Chemotaxis shapes the microscale organization of the ocean's microbiome.

The capacity of planktonic marine microorganisms to actively seek out and exploit microscale chemical hotspots has been widely theorized to affect ocean-basin scale biogeochemistry1-3, but has never been examined comprehensively in situ among natural microbial communities. Here, using a field-based microfluidic platform to quantify the behavioural responses of marine bacteria and archaea, we observed significant levels of chemotaxis towards microscale hotspots of phytoplankton-derived dissolved organic matter (DOM) at a coastal field site across multiple deployments, spanning several months. Microscale metagenomics revealed that a wide diversity of marine prokaryotes, spanning 27 bacterial and 2 archaeal phyla, displayed chemotaxis towards microscale patches of DOM derived from ten globally distributed phytoplankton species. The distinct DOM composition of each phytoplankton species attracted phylogenetically and functionally discrete populations of bacteria and archaea, with 54% of chemotactic prokaryotes displaying highly specific responses to the DOM derived from only one or two phytoplankton species. Prokaryotes exhibiting chemotaxis towards phytoplankton-derived compounds were significantly enriched in the capacity to transport and metabolize specific phytoplankton-derived chemicals, and displayed enrichment in functions conducive to symbiotic relationships, including genes involved in the production of siderophores, B vitamins and growth-promoting hormones. Our findings demonstrate that the swimming behaviour of natural prokaryotic assemblages is governed by specific chemical cues, which dictate important biogeochemical transformation processes and the establishment of ecological interactions that structure the base of the marine food web.

Rhometa: Population recombination rate estimation from metagenomic read datasets.

Prokaryotic evolution is influenced by the exchange of genetic information between species through a process referred to as recombination. The rate of recombination is a useful measure for the adaptive capacity of a prokaryotic population. We introduce Rhometa (https://github.com/sid-krish/Rhometa), a new software package to determine recombination rates from shotgun sequencing reads of metagenomes. It extends the composite likelihood approach for population recombination rate estimation and enables the analysis of modern short-read datasets. We evaluated Rhometa over a broad range of sequencing depths and complexities, using simulated and real experimental short-read data aligned to external reference genomes. Rhometa offers a comprehensive solution for determining population recombination rates from contemporary metagenomic read datasets. Rhometa extends the capabilities of conventional sequence-based composite likelihood population recombination rate estimators to include modern aligned metagenomic read datasets with diverse sequencing depths, thereby enabling the effective application of these techniques and their high accuracy rates to the field of metagenomics. Using simulated datasets, we show that our method performs well, with its accuracy improving with increasing numbers of genomes. Rhometa was validated on a real S. pneumoniae transformation experiment, where we show that it obtains plausible estimates of the rate of recombination. Finally, the program was also run on ocean surface water metagenomic datasets, through which we demonstrate that the program works on uncultured metagenomic datasets.

Defining marine bacterioplankton community assembly rules by contrasting the importance of environmental determinants and biotic interactions.

Bacterioplankton communities govern marine productivity and biogeochemical cycling, yet drivers of bacterioplankton assembly remain unclear. Here, we contrast the relative contribution of deterministic processes (environmental factors and biotic interactions) in driving temporal dynamics of bacterioplankton diversity at three different oceanographic time series locations, spanning 15° of latitude, which are each characterized by different environmental conditions and varying degrees of seasonality. Monthly surface samples (5.5 years) were analysed using 16S rRNA amplicon sequencing. The high- and mid-latitude sites of Maria Island and Port Hacking were characterized by high and intermediate levels of environmental heterogeneity, respectively, with both alpha diversity (72%; 24% of total variation) and beta diversity (32%; 30%) patterns within bacterioplankton assemblages explained by day length, ammonium, and mixed layer depth. In contrast, North Stradbroke Island, a sub-tropical location where environmental conditions are less variable, interspecific interactions were of increased importance in structuring bacterioplankton diversity (alpha: 33%; beta: 26%) with environment only contributing 11% and 13% to predicting diversity, respectively. Our results demonstrate that bacterioplankton diversity is the result of both deterministic environmental and biotic processes and that the importance of these different deterministic processes varies, potential in response to environmental heterogeneity.

Cyanorak v2.1: a scalable information system dedicated to the visualization and expert curation of marine and brackish picocyanobacteria genomes.

Cyanorak v2.1 (http://www.sb-roscoff.fr/cyanorak) is an information system dedicated to visualizing, comparing and curating the genomes of Prochlorococcus, Synechococcus and Cyanobium, the most abundant photosynthetic microorganisms on Earth. The database encompasses sequences from 97 genomes, covering most of the wide genetic diversity known so far within these groups, and which were split into 25,834 clusters of likely orthologous groups (CLOGs). The user interface gives access to genomic characteristics, accession numbers as well as an interactive map showing strain isolation sites. The main entry to the database is through search for a term (gene name, product, etc.), resulting in a list of CLOGs and individual genes. Each CLOG benefits from a rich functional annotation including EggNOG, EC/K numbers, GO terms, TIGR Roles, custom-designed Cyanorak Roles as well as several protein motif predictions. Cyanorak also displays a phyletic profile, indicating the genotype and pigment type for each CLOG, and a genome viewer (Jbrowse) to visualize additional data on each genome such as predicted operons, genomic islands or transcriptomic data, when available. This information system also includes a BLAST search tool, comparative genomic context as well as various data export options. Altogether, Cyanorak v2.1 constitutes an invaluable, scalable tool for comparative genomics of ecologically relevant marine microorganisms.

Biogeography of Southern Ocean prokaryotes: a comparison of the Indian and Pacific sectors.

We investigated the Southern Ocean (SO) prokaryote community structure via zero-radius operational taxonomic unit (zOTU) libraries generated from 16S rRNA gene sequencing of 223 full water column profiles. Samples reveal the prokaryote diversity trend between discrete water masses across multiple depths and latitudes in Indian (71-99°E, summer) and Pacific (170-174°W, autumn-winter) sectors of the SO. At higher taxonomic levels (phylum-family) we observed water masses to harbour distinct communities across both sectors, but observed sectorial variations at lower taxonomic levels (genus-zOTU) and relative abundance shifts for key taxa such as Flavobacteria, SAR324/Marinimicrobia, Nitrosopumilus and Nitrosopelagicus at both epi- and bathy-abyssopelagic water masses. Common surface bacteria were abundant in several deep-water masses and vice-versa suggesting connectivity between surface and deep-water microbial assemblages. Bacteria from same-sector Antarctic Bottom Water samples showed patchy, high beta-diversity which did not correlate well with measured environmental parameters or geographical distance. Unconventional depth distribution patterns were observed for key archaeal groups: Crenarchaeota was found across all depths in the water column and persistent high relative abundances of common epipelagic archaeon Nitrosopelagicus was observed in deep-water masses. Our findings reveal substantial regional variability of SO prokaryote assemblages that we argue should be considered in wide-scale SO ecosystem microbial modelling.

Functional characterisation of substrate-binding proteins to address nutrient uptake in marine picocyanobacteria.

Marine cyanobacteria are key primary producers, contributing significantly to the microbial food web and biogeochemical cycles by releasing and importing many essential nutrients cycled through the environment. A subgroup of these, the picocyanobacteria (Synechococcus and Prochlorococcus), have colonised almost all marine ecosystems, covering a range of distinct light and temperature conditions, and nutrient profiles. The intra-clade diversities displayed by this monophyletic branch of cyanobacteria is indicative of their success across a broad range of environments. Part of this diversity is due to nutrient acquisition mechanisms, such as the use of high-affinity ATP-binding cassette (ABC) transporters to competitively acquire nutrients, particularly in oligotrophic (nutrient scarce) marine environments. The specificity of nutrient uptake in ABC transporters is primarily determined by the peripheral substrate-binding protein (SBP), a receptor protein that mediates ligand recognition and initiates translocation into the cell. The recent availability of large numbers of sequenced picocyanobacterial genomes indicates both Synechococcus and Prochlorococcus apportion >50% of their transport capacity to ABC transport systems. However, the low degree of sequence homology among the SBP family limits the reliability of functional assignments using sequence annotation and prediction tools. This review highlights the use of known SBP structural representatives for the uptake of key nutrient classes by cyanobacteria to compare with predicted SBP functionalities within sequenced marine picocyanobacteria genomes. This review shows the broad range of conserved biochemical functions of picocyanobacteria and the range of novel and hypothetical ABC transport systems that require further functional characterisation.

Oceanographic boundaries constrain microbial diversity gradients in the South Pacific Ocean.

Marine microbes along with microeukaryotes are key regulators of oceanic biogeochemical pathways. Here we present a high-resolution (every 0.5° of latitude) dataset describing microbial pro- and eukaryotic richness in the surface and just below the thermocline along a 7,000-km transect from 66°S at the Antarctic ice edge to the equator in the South Pacific Ocean. The transect, conducted in austral winter, covered key oceanographic features including crossing of the polar front (PF), the subtropical front (STF), and the equatorial upwelling region. Our data indicate that temperature does not determine patterns of marine microbial richness, complementing the global model data from Ladau et al. [Ladau J, et al. (2013) ISME J 7:1669-1677]. Rather, NH4+, nanophytoplankton, and primary productivity were the main drivers for archaeal and bacterial richness. Eukaryote richness was highest in the least-productive ocean region, the tropical oligotrophic province. We also observed a unique diversity pattern in the South Pacific Ocean: a regional increase in archaeal and bacterial diversity between 10°S and the equator. Rapoport's rule describes the tendency for the latitudinal ranges of species to increase with latitude. Our data showed that the mean latitudinal ranges of archaea and bacteria decreased with latitude. We show that permanent oceanographic features, such as the STF and the equatorial upwelling, can have a significant influence on both alpha-diversity and beta-diversity of pro- and eukaryotes.

Comparative membrane proteomics reveal contrasting adaptation strategies for coastal and oceanic marine Synechococcus cyanobacteria.

Marine cyanobacteria genus Synechococcus are among the most abundant and widespread primary producers in the open ocean. Synechococcus strains belonging to different clades have adapted distinct strategies for growth and survival across a range of marine conditions. Clades I and IV are prevalent in colder, mesotrophic, coastal waters, while clades II and III prefer warm, oligotrophic open oceans. To gain insight into the cellular resources these unicellular organisms invest in adaptation strategies we performed shotgun membrane proteomics of four Synechococcus spp. strains namely CC9311 (clade I), CC9605 (clade II), WH8102 (clade III) and CC9902 (clade IV). Comparative membrane proteomes analysis demonstrated that CC9902 and WH8102 showed high resource allocation for phosphate uptake, accounting for 44% and 38% of overall transporter protein expression of the species. WH8102 showed high expression of the iron uptake ATP-binding cassette binding protein FutA, suggesting that a high binding affinity for iron is possibly a key adaptation strategy for some strains in oligotrophic ocean environments. One protein annotated as a phosphatase 2c (Sync_2505 and Syncc9902_0387) was highly expressed in the coastal mesotrophic strains CC9311 and CC9902, constituting 14%-16% of total membrane protein, indicating a vital, but undefined function, for strains living in temperate mesotrophic environments.

Microbial tropicalization driven by a strengthening western ocean boundary current.

Western boundary currents (WBCs) redistribute heat and oligotrophic seawater from the tropics to temperate latitudes, with several displaying substantial climate change-driven intensification over the last century. Strengthening WBCs have been implicated in the poleward range expansion of marine macroflora and fauna, however, the impacts on the structure and function of temperate microbial communities are largely unknown. Here we show that the major subtropical WBC of the South Pacific Ocean, the East Australian Current (EAC), transports microbial assemblages that maintain tropical and oligotrophic (k-strategist) signatures, to seasonally displace more copiotrophic (r-strategist) temperate microbial populations within temperate latitudes of the Tasman Sea. We identified specific characteristics of EAC microbial assemblages compared with non-EAC assemblages, including strain transitions within the SAR11 clade, enrichment of Prochlorococcus, predicted smaller genome sizes and shifts in the importance of several functional genes, including those associated with cyanobacterial photosynthesis, secondary metabolism and fatty acid and lipid transport. At a temperate time-series site in the Tasman Sea, we observed significant reductions in standing stocks of total carbon and chlorophyll a, and a shift towards smaller phytoplankton and carnivorous copepods, associated with the seasonal impact of the EAC microbial assemblage. In light of the substantial shifts in microbial assemblage structure and function associated with the EAC, we conclude that climate-driven expansions of WBCs will expand the range of tropical oligotrophic microbes, and potentially profoundly impact the trophic status of temperate waters.

Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria.

Prochlorococcus and Synechococcus are the two most abundant and widespread phytoplankton in the global ocean. To better understand the factors controlling their biogeography, a reference database of the high-resolution taxonomic marker petB, encoding cytochrome b6, was used to recruit reads out of 109 metagenomes from the Tara Oceans expedition. An unsuspected novel genetic diversity was unveiled within both genera, even for the most abundant and well-characterized clades, and 136 divergent petB sequences were successfully assembled from metagenomic reads, significantly enriching the reference database. We then defined Ecologically Significant Taxonomic Units (ESTUs)-that is, organisms belonging to the same clade and occupying a common oceanic niche. Three major ESTU assemblages were identified along the cruise transect for Prochlorococcus and eight for Synechococcus Although Prochlorococcus HLIIIA and HLIVA ESTUs codominated in iron-depleted areas of the Pacific Ocean, CRD1 and the yet-to-be cultured EnvB were the prevalent Synechococcus clades in this area, with three different CRD1 and EnvB ESTUs occupying distinct ecological niches with regard to iron availability and temperature. Sharp community shifts were also observed over short geographic distances-for example, around the Marquesas Islands or between southern Indian and Atlantic Oceans-pointing to a tight correlation between ESTU assemblages and specific physico-chemical parameters. Together, this study demonstrates that there is a previously overlooked, ecologically meaningful, fine-scale diversity within some currently defined picocyanobacterial ecotypes, bringing novel insights into the ecology, diversity, and biology of the two most abundant phototrophs on Earth.

A mobility shift assay for DNA detection using nanochannel gradient electrophoresis.

Conventional detection of pathogenic or other biological contamination relies on amplification of DNA using sequence-specific primers. Recent work in nanofluidics has shown very high concentration enhancement of biomolecules with some degree of simultaneous separation. This work demonstrates the combination of these two approaches by selectively concentrating a mobility-shifted hybridization product, potentially enabling rapid detection of rare DNA fragments such as highly specific 16S ribosomal DNA. We have performed conductivity gradient electrofocusing within nanofluidic channels and have shown concentration of hybridized peptide nucleic acids and DNA oligomers. We also show selectivity to single base-pair mismatch on 18-mer oligos. This approach may enable sensitive optical detection of small amounts of DNA.

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.

A marine heatwave drives significant shifts in pelagic microbiology.

Marine heatwaves (MHWs) cause disruption to marine ecosystems, deleteriously impacting macroflora and fauna. However, effects on microorganisms are relatively unknown despite ocean temperature being a major determinant of assemblage structure. Using data from thousands of Southern Hemisphere samples, we reveal that during an "unprecedented" 2015/16 Tasman Sea MHW, temperatures approached or surpassed the upper thermal boundary of many endemic taxa. Temperate microbial assemblages underwent a profound transition to niche states aligned with sites over 1000 km equatorward, adapting to higher temperatures and lower nutrient conditions bought on by the MHW. MHW conditions also modulate seasonal patterns of microbial diversity and support novel assemblage compositions. The most significant affects of MHWs on microbial assemblages occurred during warmer months, when temperatures exceeded the upper climatological bounds. Trends in microbial response across several MHWs in different locations suggest these are emergent properties of temperate ocean warming, which may facilitate monitoring, prediction and adaptation efforts.

Elemental composition of natural populations of key microbial groups in Atlantic waters.

Intracellular carbon (C), nitrogen (N) and phosphorus (P) content of marine phytoplankton and bacterioplankton can vary according to cell requirements or physiological acclimation to growth under nutrient limited conditions. Although such variation in macronutrient content is well known for cultured organisms, there is a dearth of data from natural populations that reside under a range of environmental conditions. Here, we compare C, N and P content of Synechococcus, Prochlorococcus, low nucleic acid (LNA) content bacterioplankton and small plastidic protists inhabiting surface waters of the North and South subtropical gyres and the Equatorial Region of the Atlantic Ocean. While intracellular C:N ratios ranged between 3.5 and 6, i.e. below the Redfield ratio of 6.6, all the C:P and N:P ratios were up to 10 times higher than the corresponding Redfield ratio of 106 and 16, respectively, reaching and in some cases exceeding maximum values reported in the literature. Similar C:P or N:P ratios in areas with different concentrations of inorganic phosphorus suggests that this is not just a response to the prevailing environmental conditions but an indication of the extremely low P content of these oceanic microbes.

Differential global distribution of marine picocyanobacteria gene clusters reveals distinct niche-related adaptive strategies.

The ever-increasing number of available microbial genomes and metagenomes provides new opportunities to investigate the links between niche partitioning and genome evolution in the ocean, especially for the abundant and ubiquitous marine picocyanobacteria Prochlorococcus and Synechococcus. Here, by combining metagenome analyses of the Tara Oceans dataset with comparative genomics, including phyletic patterns and genomic context of individual genes from 256 reference genomes, we show that picocyanobacterial communities thriving in different niches possess distinct gene repertoires. We also identify clusters of adjacent genes that display specific distribution patterns in the field (eCAGs) and are thus potentially involved in the same metabolic pathway and may have a key role in niche adaptation. Several eCAGs are likely involved in the uptake or incorporation of complex organic forms of nutrients, such as guanidine, cyanate, cyanide, pyrimidine, or phosphonates, which might be either directly used by cells, for example for the biosynthesis of proteins or DNA, or degraded to inorganic nitrogen and/or phosphorus forms. We also highlight the enrichment of eCAGs involved in polysaccharide capsule biosynthesis in Synechococcus populations thriving in both nitrogen- and phosphorus-depleted areas vs. low-iron (Fe) regions, suggesting that the complexes they encode may be too energy-consuming for picocyanobacteria thriving in the latter areas. In contrast, Prochlorococcus populations thriving in Fe-depleted areas specifically possess an alternative respiratory terminal oxidase, potentially involved in the reduction of Fe(III) to Fe(II). Altogether, this study provides insights into how phytoplankton communities populate oceanic ecosystems, which is relevant to understanding their capacity to respond to ongoing climate change.

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.

Resolving low-expression cell surface antigens by time-gated orthogonal scanning automated microscopy.

We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning automated microscopy (OSAM) technique to rapidly process a large population of target-only cells on microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events.

Correction: McLennan et al. Assessing the Use of Molecular Barcoding and qPCR for Investigating the Ecology of Prorocentrum minimum (Dinophyceae), a Harmful Algal Species. Microorganisms 2021, 9, 510.

The authors wish to make the following corrections to this paper [...].

Chemotaxis may assist marine heterotrophic bacterial diazotrophs to find microzones suitable for N2 fixation in the pelagic ocean.

Heterotrophic bacterial diazotrophs (HBDs) are ubiquitous in the pelagic ocean, where they have been predicted to carry out the anaerobic process of nitrogen fixation within low-oxygen microenvironments associated with marine pelagic particles. However, the mechanisms enabling particle colonization by HBDs are unknown. We hypothesized that HBDs use chemotaxis to locate and colonize suitable microenvironments, and showed that a cultivated marine HBD is chemotactic toward amino acids and phytoplankton-derived DOM. Using an in situ chemotaxis assay, we also discovered that diverse HBDs at a coastal site are motile and chemotactic toward DOM from various phytoplankton taxa and, indeed, that the proportion of diazotrophs was up to seven times higher among the motile fraction of the bacterial community compared to the bulk seawater community. Finally, three of four HBD isolates and 16 of 17 HBD metagenome assembled genomes, recovered from major ocean basins and locations along the Australian coast, each encoded >85% of proteins affiliated with the bacterial chemotaxis pathway. These results document the widespread capacity for chemotaxis in diverse and globally relevant marine HBDs. We suggest that HBDs could use chemotaxis to seek out and colonize low-oxygen microenvironments suitable for nitrogen fixation, such as those formed on marine particles. Chemotaxis in HBDs could therefore affect marine nitrogen and carbon biogeochemistry by facilitating nitrogen fixation within otherwise oxic waters, while also altering particle degradation and the efficiency of the biological pump.