Seasonal changes in nitrogen assimilation have been studied in the western English Channel by sampling at approximately weekly intervals for 12 months. Nitrate concentrations showed strong seasonal variations. Available nitrogen in the winter was dominated by nitrate but this was close to limit of detection from May to September, after the spring phytoplankton bloom. The 15N uptake experiments showed that nitrate was the nitrogen source for the spring phytoplankton bloom but regenerated nitrogen supported phytoplankton productivity throughout the summer. The average annual f-ratio was 0.35, which demonstrated the importance of ammonia regeneration in this dynamic temperate region. Nitrogen uptake rate measurements were related to the phytoplankton responsible by assessing the relative abundance of nitrate reductase (NR) genes and the expression of NR among eukaryotic phytoplankton. Strong signals were detected from NR sequences that are not associated with known phylotypes or cultures. NR sequences from the diatom Phaeodactylum tricornutum were highly represented in gene abundance and expression, and were significantly correlated with f-ratio. The results demonstrate that analysis of functional genes provides additional information, and may be able to give better indications of which phytoplankton species are responsible for the observed seasonal changes in f-ratio than microscopic phytoplankton identification.
f-ratio; seasonal variation; nitrate reductase gene expression; microarrays
Viruses that infect the marine cyanobacterium Prochlorococcus have the potential to impact the growth, productivity, diversity and abundance of their hosts. In this study, changes in the microdiversity of cyanomyoviruses were investigated in 10 environmental samples taken along a North–South Atlantic Ocean transect using a myoviral-specific PCR-sequencing approach. Phylogenetic analyses of 630 viral g20 clones from this study, with 786 published g20 sequences, revealed that myoviral populations in the Atlantic Ocean had higher diversity than previously reported, with several novel putative g20 clades. Some of these clades were detected throughout the Atlantic Ocean. Multivariate statistical analyses did not reveal any significant correlations between myoviral diversity and environmental parameters, although myoviral diversity appeared to be lowest in samples collected from the north and south of the transect where Prochlorococcus diversity was also lowest. The results were correlated to the abundance and diversity of the co-occurring Prochlorococcus and Synechococcus populations, but revealed no significant correlations to either of the two potential host genera. This study provides evidence that cyanophages have extremely high and variable diversity and are distributed over large areas of the Atlantic Ocean.
cyanophage; virus; Prochlorococcus; marine; g20; genetic diversity
Here we describe, the longest microbial time-series analyzed to date using high-resolution 16S rRNA tag pyrosequencing of samples taken monthly over 6 years at a temperate marine coastal site off Plymouth, UK. Data treatment effected the estimation of community richness over a 6-year period, whereby 8794 operational taxonomic units (OTUs) were identified using single-linkage preclustering and 21 130 OTUs were identified by denoising the data. The Alphaproteobacteria were the most abundant Class, and the most frequently recorded OTUs were members of the Rickettsiales (SAR 11) and Rhodobacteriales. This near-surface ocean bacterial community showed strong repeatable seasonal patterns, which were defined by winter peaks in diversity across all years. Environmental variables explained far more variation in seasonally predictable bacteria than did data on protists or metazoan biomass. Change in day length alone explains >65% of the variance in community diversity. The results suggested that seasonal changes in environmental variables are more important than trophic interactions. Interestingly, microbial association network analysis showed that correlations in abundance were stronger within bacterial taxa rather than between bacteria and eukaryotes, or between bacteria and environmental variables.
16S rRNA; microbial; bacteria; community; diversity; model
The pH of the surface ocean is changing as a result of increases in atmospheric carbon dioxide (CO2), and there are concerns about potential impacts of lower pH and associated alterations in seawater carbonate chemistry on the biogeochemical processes in the ocean. However, it is important to place these changes within the context of pH in the present-day ocean, which is not constant; it varies systematically with season, depth and along productivity gradients. Yet this natural variability in pH has rarely been considered in assessments of the effect of ocean acidification on marine microbes. Surface pH can change as a consequence of microbial utilization and production of carbon dioxide, and to a lesser extent other microbially mediated processes such as nitrification. Useful comparisons can be made with microbes in other aquatic environments that readily accommodate very large and rapid pH change. For example, in many freshwater lakes, pH changes that are orders of magnitude greater than those projected for the twenty second century oceans can occur over periods of hours. Marine and freshwater assemblages have always experienced variable pH conditions. Therefore, an appropriate null hypothesis may be, until evidence is obtained to the contrary, that major biogeochemical processes in the oceans other than calcification will not be fundamentally different under future higher CO2/lower pH conditions.
ocean acidification; rapid pH change; biogeochemical processes
The effect of ocean acidification conditions has been investigated in cultures of the diatom Thalassiosira pseudonana CCMP1335. Expected end-of-the-century pCO2 (aq) concentrations of 760 µatm (equivalent to pH 7.8) were compared with present-day condition (380 µatm CO2, pH 8.1). Batch culture pH changed rapidly because of CO2 (aq) assimilation and pH targets of 7.8 and 8.1 could not be sustained. Long-term (∼100 generation) pH-auxostat, continuous cultures could be maintained at target pH when cell density was kept low (<2×105 cells mL−1). After 3 months continuous culture, the C:N ratio was slightly decreased under high CO2 conditions and red fluorescence per cell was slightly increased. However, no change was detected in photosynthetic efficiency (Fv/Fm) or functional cross section of PS II (σPSII). Elevated pCO2 has been predicted to be beneficial to diatoms due to reduced cost of carbon concentration mechanisms. There was reduced transcription of one putative δ-carbonic anhydrase (CA-4) after 3 months growth at increased CO2 but 3 other δ-CAs and the small subunit of RUBISCO showed no change. There was no evidence of adaptation or clade selection of T. pseudonana after ∼100 generations at elevated CO2. On the basis of this long-term culture, pH change of this magnitude in the future ocean may have little effect on T. pseudonana in the absence of genetic adaption.
How microbial communities change over time in response to the environment is poorly understood. Previously a six-year time series of 16S rRNA V6 data from the Western English Channel demonstrated robust seasonal structure within the bacterial community, with diversity negatively correlated with day-length. Here we determine whether metagenomes and metatranscriptomes follow similar patterns. We generated 16S rRNA datasets, metagenomes (1.2 GB) and metatranscriptomes (157 MB) for eight additional time points sampled in 2008, representing three seasons (Winter, Spring, Summer) and including day and night samples. This is the first microbial ‘multi-omic’ study to combine 16S rRNA amplicon sequencing with metagenomic and metatranscriptomic profiling. Five main conclusions can be drawn from analysis of these data: 1) Archaea follow the same seasonal patterns as Bacteria, but show lower relative diversity; 2) Higher 16S rRNA diversity also reflects a higher diversity of transcripts; 3) Diversity is highest in winter and at night; 4) Community-level changes in 16S-based diversity and metagenomic profiles are better explained by seasonal patterns (with samples closest in time being most similar), while metatranscriptomic profiles are better explained by diel patterns and shifts in particular categories (i.e., functional groups) of genes; 5) Changes in key genes occur among seasons and between day and night (i.e., photosynthesis); but these samples contain large numbers of orphan genes without known homologues and it is these unknown gene sets that appear to contribute most towards defining the differences observed between times. Despite the huge diversity of these microbial communities, there are clear signs of predictable patterns and detectable stability over time. Renewed and intensified efforts are required to reveal fundamental deterministic patterns in the most complex microbial communities. Further, the presence of a substantial proportion of orphan sequences underscores the need to determine the gene products of sequences with currently unknown function.
Both metagenomic data and metatranscriptomic data were collected from surface water (0-2m) of the L4 sampling station (50.2518 N, 4.2089 W), which is part of the Western Channel Observatory long-term coastal-marine monitoring station. We previously generated from this area a six-year time series of 16S rRNA V6 data, which demonstrated robust seasonal structure for the bacterial community, with diversity correlated with day length. Here we describe the features of these metagenomes and metatranscriptomes. We generated 8 metagenomes (4.5 million sequences, 1.9 Gbp, average read-length 350 bp) and 7 metatranscriptomes (392,632 putative mRNA-derived sequences, 159 Mbp, average read-length 272 bp) for eight time-points sampled in 2008. These time points represent three seasons (winter, spring, and summer) and include both day and night samples. These data demonstrate the major differences between genetic potential and actuality, whereby genomes follow general seasonal trends yet with surprisingly little change in the functional potential over time; transcripts tended to be far more structured by changes occurring between day and night.
Marine; aerobic; surface water; coastal; temperate; metagenome; metatranscriptome; pyrosequencing; time-series; diel; seasonal
Sequencing the expressed genetic information of an ecosystem (metatranscriptome) can provide information about the response of organisms to varying environmental conditions. Until recently, metatranscriptomics has been limited to microarray technology and random cloning methodologies. The application of high-throughput sequencing technology is now enabling access to both known and previously unknown transcripts in natural communities.
We present a study of a complex marine metatranscriptome obtained from random whole-community mRNA using the GS-FLX Pyrosequencing technology. Eight samples, four DNA and four mRNA, were processed from two time points in a controlled coastal ocean mesocosm study (Bergen, Norway) involving an induced phytoplankton bloom producing a total of 323,161,989 base pairs. Our study confirms the finding of the first published metatranscriptomic studies of marine and soil environments that metatranscriptomics targets highly expressed sequences which are frequently novel. Our alternative methodology increases the range of experimental options available for conducting such studies and is characterized by an exceptional enrichment of mRNA (99.92%) versus ribosomal RNA. Analysis of corresponding metagenomes confirms much higher levels of assembly in the metatranscriptomic samples and a far higher yield of large gene families with >100 members, ∼91% of which were novel.
This study provides further evidence that metatranscriptomic studies of natural microbial communities are not only feasible, but when paired with metagenomic data sets, offer an unprecedented opportunity to explore both structure and function of microbial communities – if we can overcome the challenges of elucidating the functions of so many never-seen-before gene families.
The green seaweed Ulva has been shown to detect signal molecules produced by bacteria. Biofilms that release N-acylhomoserine lactones (AHLs) attract zoospores—the motile reproductive stages of Ulva. The evidence for AHL involvement is based on several independent lines of evidence, including the observation that zoospores are attracted to wild-type bacteria that produce AHLs but are not attracted to mutants that do not produce signal molecules. Synthetic AHL also attracts zoospores and the attraction is lost in the presence of autoinducer inactivation (AiiA) protein. The mechanism of attraction is not chemotactic but involves chemokinesis. When zoospores detect AHLs, the swimming rate is reduced and this results in accumulation of cells at the source of the AHL. It has been demonstrated that the detection of AHLs results in calcium influx into the zoospore. This is the first example of a calcium signalling event in a eukaryote in response to bacterial quorum sensing molecules. The role of AHLs in the ecology of Ulva is discussed. It is probable that AHLs act as cues for the settlement of zoospores, rather than being directly involved as a signalling mechanism.
cross-kingdom signalling; Ulva; N-acylhomoserine lactones; chemokinesis; calcium ion influx
With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the ‘transparency’ of the information contained in existing genomic databases.
quorum sensing; plant pathogen; human pathogen; quorum quenching; prokaryote/eukaryote signalling; wastewater treatment
Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.
Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.
A fundamental problem in estuarine microbiology studies is the accurate determination of the density in the water column of both free-living bacteria and those attached to suspended particulate matter. When a water sample is filtered and the filter is viewed by epifluorescence microscopy, counts can be made of the numbers of bacteria which are seen on the filter background (free-living) and those which appear to lie on sediment particles (both free-living and attached). With only the additional knowledge of the proportion of the filter area covered by particles (a quantity that is straightforwardly determined by stereological point counting), results from geometric probability were used to determine the expected number of bacteria which are hidden by particles and hence to provide an estimation scheme for the true densities of free-living and attached bacteria. Variance equations based on a Taylor series are given, and a partial check of the method is attempted with controlled mixtures of bacteria and sediment. An alternative procedure is also proposed, in which the natural attached/free-living ratio is altered by an intervention experiment, allowing an estimation which is less model dependent but more labor intensive. Both methods are applied to a series of samples from the Tamar estuary, United Kingdom, taken in April 1985. A notable conclusion is that there are always more free-living than attached bacteria in the water column throughout the estuary.
The oceans are the Earth's largest ecosystem, covering 70% of our planet and providing goods and services for the majority of the world's population. Understanding the complex abiotic and biotic processes on the micro- to macroscale is the key to protect and sustain the marine ecosystem. Marine microorganisms are the ‘gatekeepers’ of the biotic processes that control the global cycles of energy and organic matter. A multinational, multidisciplinary approach, bringing together research on oceanography, biodiversity and genomics, is now needed to understand and finally predict the complex responses of the marine ecosystem to ongoing global changes. Such an integrative approach will not only bring better understanding of the complex interplay of the organisms with their environment, but will reveal a wealth of new metabolic processes and functions, which have a high potential for biotechnological applications. This potential has already been recognized by the European commission which funded a series of workshops and projects on marine genomics in the sixth and seventh framework programme. Nevertheless, there remain many obstacles to achieving the goal – such as a lack of bioinformatics tailored for the marine field, consistent data acquisition and exchange, as well as continuous monitoring programmes and a lack of relevant marine bacterial models. Marine ecosystems research is complex and challenging, but it also harbours the opportunity to cross the borders between disciplines and countries to finally create a rewarding marine research era that is more than the sum of its parts.
The composition and activities of microbes from diverse habitats have been the focus of intense research during the past decade with this research being spurred on largely by advances in molecular biology and genomic technologies. In recent years environmental microbiology has entered very firmly into the age of the ‘omics’– (meta)genomics, proteomics, metabolomics, transcriptomics – with probably others on the rise. Microbes are essential participants in all biogeochemical processes on our planet, and the practical applications of what we are learning from the use of molecular approaches has altered how we view biological systems. In addition, there is considerable potential to use information about uncultured microbes in biodiscovery research as microbes provide a rich source of discovery for novel genes, enzymes and metabolic pathways. This review explores the brief history of genomic and metagenomic approaches to study environmental microbial assemblages and describes some of the future challenges involved in broadening our approaches – leading to new insights for understanding environmental problems and enabling biodiscovery research.