The Bacterial Microbiome of the Coral Skeleton Algal Symbiont Ostreobium Shows Preferential Associations and Signatures of Phylosymbiosis.

Ostreobium, the major algal symbiont of the coral skeleton, remains understudied despite extensive research on the coral holobiont. The enclosed nature of the coral skeleton might reduce the dispersal and exposure of residing bacteria to the outside environment, allowing stronger associations with the algae. Here, we describe the bacterial communities associated with cultured strains of 5 Ostreobium clades using 16S rRNA sequencing. We shed light on their likely physical associations by comparative analysis of three datasets generated to capture (1) all algae associated bacteria, (2) enriched tightly attached and potential intracellular bacteria, and (3) bacteria in spent media. Our data showed that while some bacteria may be loosely attached, some tend to be tightly attached or potentially intracellular. Although colonised with diverse bacteria, Ostreobium preferentially associated with 34 bacterial taxa revealing a core microbiome. These bacteria include known nitrogen cyclers, polysaccharide degraders, sulphate reducers, antimicrobial compound producers, methylotrophs, and vitamin B12 producers. By analysing co-occurrence networks of 16S rRNA datasets from Porites lutea and Paragoniastrea australensis skeleton samples, we show that the Ostreobium-bacterial associations present in the cultures are likely to also occur in their natural environment. Finally, our data show significant congruence between the Ostreobium phylogeny and the community composition of its tightly associated microbiome, largely due to the phylosymbiotic signal originating from the core bacterial taxa. This study offers insight into the Ostreobium microbiome and reveals preferential associations that warrant further testing from functional and evolutionary perspectives.

Unravelling microalgal-bacterial interactions in aquatic ecosystems through 16S rRNA gene-based co-occurrence networks.

Interactions between microalgae and bacteria can directly influence the global biogeochemical cycles but the majority of such interactions remain unknown. 16S rRNA gene-based co-occurrence networks have potential to help identify microalgal-bacterial interactions. Here, we used data from 10 Earth microbiome projects to identify potential microalgal-bacterial associations in aquatic ecosystems. A high degree of clustering was observed in microalgal-bacterial modules, indicating densely connected neighbourhoods. Proteobacteria and Bacteroidetes predominantly co-occurred with microalgae and represented hubs of most modules. Our results also indicated that species-specificity may be a global characteristic of microalgal associated microbiomes. Several previously known associations were recovered from our network modules, validating that biologically meaningful results can be inferred using this approach. A range of previously unknown associations were recognised such as co-occurrences of Bacillariophyta with uncultured Planctomycetes OM190 and Deltaproteobacteria order NB1-j. Planctomycetes and Verrucomicrobia were identified as key associates of microalgae due to their frequent co-occurrences with several microalgal taxa. Despite no clear taxonomic pattern, bacterial associates appeared functionally similar across different environments. To summarise, we demonstrated the potential of 16S rRNA gene-based co-occurrence networks as a hypothesis-generating framework to guide more focused research on microalgal-bacterial associations.

Chrysosporum ovalisporum is synonymous with the true-branching cyanobacterium Umezakia natans (Nostocales/Aphanizomenonaceae).

True branching is a facultative characteristic only known from two cyanobacteria in the Aphanizomenonaceae, Umezakia natans and Dolichospermum brachiatum. In both cases, its expression has been associated with environmental stress, and its practical use as a diacritical feature has been previously evaluated. In this study, we undertook further evaluation of the phylogeny of Umezakia natans and its relationship to Chrysosporum ovalisporum as a previous study suggested the two were potentially congeneric. We used combined morphological, phylogenetic, and phylogenomic approaches to determine their relatedness using new strains available from a broad geographic range. Phylogenetic analysis based on 16S rRNA gene sequences showed that Australian C. ovalisporum and Japanese U. natans strains clustered together with accessions of C. ovalisporum originating from Australia, Israel, and Spain, with high p-distance similarity values (99.5%-99.9%). Additionally, differences between the two species in the 16S-23S ITS region was low (0%-2.5%). The average nucleotide identity of the U. natans and C. ovalisporum strains was also high (ANI of > 99.5 and AF > 0.9) and supported a genus-level separation from Chrysosporum bergii (83 ANI between clusters). Furthermore, in culture, strains of both species grown in vitamin-free media showed facultative true branching, a feature not previously known in C. ovalisporum. Collectively, the results support unification of C. ovalisporum and U. natans according to the principle of priority as Umezakia ovalisporum.

Precision early detection of invasive and toxic cyanobacteria: A case study of Raphidiopsis raciborskii.

Blooms of the toxic cyanobacterium, Raphidiopsis raciborskii (basionym Cylindrospermopsis raciborskii), are becoming a major environmental issue in freshwater ecosystems globally. Our precision prevention and early detection of R. raciborskii blooms rely upon the accuracy and speed of the monitoring method. A duplex digital PCR (dPCR) monitoring approach was developed and validated to detect the abundance and toxin-producing potential of R. raciborskii simultaneously in both laboratory spiked and environmental samples. Results of dPCR were strongly correlated with traditional real time quantitative PCR (qPCR) and microscopy for both laboratory and environmental samples. However, discrepancies between methods were observed when measuring R. raciborskii at low abundance (1 - 105 cells L - 1), with dPCR showing a higher precision compared to qPCR at low cell concentration. Furthermore, the dPCR assay had the highest detection rate for over two hundred environmental samples especially under low abundance conditions, followed by microscopy and qPCR. dPCR assay had the advantages of simple operation, time-saving, high sensitivity and excellent reproducibility. Therefore, dPCR would be a fast and precise monitoring method for the early warning of toxic bloom-forming cyanobacterial species and assessment of water quality risks, which can improve prediction and prevention of the impacts of harmful cyanobacterial bloom events in inland waters.

Are laboratory growth rate experiments relevant to explaining bloom-forming cyanobacteria distributions at global scale?

Predicting algal population dynamics using models informed by experimental data has been used as a strategy to inform the management and control of harmful cyanobacterial blooms. We selected toxic bloom-forming species Microcystis spp. and Raphidiopsis raciborskii (basionym Cylindrospermopsis raciborskii) for further examination as they dominate in 78 % and 17 %, respectively, of freshwater cyanobacterial blooms (cyanoHABs) reported globally over the past 30 years. Field measurements of cyanoHABs are typically based on biomass accumulation, but laboratory experiments typically measure growth rates, which are an important variable in cyanoHAB models. Our objective was to determine the usefulness of laboratory studies of these cyanoHAB growth rates for simulating the species dominance at a global scale. We synthesized growth responses of M. aeruginosa and R. raciborskii from 20 and 16 culture studies, respectively, to predict growth rates as a function of two environmental variables, light and temperature. Predicted growth rates of R. raciborskii exceeded those of M. aeruginosa at temperatures ≳ 25 °C and light intensities ≳ 150 µmol photons m-2 s-1. Field observations of biomass accumulation, however, show that M. aeruginosa dominates over R. raciborskii, irrespective of climatic zones. The mismatch between biomass accumulation measured in the field, and what is predicted from growth rate measured in the laboratory, hinders effective use of culture studies to predict formation of cyanoHABs in the natural environment. The usefulness of growth rates measured may therefore be limited, and field experiments should instead be designed to examine key physiological attributes such as colony formation, buoyancy regulation and photoadaptation. Improving prediction of cyanoHABs in a changing climate requires a more effective integration of field and laboratory approaches, and an explicit consideration of strain-level variability.

Subtropical freshwater phytoplankton show a greater response to increased temperature than to increased pCO2.

Global increases in atmospheric CO2 and temperatures will impact aquatic systems, with freshwater habitats being affected. Some studies suggest that these conditions will promote cyanobacterial dominance. There is a need for a clearer picture of how algal species and strains within species will respond to higher temperatures and CO2, especially in combination. This study examined two chlorophytes (Monoraphidium and Staurastrum), and two strains of the cyanobacterium Raphidiopsis raciborskii (straight S07 and coiled C03), to determine how the combination of higher temperature and CO2 levels will affect their growth and maximum cell concentrations. Continuous cultures were used to compare the steady state cell concentrations at 28 °C and 30 °C, and CO2 partial pressures (pCO2), 400 and 750 ppm for all cultures, and in addition 1000 ppm at 28 °C for R. raciborskii strains. This study showed that, for all species, water temperature had a greater effect than higher pCO2 on cell concentrations. There were clear differences in response between the chlorophyte species, with Monoraphidium preferring 28 °C and Staurastrum preferring 30 °C. There were also differences in response of the R. raciborskii strains to increasing temperature and pCO2, with S07 having a greater increase in cell concentration. Genome analysis of R. raciborskii showed that the straight strain has five additional carbon acquisition genes (ß-CA, chpY, cmpB, cmpD and NdhD4), indicative of increased carbon metabolism. These differences in the strains' response to elevated pCO2 will lead to changes in the species population structure and distribution in the water column. This study shows that it is important to examine the effects of both pCO2 and temperature, and to consider strain variation, to understand how species composition of natural systems may change under future climate conditions.

Differential expression of phosphorus acquisition genes in response to phosphorus stress in two Raphidiopsis raciborskii strains.

The cyanobacterium Raphidiopsis raciborskii is a nuisance in freshwater ecosystems. Strains vary in their physiological responses to environmental drivers, thus a greater understanding of the magnitude of strain variation is required to characterize the species. In this study, two strains of R. raciborskii isolated from a tropical Australian water reservoir were grown with and without phosphorus (P) to determine any relative response to P stress. The strains had the same growth rates and under P free conditions, cells grew at the same rate as P replete conditions until day 9 when cell growth ceased. There was no difference in the alkaline phosphatase activity per cell for the P replete and P free conditions, but the level of activity per cell was greater in CS-505 than CS-506. P acquisition genes were identified from the sequenced genomes; both strains contained the same genes, but with differences in copy number of phoA (7 and 6), phnK (3 and 1) and phnH (2 and 1) between CS-505 and CS-506 (respectively). The expression of P acquisition genes under P stress was measured throughout the experiment and shown to vary in magnitude and timing across strains, and in P replete versus P free cultures. In strain CS-505, upregulation of the pstS1 and phoA genes occurred late in the growth phase and into senescence. These genes are involved in phosphate uptake and use of various forms of organic P. For strain CS-506, there was upregulation of the phosphate uptake gene, pit, and organic P utilization genes, phoA, phoU, phnD and phnK, commencing late in the growth phase. Our study shows that despite the fact that these two strains were isolated from the same waterbody, they differed markedly in their gene expression response to P free conditions. This capacity of R. raciborskii to vary in strain responses to P conditions gives the organism flexibility in responding to environmental change, particularly P stress conditions.

Cylindrospermopsis raciborskii Virus and host: genomic characterization and ecological relevance.

Cylindrospermopsis (Raphidiopsis) raciborskii is an invasive, filamentous, nitrogen-fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology of C. raciborskii is almost unexplored. Progress in this field has been hindered by the absence of a characterized virus-host system in C. raciborskii. To bridge this gap, we sequenced the genome of CrV-01T, a previously isolated cyanosiphovirus, and its host, C. raciborskii strain Cr2010. Analyses suggest that CrV-01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12 kb inverted duplication. Evidence suggests CrV-01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR-Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV-01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV-01T, but lacking the inverted duplication, are present in C. raciborskii blooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus-host interactions in an ecologically important bloom-forming cyanobacterium.

Genome variation in nine co-occurring toxic Cylindrospermopsis raciborskii strains.

Cyanobacteria form harmful algal blooms and are highly adapted to a range of habitats, in part due to their phenotype plasticity. This plasticity is partially the result of co-existence of multiple strains within a single population. The toxic cyanobacterium Cylindrospermopsis raciborskii has remarkable phenotypic plasticity, strain variation and environmental adaptation resulting in an expansion of its global range. To understand the genetic basis of the high level of plasticity within a C. raciborskii population, the genomes of nine co-occurring strains were compared. The strains differed in morphology, toxin cell quotas and physiology, despite being obtained from a single water sample. Comparative genomics showed that three coiled strains were 3.9 Mbp in size, with 3544 ±â€¯11 genes, while straight strains were 3.8 Mbp in size, with 3485 ±â€¯20 genes. The core proteome comprised 86% of the genome and consisted of 2891 orthologous groups (OGs), whereas the variable genome comprised ∼14% (847 OGs), and the strain specific genome only ∼1% (433 OGs).There was a high proportion of variable strain-specific genes for the very closely related strains, which may underpin strain differentiation. The variable genes were associated with environmental responses and adaptation, particularly phage defence, DNA repair, membrane transport, and stress, illustrative of the adaptability of the strains in response to environmental and biological stressors. This study shows that high genomic variability exists between co-occurring strains and may be the basis of strain phenotypic differences and plasticity of populations. Therefore management and prediction of blooms of this harmful species requires different approaches to capture this strain variability.

Variation within and between cyanobacterial species and strains affects competition: Implications for phytoplankton modelling.

Cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii are two harmful species which co-occur and successively dominate in freshwaters globally. Within-species strain variability affects cyanobacterial population responses to environmental conditions, and it is unclear which species/strain would dominate under different environmental conditions. This study applied a Monte Carlo approach to a phytoplankton dynamic growth model to identify how growth variability of multiple strains of these two species affects their competition. Pairwise competition between four M. aeruginosa and eight C. raciborskii strains was simulated using a deterministic model, parameterized with laboratory measurements of growth and light attenuation for all strains, and run at two temperatures and light intensities. 17 000 runs were simulated for each pair using a statistical distribution with Monte Carlo approach. The model results showed that cyanobacterial competition was highly variable, depending on strains present, light and temperature conditions. There was no absolute 'winner' under all conditions as there were always strains predicted to coexist with the dominant strains, which were M. aeruginosa strains at 20°C and C. raciborskii strains at 28°C. The uncertainty in prediction of species competition outcomes was due to the substantial variability of growth responses within and between strains. Overall, this study demonstrates that within-species strain variability has a potentially large effect on cyanobacterial population dynamics, and therefore this variability may substantially reduce confidence in predicting outcomes of phytoplankton competition in deterministic models, that are based on only one set of parameters for each species or strain.

Review: a meta-analysis comparing cell-division and cell-adhesion in Microcystis colony formation.

The freshwater cyanobacterium Microcystis is a nuisance species. It forms large blooms on the water surface and overwhelmingly dominates the ecosystem through the formation of colonies from single cells surrounded by mucilage; however, the mechanism of colony formation is poorly understood. Two mechanisms of Microcystis colony formation have been proposed: cell-division, where cells remain attached after binary fission; and cell-adhesion, where single cells stick together. This paper examined the published literature on Microcystis colony formation to clarify the mechanism of colony formation and its relationship to environmental drivers. This meta-analysis showed that in laboratory experiments, colony formation by cell-division was mainly induced by zooplankton filtrate, high Pb2+ concentrations, the presence of the cyanobacterium Cylindrospermopsis raciborskii, heterotrophic bacteria, and low temperature and low light intensities. Alternatively, colony formation by cell-adhesion was mainly induced by zooplankton grazing, high Ca2+ concentrations, and microcystins. Therefore, colony formation by cell-division appears to be a slower process and to occur under an environmental stress factor, while cell-adhesion occurs more quickly to an environmental threat. Applying the criteria to the different morphospecies of Microcystis, it was found that under natural conditions M. ichthyoblabe colonies formed predominantly through cell-division, whereas M. wesenbergii colonies formed predominantly through cell-adhesion. This study provides new insights into the mechanisms and environmental drivers of colony formation by Microcystis.

Differences in cyanobacterial strain responses to light and temperature reflect species plasticity.

Microcystis aeruginosa and Cylindrospermopsis raciborskii are two cyanobacterial species that dominate freshwaters globally. Multiple strains of each species with different physiology occur, however, many studies have focused only on one or two strains, limiting our understanding of both strain variation and characterisation of the species. Therefore, in this study we examined the variation in growth and morphology of multiple isolates of both species, isolated from two adjacent Australian reservoirs. Four M. aeruginosa strains (=isolates) (one colony-forming, three single-celled morphology) and eight C. raciborskii isolates (five with straight trichomes, three with coiled trichomes) were cultured individually in a factorial designed experiment with four light intensities (L: 10, 30, 50 and 100µmol photons m-2s-1) and two temperatures (T: 20 and 28°C). The specific growth rate (µ), cell volume, and final cell concentration was measured. The light attenuation coefficient (kj), a measure of self-shading, was calculated. The results showed that the intraspecific variation was greater than the interspecific variation. The µ of all isolates of M. aeruginosa and C. raciborskii ranged from 0.16 to 0.55d-1 and 0.15 to 0.70d-1, respectively. However, at a specific light and temperature the mean µ of all M. aeruginosa isolates and C. raciborskii isolates were similar. At the species level, M. aeruginosa had higher growth rates at higher light intensity but lower temperature (L100T20), while straight C. raciborskii had higher growth rates at lower light intensity but higher temperature (L50T28), and coiled C. raciborskii had higher growth rates at higher light intensity and higher temperature (L100T28). The final cell concentrations of M. aeruginosa were higher than C. raciborskii. However, C. raciborskii isolates had greater variation in µ, kj and cell volume than M. aeruginosa. kj varied with light and temperature, and decreased with surface-to-volume ratio within each species. kj was lower for M. aeruginosa compared to C. raciborskii as expected based on cell size, but interestingly, C. raciborskii coiled isolates had lower kj than the straight isolates suggesting lower effect of self-shading. This study highlights the extent of strain variation to environmental conditions and to species variability.

Intraspecific variation in growth, morphology and toxin quotas for the cyanobacterium, Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a bloom forming cyanobacterium with complex population dynamics and toxicity. In January of 2013 a single sample was collected from surface waters in Lake Wivenhoe, Australia, and twenty-four individual trichomes were isolated. Each isolate exhibited differences in growth rate, toxin cell quota and morphology, in the absence of phylogenetic heterogeneity. This study demonstrates substantial intraspecific isolate variation within a small sample and this has implications for understanding the population dynamics of this species.

Nitrogen fixation by the diazotroph Cylindrospermopsis raciborskii (Cyanophyceae).

Nitrogen fixation has been proposed as a mechanism that allows the diazotrophic cyanobacterium, Cylindrospermopsis raciborskii, to bloom in nitrogen-limited freshwater systems. However, it is unclear whether dinitrogen fixation (N2 fixation) can supplement available dissolved inorganic nitrogen (DIN) for growth, or only provides minimum nitrogen (N) for cell maintenance under DIN deplete conditions. Additionally, the rate at which cells can switch between DIN use and N2 fixation is unknown. This study investigated N2 fixation under a range of nitrate concentrations. Cultures were grown with pretreatments of nitrate replete (single dose 941 µmol NO3- · L-1 ) and N-free conditions and then either received a single dose of 941 µmol NO3- · L-1 (N941), 118 µmol NO3- · L-1 (N118) or 0 N. Heterocysts appeared from days 3 to 5 when treatments of high NO3- were transferred to N free media (N941:N0), and from day 5 in N941 transferred to N118 treatments. Conversely, transferring cells from N0 to N941 resulted in heterocysts being discarded from day 3 and day 5 for N0:N118. Heterocyst appearance correlated with a detectable rate of N2 fixation and up-regulation of nifH gene expression, the discard of heterocysts occurred after sequential reduction of nifH expression and N2 fixation. Nitrate uptake rates were not affected by pretreatment, suggesting no regulation or saturation of this uptake pathway. These data demonstrate that for C. raciborskii, N2 fixation is regulated by the production or discard of heterocysts. In conclusion, this study has shown that N2 fixation only provides enough N to support relatively low growth under N-limited conditions, and does not supplement available nitrate to increase growth rates.

Understanding the winning strategies used by the bloom-forming cyanobacterium Cylindrospermopsis raciborskii.

The cyanobacterium Cylindrospermopsis raciborskii is a widespread species increasingly being recorded in freshwater systems around the world. It is of particular concern because strains in some geographic areas are capable of producing toxins with implications for human and animal health. Studies of this species have increased rapidly in the last two decades, especially in the southern hemisphere where toxic strains are prevalent. A clearer picture is emerging of the strategies adopted by this species to bloom and out-compete other species. This species has a high level of flexibility with respect to light and nutrients, with higher temperatures and carbon dioxide also promoting growth. There are two types of toxins produced by C. raciborskii: cylindrospermopsins (CYNs) and saxitoxins (STXs). The toxins CYNs are constitutively produced irrespective of environmental conditions and the ecological or physiological role is unclear, while STXs appear to serve as protection against high salinity and/or water hardness. It is also apparent that strains of this species can vary substantially in their physiological responses to environmental conditions, including CYNs production, and this may explain discrepancies in findings from studies in different geographical areas. The combination of a flexible strategy with respect to environmental conditions, and variability in strain response makes it a challenging species to manage. Our ability to improve bloom prediction will rely on a more detailed understanding of the complex physiology of this species.

Draft Genome Assembly of Filamentous Brackish Cyanobacterium Limnoraphis robusta Strain CS-951.

Limnoraphis robusta CS-951 is a sheathed, filamentous benthic, nonheterocystous cyanobacterium. It was isolated from brackish water and identified morphologically as Lyngbya majuscula. We report the draft genome of L. robusta CS-951, with a genome size of 7,314,117 bp, a 41.6% GC content, and 6,791 putative protein-coding genes assembled into 361contigs.

Constitutive cylindrospermopsin pool size in Cylindrospermopsis raciborskii under different light and CO2 partial pressure conditions.

Cylindrospermopsin (CYN) and 7-deoxy-cylindrospermopsin (dCYN) are potent hepatotoxic alkaloids produced by numerous species of cyanobacteria, including the freshwater Cylindrospermopsis raciborskii. C. raciborskii is an invasive cyanobacterium, and the study of how environmental parameters drive CYN production has received significant interest from water managers and health authorities. Light and CO2 affect cell growth and physiology in photoautotrophs, and these are potential regulators of cyanotoxin biosynthesis. In this study, we investigated how light and CO2 affect CYN and dCYN pool size as well as the expression of the key genes, cyrA and cyrK, involved in CYN biosynthesis in a toxic C. raciborskii strain. For cells growing at different light intensities (10 and 100 µmol photons m(-2) s(-1)), we observed that the rate of CYN pool size production (µCYN) was coupled to the cell division rate (µc) during batch culture. This indicated that CYN pool size under our experimental conditions is constant and cell quotas of CYN (QCYN) and dCYN (QdCYN) are fixed. Moreover, a lack of correlation between expression of cyrA and total CYN cell quotas (QCYNs) suggests that the CYN biosynthesis is regulated posttranscriptionally. Under elevated CO2 (1,300 ppm), we observed minor effects on QCYN and no effects on expression of cyrA and cyrK. We conclude that the CYN pool size is constitutive and not affected by light and CO2 conditions. Thus, C. raciborskii bloom toxicity is determined by the absolute abundance of C. raciborskii cells within the water column and the relative abundance of toxic and nontoxic strains.

Nutrient-related changes in the toxicity of field blooms of the cyanobacterium, Cylindrospermopsis raciborskii.

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3 ), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS ) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions.

Adhesion molecules from the diatom Phaeodactylum tricornutum (Bacillariophyceae): genomic identification by amino-acid profiling and in vivo analysis.

Cell adhesion molecules (CAMs) are important in prokaryotes and eukaryotes for cell-cell and cell-substratum interactions. The characteristics of adhesive proteins in the model diatom Phaeodactylum tricornutum were investigated by bioinformatic analysis and in vivo characterization. Bioinformatic analysis of the protein coding potential of the P. tricornutum genome used an amino-acid profile that we developed as a new system to identify uncharacterized or novel CAMs. Putative diatom CAMs were identified and seven were characterized in vivo, by generation of transgenic diatom lines overexpressing genes encoding C-terminal yellow fluorescent protein (YFP) fusion proteins. Three of these selected genes encode proteins with weak similarity to characterized proteins, a c-type lectin and two fasciclins, whereas the others are novel. The resultant cell lines were investigated for alterations in their adhesive ability. Whole cell-substratum adhesion strength was measured in a fully turbulent flow chamber, while atomic force microscopy was used to quantify the relative frequency of adhesion, as well as the length and strength of single molecules in the secreted mucilage. Finally, quartz crystal microbalance analysis characterized the visco-elastic properties and interaction of the mucilage-substratum interface. These combined studies revealed a range of phenotypes affecting adhesion, and led to the identification of candidate proteins involved in diatom adhesion. In summary, our study has for the first time combined bioinformatics and molecular physiological studies to provide new insights into diatom adhesive molecules.

Characterization of the extracellular matrix of Phaeodactylum tricornutum (Bacillariophyceae): structure, composition, and adhesive characteristics.

The extracellular matrix of the ovoid and fusiform morphotypes of Phaeodactylum tricornutum (Bohlin) was characterized in detail. The structural and nanophysical properties were analyzed by microscopy. Of the two morphotypes, only the ovoid form secretes adhesive mucilage; light microscopy and scanning electron microscopy images showed that the mucilage was secreted from the girdle band region of the cell as cell-substratum tethers, accumulating on the surface forming a biofilm. After 7 d, the secreted mucilage became entangled, forming adhesive strands that crisscrossed the substratum surface. In the initial secreted mucilage atomic force microscopy identified a high proportion of adhesive molecules without regular retraction curves and some modular-like adhesive molecules, in the 7 d old biofilm, the adhesive molecules were longer with fewer adhesive events but greater adhesive strength. Chemical characterization was carried out on extracted proteins and polysaccharides. Differences in protein composition, monosaccharide composition, and linkage analysis are discussed in relation to the composition of the frustule and secreted adhesive mucilage. Polysaccharide analysis showed a broad range of monosaccharides and linkages across all fractions with idiosyncratic enrichment of particular monosaccharides and linkages in each fraction. 3-linked Mannan was highly enriched in the cell frustule fractions indicating a major structural role, while Rhamnose and Fucose derivatives were enriched in the secreted fractions of the ovoid morphotype suggesting involvement in cell adhesion. Comparison of SDS-PAGE of extracellular proteins showed two major bands for the ovoid morphotype and four for the fusiform morphotype of which only one appeared to be common to both morphotypes.