Stormwater influences phytoplankton assemblages within the diverse, but impacted Sydney Harbour estuary.

Sydney Harbour is subjected to persistent stress associated with anthropogenic activity and global climate change, but is particularly subjected to pulse stress events associated with stormwater input during episodic periods of high rainfall. Photosynthetic microbes underpin metazoan diversity within estuarine systems and are therefore important bioindicators of ecosystem health; yet how stormwater input affects their occurrence and distribution in Sydney Harbour remains poorly understood. We utilised molecular tools (16S/18S rRNA and petB genes) to examine how the phytoplankton community structure (both prokaryotes and eukaryotes) within Sydney Harbour varies between high and low rainfall periods. The relative proportion of phytoplankton sequences was more abundant during the high rainfall period, comprising mainly of diatoms, an important functional group supporting increased productivity within estuarine systems, together with cyanobacteria. Increased spatial variability in the phytoplankton community composition was observed, potentially driven by the steepened physico-chemical gradients associated with stormwater inflow. Conversely, during a low rainfall period, the proportion of planktonic photosynthetic microbes was significantly lower and the persistent phytoplankton were predominantly represented by chlorophyte and dinoflagellate sequences, with lower overall diversity. Differences in phytoplankton composition between the high and low rainfall periods were correlated with temperature, salinity, total nitrogen and silicate. These results suggest that increased frequency of high-rainfall events may change the composition, productivity and health of the estuary. Our study begins to populate the knowledge gap in the phytoplankton community structure and substantial changes associated with transient environmental perturbations, an essential step towards unravelling the dynamics of primary production in a highly urbanised estuarine ecosystem in response to climate change and other anthropogenic stressors.

Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere.

Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.

Unicellular Cyanobacteria Are Important Components of Phytoplankton Communities in Australia's Northern Oceanic Ecoregions.

The tropical marine environments of northern Australia encompasses a diverse range of geomorphological and oceanographic conditions and high levels of productivity and nitrogen fixation. However, efforts to characterize phytoplankton assemblages in these waters have been restricted to studies using microscopic and pigment analyses, leading to the current consensus that this region is dominated by large diatoms, dinoflagellates, and the marine cyanobacterium Trichodesmium. During an oceanographic transect from the Arafura Sea through the Torres Strait to the Coral Sea, we characterized prokaryotic and eukaryotic phytoplankton communities in surface waters using a combination of flow cytometry and Illumina based 16S and 18S ribosomal RNA amplicon sequencing. Similar to observations in other marine regions around Australian, phytoplankton assemblages throughout this entire region were rich in unicellular picocyanobacterial primary producers while picoeukaryotic phytoplankton formed a consistent, though smaller proportion of the photosynthetic biomass. Major taxonomic groups displayed distinct biogeographic patterns linked to oceanographic and nutrient conditions. Unicellular picocyanobacteria dominated in both flow cytometric abundance and carbon biomass, with members of the Synechococcus genus dominating in the shallower Arafura Sea and Torres Strait where chlorophyll a was relatively higher (averaging 0.4 ± 0.2 mg m-3), and Prochlorococcus dominating in the oligotrophic Coral Sea where chlorophyll a averaged 0.13 ± 0.07 mg m-3. Consistent with previous microscopic and pigment-based observations, we found from sequence analysis that a variety of diatoms (Bacillariophyceae) exhibited high relative abundance in the Arafura Sea and Torres Strait, while dinoflagellates (Dinophyceae) and prymnesiophytes (Prymnesiophyceae) were more abundant in the Coral Sea. Ordination analysis identified temperature, nutrient concentrations and water depth as key drivers of the region's assemblage composition. This is the first molecular and flow cytometric survey of the abundance and diversity of both prokaryotic and picoeukaryotic phytoplankton in this region, and points to the need to include the picocyanobacterial populations as an essential oceanic variable for sustained monitoring in order to better understand the health of these important coastal waters as global oceans change.

A Sample-to-Sequence Protocol for Genus Targeted Transcriptomic Profiling: Application to Marine Synechococcus.

Recent studies using whole community metagenomic and metatranscriptomic approaches are revealing important new insights into the functional potential and activity of natural marine microbial communities. Here, we complement these approaches by describing a complete ocean sample-to-sequence protocol, specifically designed to target a single bacterial genus for purposes of both DNA and RNA profiling using fluorescence activated cell sorting (FACS). The importance of defining and understanding the effects of a sampling protocol are critical if we are to gain meaningful data from environmental surveys. Rigorous pipeline trials with a cultured isolate, Synechococcus sp. BL107 demonstrate that water filtration has a well-defined but limited impact on the transcriptomic profile of this organism, whilst sample storage and multiple rounds of cell sorting have almost no effect on the resulting RNA sequence profile. Attractively, the means to replicate the sampling strategy is within the budget and expertise of most researchers.

Tiny Microbes with a Big Impact: The Role of Cyanobacteria and Their Metabolites in Shaping Our Future.

Cyanobacteria are among the first microorganisms to have inhabited the Earth. Throughout the last few billion years, they have played a major role in shaping the Earth as the planet we live in, and they continue to play a significant role in our everyday lives. Besides being an essential source of atmospheric oxygen, marine cyanobacteria are prolific secondary metabolite producers, often despite the exceptionally small genomes. Secondary metabolites produced by these organisms are diverse and complex; these include compounds, such as pigments and fluorescent dyes, as well as biologically-active compounds with a particular interest for the pharmaceutical industry. Cyanobacteria are currently regarded as an important source of nutrients and biofuels and form an integral part of novel innovative energy-efficient designs. Being autotrophic organisms, cyanobacteria are well suited for large-scale biotechnological applications due to the low requirements for organic nutrients. Recent advances in molecular biology techniques have considerably enhanced the potential for industries to optimize the production of cyanobacteria secondary metabolites with desired functions. This manuscript reviews the environmental role of marine cyanobacteria with a particular focus on their secondary metabolites and discusses current and future developments in both the production of desired cyanobacterial metabolites and their potential uses in future innovative projects.

Effects of low temperature on tropical and temperate isolates of marine Synechococcus.

Temperature is an important factor influencing the distribution of marine picocyanobacteria. However, molecular responses contributing to temperature preferences are poorly understood in these important primary producers. We compared the temperature acclimation of a tropical Synechococcus strain WH8102 with temperate strain BL107 at 18 °C relative to 22 °C and examined their global protein expression, growth patterns, photosynthetic efficiency and lipid composition. Global protein expression profiles demonstrate the partitioning of the proteome into major categories: photosynthesis (>40%), translation (10-15%) and membrane transport (2-8%) with distinct differences between and within strains grown at different temperatures. At low temperature, growth and photosynthesis of strain WH8102 was significantly decreased, while BL107 was largely unaffected. There was an increased abundance of proteins involved in protein biosynthesis at 18 °C for BL107. Each strain showed distinct differences in lipid composition with higher unsaturation in strain BL107. We hypothesize that differences in membrane fluidity, abundance of protein biosynthesis machinery and the maintenance of photosynthesis efficiency contribute to the acclimation of strain BL107 to low temperature. Additional proteins unique to BL107 may also contribute to this strain's improved fitness at low temperature. Such adaptive capacities are likely important factors favoring growth of temperate strains over tropical strains in high latitude niches.

Development of a targeted metagenomic approach to study a genomic region involved in light harvesting in marine Synechococcus.

Synechococcus, one of the most abundant cyanobacteria in marine ecosystems, displays a broad pigment diversity. However, the in situ distribution of pigment types remains largely unknown. In this study, we combined flow cytometry cell sorting, whole-genome amplification, and fosmid library construction to target a genomic region involved in light-harvesting complex (phycobilisome) biosynthesis and regulation. Synechococcus community composition and relative contamination by heterotrophic bacteria were assessed at each step of the pipeline using terminal restriction fragment length polymorphism targeting the petB and 16S rRNA genes, respectively. This approach allowed us to control biases inherent to each method and select reliable WGA products to construct a fosmid library from a natural sample collected off Roscoff (France). Sequencing of 25 fosmids containing the targeted region led to the assembly of whole or partial phycobilisome regions. Most contigs were assigned to clades I and IV consistent with the known dominance of these clades in temperate coastal waters. However, one of the fosmids contained genes distantly related to their orthologs in reference genomes, suggesting that it belonged to a novel phylogenetic clade. Altogether, this study provides novel insights into Synechococcus community structure and pigment type diversity at a representative coastal station of the English Channel.

Stable isotope probing to study functional components of complex microbial ecosystems.

This protocol presents a method of dissecting the DNA or RNA of key organisms involved in a specific biochemical process within a complex ecosystem. Stable isotope probing (SIP) allows the labelling and separation of nucleic acids from community members that are involved in important biochemical transformations, yet are often not the most numerically abundant members of a community. This pure culture-independent technique circumvents limitations of traditional microbial isolation techniques or data mining from large-scale whole-community metagenomic studies to tease out the identities and genomic repertoires of microorganisms participating in biological nutrient cycles. SIP experiments can be applied to virtually any ecosystem and biochemical pathway under investigation provided a suitable stable isotope substrate is available. This versatile methodology allows a wide range of analyses to be performed, from fatty-acid analyses, community structure and ecology studies, and targeted metagenomics involving nucleic acid sequencing. SIP experiments provide an effective alternative to large-scale whole-community metagenomic studies by specifically targeting the organisms or biochemical transformations of interest, thereby reducing the sequencing effort and time-consuming bioinformatics analyses of large datasets.

Targeted genomics of flow cytometrically sorted cultured and uncultured microbial groups.

High throughput sequencing of genetic material recovered from environmental samples (i.e., metagenomics) is becoming the method of choice for either medical or environmental genomic studies. However, the large amount of data and complexity of the sequenced "biomes" present challenges for teasing meaningful results out of the mass. Here, we describe a targeted genomic pipeline which uses fluorescence-activated cell sorting (FACS) in combination with multiple displacement amplification (MDA) of nucleic acids that allows to dissect a complex system into its component parts to facilitate high-quality single-cell, or targeted population, genomic reconstructions of microbial communities. This pipeline is presented with methods for collecting, concentrating, and preserving cells from aquatic and marine environments suitable for flow cytometric processing at a later date.

Multi-locus sequence analysis, taxonomic resolution and biogeography of marine Synechococcus.

Conserved markers such as the 16S rRNA gene do not provide sufficient molecular resolution to identify spatially structured populations of marine Synechococcus, or 'ecotypes' adapted to distinct ecological niches. Multi-locus sequence analysis targeting seven 'core' genes was employed to taxonomically resolve Synechococcus isolates and correlate previous phylogenetic analyses encompassing a range of markers. Despite the recognized importance of lateral gene transfer in shaping the genomes of marine cyanobacteria, multi-locus sequence analysis of more than 120 isolates reflects a clonal population structure of major lineages and subgroups. A single core genome locus, petB, encoding the cytochrome b(6) subunit of the cytochrome b(6) f complex, was selected to expand our understanding of the diversity and ecology of marine Synechococcus populations. Environmental petB sequences cloned from contrasting sites highlight numerous genetically and ecologically distinct clusters, some of which represent novel, environmentally abundant clades without cultured representatives. With a view to scaling ecological analyses, the short sequence, taxonomic resolution and accurate automated alignment of petB is ideally suited to high-throughput and high-resolution sequencing projects to explore links between the ecology, evolution and biology of marine Synechococcus.

DISSECTING THE PHYSIOLOGICAL RESPONSE TO PHOSPHORUS STRESS IN MARINE SYNECHOCOCCUS ISOLATES (CYANOPHYCEAE)(1).

Marine Synechococcus is ubiquitous in aquatic environments. However, distinct phylogenetic lineages of this genus have a complex ecological distribution that is not fully explained. Here, we undertook a broad study of the phosphorus (P)-related behavior of marine Synechococcus isolates from all previously described ribotypes (sensu Fuller et al. 2003). A wide variability in P-related physiology was noted among members of this genus, particularly in the utilization of organic P sources. However, some characteristics (e.g., cell size change during P limitation and the ability to accumulate polyphosphate) were largely consistent with their phylogenetic lineage and inferred ecology, with clear distinctions between oligotrophic, mesotrophic, and opportunistic lineages. Similarly, the ability to induce protein expression in response to P limitation was consistent with the presence/absence of phoB/R regulatory capacity of the corresponding strain. Taxonomic differences in P uptake, storage, and utilization strategies could explain the ubiquitous distribution of marine Synechococcus throughout the world's oceans and explain the coexistence and/or ecological partitioning of multiple phototrophic taxa in the photic zone of tropical and subtropical oligotrophic oceans.

Functional characterization of Synechocystis sp. strain PCC 6803 pst1 and pst2 gene clusters reveals a novel strategy for phosphate uptake in a freshwater cyanobacterium.

Synechocystis sp. strain PCC 6803 possesses two putative ABC-type inorganic phosphate (P(i)) transporters with three associated P(i)-binding proteins (PBPs), SphX (encoded by sll0679), PstS1 (encoded by sll0680), and PstS2 (encoded by slr1247), organized in two spatially discrete gene clusters, pst1 and pst2. We used a combination of mutagenesis, gene expression, and radiotracer uptake analyses to functionally characterize the role of these PBPs and associated gene clusters. Quantitative PCR (qPCR) demonstrated that pstS1 was expressed at a high level in P(i)-replete conditions compared to sphX or pstS2. However, a P(i) stress shift increased expression of pstS2 318-fold after 48 h, compared to 43-fold for pstS1 and 37-fold for sphX. A shift to high-light conditions caused a transient increase of all PBPs, whereas N stress primarily increased expression of sphX. Interposon mutagenesis of each PBP demonstrated that disruption of pstS1 alone caused constitutive expression of pho regulon genes, implicating PstS1 as a major component of the P(i) sensing machinery. The pstS1 mutant was also transformation incompetent. (32)P(i) radiotracer uptake experiments using pst1 and pst2 deletion mutants showed that Pst1 acts as a low-affinity, high-velocity transporter (K(s), 3.7 + or - 0.7 microM; V(max), 31.18 + or - 3.96 fmol cell(-1) min(-1)) and Pst2 acts as a high-affinity, low-velocity system (K(s), 0.07 + or - 0.01 microM; V(max), 0.88 + or - 0.11 fmol cell(-1) min(-1)). These P(i) ABC transporters thus exhibit differences in both kinetic and regulatory properties, the former trait potentially dramatically increasing the dynamic range of P(i) transport into the cell, which has potential implications for our understanding of the ecological success of this key microbial group.

PtrA is required for coordinate regulation of gene expression during phosphate stress in a marine Synechococcus.

Previous microarray analyses have shown a key role for the two-component system PhoBR (SYNW0947, SYNW0948) in the regulation of P transport and metabolism in the marine cyanobacterium Synechococcus sp. WH8102. However, there is some evidence that another regulator, SYNW1019 (PtrA), probably under the control of PhoBR, is involved in the response to P depletion. PtrA is a member of the cAMP receptor protein transcriptional regulator family that shows homology to NtcA, the global nitrogen regulator in cyanobacteria. To define the role of this regulator, we constructed a mutant by insertional inactivation and compared the physiology of wild-type Synechcococcus sp. WH8102 with the ptrA mutant under P-replete and P-stress conditions. In response to P stress the ptrA mutant failed to upregulate phosphatase activity. Microarrays and quantitative RT-PCR indicate that a subset of the Pho regulon is controlled by PtrA, including two phosphatases, a predicted phytase and a gene of unknown function psip1 (SYNW0165), all of which are highly upregulated during P limitation. Electrophoretic mobility shift assays indicate binding of overexpressed PtrA to promoter sequences upstream of the induced genes. This work suggests a two-tiered response to P depletion in this strain, the first being PhoB-dependent induction of high-affinity PO(4) transporters, and the second the PtrA-dependent induction of phosphatases for scavenging organic P. The levels of numerous other transcripts are also directly or indirectly influenced by PtrA, including those involved in cell-surface modification, metal uptake, photosynthesis, stress responses and other metabolic processes, which may indicate a wider role for PtrA in cellular regulation in marine picocyanobacteria.

Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria.

BACKGROUND: The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group. RESULTS: Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance. CONCLUSION: We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data.

Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes.

Marine cyanobacteria of the genera Prochlorococcus and Synechococcus are important contributors to global primary production occupying a key position at the base of marine food webs. The genetically diverse nature of each genus is likely an important reason for their successful colonization of vast tracts of the world's oceans, a feature that has led to detailed analysis of the distribution of these genetic lineages at the local and ocean basin scale. Here, we extend these analyses to the global dimension, using new data from cruises in the Pacific, Indian and Arctic Oceans in combination with data from previous studies in the Atlantic Ocean, Arabian Sea, Red Sea and a circumnavigation of the southern hemisphere to form a data set which comprises most of the world's major ocean systems. We show that the distribution patterns of Prochlorococcus and Synechococcus lineages are remarkably similar in different ocean systems with comparable environmental conditions, but producing a strikingly different 'signature' in the four major ocean domains or biomes (the Polar Domain, Coastal Boundary Domain, Trade Winds Domain and Westerly Winds Domain). This clearly reiterates the idea of spatial partitioning of individual cyanobacterial lineages, but at the global scale.

High vertical and low horizontal diversity of Prochlorococcus ecotypes in the Mediterranean Sea in summer.

Natural populations of the marine cyanobacterium Prochlorococcus exist as two main ecotypes, inhabiting different layers of the ocean's photic zone. These so-called high light- (HL-) and low light (LL-) adapted ecotypes are both physiologically and genetically distinct. HL strains can be separated into two major clades (HLI and HLII), whereas LL strains are more diverse. Here, we used several molecular techniques to study the genetic diversity of natural Prochlorococcus populations during the Prosope cruise in the Mediterranean Sea in the summer of 1999. Using a dot blot hybridization technique, we found that HLI was the dominant HL group and was confined to the upper mixed layer. In contrast, LL ecotypes were only found below the thermocline. Secondly, a restriction fragment length polymorphism analysis of PCR-amplified pcb genes (encoding the major light-harvesting proteins of Prochlorococcus) suggested that there were at least four genetically different ecotypes, occupying distinct but overlapping light niches in the photic zone. At comparable depths, similar banding patterns were observed throughout the sampled area, suggesting a horizontal homogenization of ecotypes. Nevertheless, environmental pcb gene sequences retrieved from different depths at two stations proved all different at the nucleotide level, suggesting a large genetic microdiversity within those ecotypes.

PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian Sea.

An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N2 fixers was developed based on newly derived sequences from Crocosphaera sp. strain WH 8501 and Cyanothece sp. strains WH 8902 and WH 8904 as well as several previously described sequences of Cyanothece sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom Climacodium frauenfeldianum. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium- and plastid-specific forward primer CYA359F (U. Nubel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol. 63:3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67 degrees E meridian from Victoria, Seychelles, to Muscat, Oman (0.5 degrees S to 26 degrees N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29 degrees C) oligotrophic subsurface waters between 0 and 7 degrees N, but they were also found at a station north of Oman at 26 degrees N, 56 degrees 35'E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29 degrees C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.