully maturate during late summer, where after growth ceases and subsequently the mat deteriorates by erosion and decomposition in winter. Here, the composition of the microbial community of three different mats developing along the tidal gradient of the North Sea beach of the Dutch barrier island Schiermonnikoog was analysed. The 16S ribosomal RNA molecules and the associated gene were sequenced in order to obtain the active (RNA) and resident (DNA) community members, respectively. Proteobacteria, Cyanobacteria, and Bacteroidetes dominated the mats during the whole year but considerable differences among these groups were found along the tidal gradient and seasonally when observed at a finer taxonomic resolution. Richness and diversity increased during the year starting from a pioneering community that is gradually succeeded by a more diverse climax community. The initial pioneers consisted of the cold-adapted photoautotrophic cyanobacterium Nodularia sp. and potential cold adapted members of the alphaproteobacterial Loktanella genus. These pioneers were succeeded by, amongst others, cyanobacteria belonging to the genera Leptolyngbya, Lyngbya, and Phormidium. At the upper littoral (Dune site), which was characterized by an extensive salt marsh vegetation, the mats contained a distinct bacterial community that potentially contribute to or benefit from plant decay. This study reports in detail on the seasonal changes and succession of these coastal microbial mat communities and discusses the potential forces that drive these changes.

Diversity and dynamics of Antarctic marine microbial eukaryotes under manipulated environmental UV radiation.

In the light of the predicted global climate change, it is essential that the status and diversity of polar microbial communities is described and understood. In the present study, molecular tools were used to investigate the marine eukaryotic communities of Prydz Bay, Eastern Antarctica, from November 2002 to January 2003. Additionally, we conducted four series of minicosm experiments, where natural Prydz Bay communities were incubated under six different irradiation regimes, in order to investigate the effects of natural UV radiation on marine microbial eukaryotes. Denaturing gradient gel electrophoresis (DGGE) and 18S rRNA gene sequencing revealed a eukaryotic Shannon diversity index averaging 2.26 and 2.12, respectively. Phylogenetic analysis of 472 sequenced clones revealed 47 phylotypes, belonging to the Dinophyceae, Stramenopiles, Choanoflagellidae, Ciliophora, Cercozoa and Metazoa. Throughout the studied period, three communities were distinguished: a postwinter/early spring community comprising dinoflagellates, ciliates, cercozoans, stramenopiles, viridiplantae, haptophytes and metazoans; a dinoflagellate-dominated community; and a diatom-dominated community that developed after sea ice breakup. DGGE analysis showed that size fraction and time had a strong shaping effect on the community composition; however, a significant contribution of natural UV irradiance towards microeukaryotic community composition could not be detected. Overall, dinoflagellates dominated our samples and their diversity suggests that they fulfill an important role in Antarctic coastal marine ecosystems preceding ice breakup as well as between phytoplankton bloom events.

Daily rhythmicity in coastal microbial mats.

Cyanobacteria are major primary producers in coastal microbial mats and provide biochemical energy, organic carbon, and bound nitrogen to the mat community through oxygenic photosynthesis and dinitrogen fixation. In order to anticipate the specific requirements to optimize their metabolism and growth during a day-and-night cycle, Cyanobacteria possess a unique molecular timing mechanism known as the circadian clock that is well-studied under laboratory conditions but little is known about its function in a natural complex community. Here, we investigated daily rhythmicity of gene expression in a coastal microbial mat community sampled at 6 time points during a 24-h period. In order to identify diel expressed genes, meta-transcriptome data was fitted to periodic functions. Out of 24,035 conserved gene transcript clusters, approximately 7% revealed a significant rhythmic expression pattern. These rhythmic genes were assigned to phototrophic micro-eukaryotes, Cyanobacteria but also to Proteobacteria and Bacteroidetes. Analysis of MG-RAST annotated genes and mRNA recruitment analysis of two cyanobacterial and three proteobacterial microbial mat members confirmed that homologs of the cyanobacterial circadian clock genes were also found in other bacterial members of the microbial mat community. These results suggest that various microbial mat members other than Cyanobacteria have their own molecular clock, which can be entrained by a cocktail of Zeitgebers such as light, temperature or metabolites from neighboring species. Hence, microbial mats can be compared to a complex organism consisting of multiple sub-systems that have to be entrained in a cooperative way such that the corpus functions optimally.