Metaproteomic analysis of a winter to spring succession in coastal northwest Atlantic Ocean microbial plankton

In this study, we used comparative metaproteomics to investigate the metabolic activity of microbial plankton inhabiting a seasonally hypoxic basin in the Northwest Atlantic Ocean (Bedford Basin). From winter to spring, we observed a seasonal increase in high-affinity membrane transport proteins involved in scavenging of organic substrates; Rhodobacterales transporters were strongly associated with the spring phytoplankton bloom, whereas SAR11 transporters were abundant in the underlying waters. A diverse array of transporters for organic compounds were similar to the SAR324 clade, revealing an active heterotrophic lifestyle in coastal waters. Proteins involved in methanol oxidation (from the OM43 clade) and carbon monoxide (from a wide variety of bacteria) were identified throughout Bedford Basin. Metabolic niche partitioning between the SUP05 and ARCTIC96BD-19 clades, which together comprise the Gamma-proteobacterial sulfur oxidizers group was apparent. ARCTIC96BD-19 proteins involved in the transport of organic compounds indicated that in productive coastal waters this lineage tends toward a heterotrophic metabolism. In contrast, the identification of sulfur oxidation proteins from SUP05 indicated the use of reduced sulfur as an energy source in hypoxic bottom water. We identified an abundance of Marine Group I Thaumarchaeota proteins in the hypoxic deep layer, including proteins for nitrification and carbon fixation. No transporters for organic compounds were detected among the thaumarchaeal proteins, suggesting a reliance on autotrophic carbon assimilation. In summary, our analyses revealed the spatiotemporal structure of numerous metabolic activities in the coastal ocean that are central to carbon, nitrogen and sulfur cycling in the sea.     

Comparable light stimulation of organic nutrient uptake by SAR11 and Prochlorococcus in the North Atlantic subtropical gyre

Subtropical oceanic gyres are the most extensive biomes on Earth where SAR11 and Prochlorococcus bacterioplankton numerically dominate the surface waters depleted in inorganic macronutrients as well as in dissolved organic matter. In such nutrient poor conditions bacterioplankton could become photoheterotrophic, that is, potentially enhance uptake of scarce organic molecules using the available solar radiation to energise appropriate transport systems. Here, we assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions using ³³P-ATP, ³H-ATP and ³⁵S-methionine tracers. Light-stimulated uptake of these substrates was assessed in two dominant bacterioplankton groups discriminated by flow cytometric sorting of tracer-labelled cells and identified using catalysed reporter deposition fluorescence in situ hybridisation. One group of cells, encompassing 48% of all bacterioplankton, were identified as members of the SAR11 clade, whereas the other group (24% of all bacterioplankton) was Prochlorococcus. When exposed to light, SAR11 cells took 31% more ATP and 32% more methionine, whereas the Prochlorococcus cells took 33% more ATP and 34% more methionine. Other bacterioplankton did not demonstrate light stimulation. Thus, the SAR11 and Prochlorococcus groups, with distinctly different light-harvesting mechanisms, used light equally to enhance, by approximately one-third, the uptake of different types of organic molecules. Our findings indicate the significance of light-driven uptake of essential organic nutrients by the dominant bacterioplankton groups in the surface waters of one of the less productive, vast regions of the world''s oceans—the oligotrophic North Atlantic subtropical gyre.     

Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria

Marine planktonic cyanobacteria capable of fixing molecular nitrogen (termed ‘diazotrophs'') are key in biogeochemical cycling, and the nitrogen fixed is one of the major external sources of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution in tropical and subtropical oligotrophic oceans, unusually reduced genome and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism was also detected in coastal waters sampled from the Scripps Institute of Oceanography pier. We analyzed the metagenome of this UCYN-A2 population by concentrating cells by flow cytometry. Phylogenomic analysis provided strong bootstrap support for the monophyly of UCYN-A (here called UCYN-A1) and UCYN-A2 within the marine Crocosphaera sp. and Cyanothece sp. clade. UCYN-A2 shares 1159 of the 1200 UCYN-A1 protein-coding genes (96.6%) with high synteny, yet the average amino-acid sequence identity between these orthologs is only 86%. UCYN-A2 lacks the same major pathways and proteins that are absent in UCYN-A1, suggesting that both strains can be grouped at the same functional and ecological level. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction. These two variants may reflect adaptation of the host to different niches, which could be coastal and open ocean habitats.     

Influence of nutrients and currents on the genomic composition of microbes across an upwelling mosaic

Metagenomic data sets were generated from samples collected along a coastal to open ocean transect between Southern California Bight and California Current waters during a seasonal upwelling event, providing an opportunity to examine the impact of episodic pulses of cold nutrient-rich water into surface ocean microbial communities. The data set consists of ∼5.8 million predicted proteins across seven sites, from three different size classes: 0.1–0.8, 0.8–3.0 and 3.0–200.0 μm. Taxonomic and metabolic analyses suggest that sequences from the 0.1–0.8 μm size class correlated with their position along the upwelling mosaic. However, taxonomic profiles of bacteria from the larger size classes (0.8–200 μm) were less constrained by habitat and characterized by an increase in Cyanobacteria, Bacteroidetes, Flavobacteria and double-stranded DNA viral sequences. Functional annotation of transmembrane proteins indicate that sites comprised of organisms with small genomes have an enrichment of transporters with substrate specificities for amino acids, iron and cadmium, whereas organisms with larger genomes have a higher percentage of transporters for ammonium and potassium. Eukaryotic-type glutamine synthetase (GS) II proteins were identified and taxonomically classified as viral, most closely related to the GSII in Mimivirus, suggesting that marine Mimivirus-like particles may have played a role in the transfer of GSII gene functions. Additionally, a Planctomycete bloom was sampled from one upwelling site providing a rare opportunity to assess the genomic composition of a marine Planctomycete population. The significant correlations observed between genomic properties, community structure and nutrient availability provide insights into habitat-driven dynamics among oligotrophic versus upwelled marine waters adjoining each other spatially.     

Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean

Methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. We report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d⁻¹ (∼10 nmol l⁻¹d⁻¹). On average, coastal upwelling waters were 11 times greater than open ocean northern temperate (NT) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (EU) waters; suggesting that all upwelling waters upon reaching the surface (⩽20 m), contain a microbial population that uses a relatively high amount of carbon (0.3–10 nmol l⁻¹d⁻¹), derived from methanol, to support their growth. In open ocean Atlantic regions, microbial uptake of methanol into biomass was significantly lower, ranging between 0.04–0.68 nmol l⁻¹d⁻¹. Microbes in the Mauritanian coastal upwelling used up to 57% of the total methanol for assimilation of the carbon into cells, compared with an average of 12% in the EU, and 1% in NT and gyre waters. Several methylotrophic bacterial species were identified from open ocean Atlantic waters using PCR amplification of mxaF encoding methanol dehydrogenase, the key enzyme in bacterial methanol oxidation. These included Methylophaga sp., Burkholderiales sp., Methylococcaceae sp., Ancylobacter aquaticus, Paracoccus denitrificans, Methylophilus methylotrophus, Methylobacterium oryzae, Hyphomicrobium sp. and Methylosulfonomonas methylovora. Statistically significant correlations for upwelling waters between methanol uptake into cells and both chlorophyll a concentrations and methanol oxidation rates suggest that remotely sensed chlorophyll a images, in these productive areas, could be used to derive total methanol biological loss rates, a useful tool for atmospheric and marine climatically active gas modellers, and air–sea exchange scientists.     

Global distribution patterns of distinct clades of the photosynthetic picoeukaryote Ostreococcus

Ostreococcus is a marine picophytoeukaryote for which culture studies indicate there are ‘high-light'' and ‘low-light'' adapted ecotypes. Representatives of these ecotypes fall within two to three 18S ribosomal DNA (rDNA) clades for the former and one for the latter. However, clade distributions and relationships to this form of niche partitioning are unknown in nature. We developed two quantitative PCR primer-probe sets and enumerated the proposed ecotypes in the Pacific Ocean as well as the subtropical and tropical North Atlantic. Statistical differences in factors such as salinity, temperature and NO₃ indicated the ecophysiological parameters behind clade distributions are more complex than irradiance alone. Clade OII, containing the putatively low-light adapted strains, was detected at warm oligotrophic sites. In contrast, Clade OI, containing high-light adapted strains, was present in cooler mesotrophic and coastal waters. Maximal OI abundance (19 555±37 18S rDNA copies per ml) was detected in mesotrophic waters at 40 m depth, approaching the nutricline. OII was often more abundant at the deep chlorophyll maximum, when nutrient concentrations were significantly higher than at the surface (stratified euphotic zone waters). However, in mixed euphotic-zone water columns, relatively high numbers (for example, 891±107 18S rDNA copies per ml, Sargasso Sea, springtime) were detected at the surface. Both Clades OI and OII were found at multiple euphotic zone depths, but co-occurrence at the same geographical location appeared rare and was detected only in continental slope waters. In situ growth rate estimates using these primer-probes and better comprehension of physiology will enhance ecological understanding of Ostreococcus Clades OII and OI which appear to be oceanic and coastal clades, respectively.     

Bifidobacteria in Feces and Environmental Waters

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.     

Microbial diversity in arctic freshwaters is structured by inoculation of microbes from soils

Microbes are transported in hydrological networks through many environments, but the nature and dynamics of underlying microbial metacommunities and the impact of downslope inoculation on patterns of microbial diversity across landscapes are unknown. Pyrosequencing of small subunit ribosomal RNA gene hypervariable regions to characterize microbial communities along a hydrological continuum in arctic tundra showed a pattern of decreasing diversity downslope, with highest species richness in soil waters and headwater streams, and lowest richness in lake water. In a downstream lake, 58% and 43% of the bacterial and archaeal taxa, respectively, were also detected in diverse upslope communities, including most of the numerically dominant lake taxa. In contrast, only 18% of microbial eukaryotic taxa in the lake were detected upslope. We suggest that patterns of diversity in surface waters are structured by initial inoculation from microbial reservoirs in soils followed by a species-sorting process during downslope dispersal of both common and rare microbial taxa. Our results suggest that, unlike for metazoans, a substantial portion of bacterial and archaeal diversity in surface freshwaters may originate in complex soil environments.     

Proteomic insights into the lifestyle of an environmentally relevant marine bacterium

In terms of lifestyle, free-living bacteria are classified as either oligotrophic/specialist or opportunist/generalist. Heterogeneous marine environments such as coastal waters favour the establishment of marine generalist bacteria, which code for a large pool of functions. This is basically foreseen to cope with the heterogeneity of organic matter supplied to these systems. Nevertheless, it is not known what fraction of a generalist proteome is needed for house-keeping functions or what fraction is modified to cope with environmental changes. Here, we used high-throughput proteomics to define the proteome of Ruegeria pomeroyi DSS-3, a model marine generalist bacterium of the Roseobacter clade. We evaluated its genome expression under several natural environmental conditions, revealing the versatility of the bacterium to adapt to anthropogenic influence, poor nutrient concentrations or the presence of the natural microbial community. We also assayed 30 different laboratory incubations to increase proteome coverage and to dig further into the functional genomics of the bacterium. We established its core proteome and the proteome devoted to adaptation to general cellular physiological variations (almost 50%). We suggest that the other half of its theoretical proteome is the opportunist genetic pool devoted exclusively to very specific environmental conditions.     

Cultivation and Growth Characteristics of a Diverse Group of Oligotrophic Marine Gammaproteobacteria

Forty-four novel strains of Gammaproteobacteria were cultivated from coastal and pelagic regions of the Pacific Ocean using high-throughput culturing methods that rely on dilution to extinction in very low nutrient media. Phylogenetic analysis showed that the isolates fell into five rRNA clades, all of which contained rRNA gene sequences reported previously from seawater environmental gene clone libraries (SAR92, OM60, OM182, BD1-7, and KI89A). Bootstrap analyses of phylogenetic reliability did not support collapsing these five clades into a single clade, and they were therefore named the oligotrophic marine Gammaproteobacteria (OMG) group. Twelve cultures chosen to represent the five clades were successively purified in liquid culture, and their growth characteristics were determined at different temperatures and dissolved organic carbon concentrations. The isolates in the OMG group were physiologically diverse heterotrophs, and their physiological properties generally followed their phylogenetic relationships. None of the isolates in the OMG group formed colonies on low- or high-nutrient agar upon their first isolation from seawater, while 7 of 12 isolates that were propagated for laboratory testing eventually produced colonies on 1/10 R2A agar. The isolates grew relatively slowly in natural seawater media (1.23 to 2.63 day⁻¹), and none of them grew in high-nutrient media (>351 mg of C liter⁻¹). The isolates were psychro- to mesophilic and obligately oligotrophic; many of them were of ultramicrobial size (<0.1 μm³). This cultivation study revealed that sporadically detected Gammaproteobacteria gene clones from seawater are part of a phylogenetically diverse constellation of organisms mainly composed of oligotrophic and ultramicrobial lineages that are culturable under specific cultivation conditions.     

High molecular weight dissolved organic matter enrichment selects for methylotrophs in dilution to extinction cultures

The role of bacterioplankton in the cycling of marine dissolved organic matter (DOM) is central to the carbon and energy balance in the ocean, yet there are few model organisms available to investigate the genes, metabolic pathways, and biochemical mechanisms involved in the degradation of this globally important carbon pool. To obtain microbial isolates capable of degrading semi-labile DOM for growth, we conducted dilution to extinction cultivation experiments using seawater enriched with high molecular weight (HMW) DOM. In total, 93 isolates were obtained. Amendments using HMW DOM to increase the dissolved organic carbon concentration 4x (280 μM) or 10x (700 μM) the ocean surface water concentrations yielded positive growth in 4–6% of replicate dilutions, whereas <1% scored positive for growth in non-DOM-amended controls. The majority (71%) of isolates displayed a distinct increase in cell yields when grown in increasing concentrations of HMW DOM. Whole-genome sequencing was used to screen the culture collection for purity and to determine the phylogenetic identity of the isolates. Eleven percent of the isolates belonged to the gammaproteobacteria including Alteromonadales (the SAR92 clade) and Vibrio. Surprisingly, 85% of isolates belonged to the methylotrophic OM43 clade of betaproteobacteria, bacteria thought to metabolically specialize in degrading C1 compounds. Growth of these isolates on methanol confirmed their methylotrophic phenotype. Our results indicate that dilution to extinction cultivation enriched with natural sources of organic substrates has a potential to reveal the previously unsuspected relationships between naturally occurring organic nutrients and the microorganisms that consume them.     

Biogeography and phylogeny of the NOR5/OM60 clade of Gammaproteobacteria

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes “Congregibacter litoralis” strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. More than 500 16S rRNA sequences affiliated with this clade were retrieved from public databases. By comparative sequence analysis, 13 subclades could be identified, some of which are currently restricted to discrete habitat types. Almost all sequences in the largest subclade NOR5-1 and related subclade NOR5-4 originated from marine surface water samples. Overall, most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil.     

Rapid Virus Production and Removal as Measured with Fluorescently Labeled Viruses as Tracers

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h⁻¹ and production rates in the dark ranging from 1.9 to 6.1% h⁻¹, corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.     

Quantitative Evaluation of the Impact of Bather Density on Levels of Human-Virulent Microsporidian Spores in Recreational Water

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.     

Comparison of the Seasonal Variations of Synechococcus Assemblage Structures in Estuarine Waters and Coastal Waters of Hong Kong

Seasonal variation in the phylogenetic composition of Synechococcus assemblages in estuarine and coastal waters of Hong Kong was examined through pyrosequencing of the rpoC1 gene. Sixteen samples were collected in 2009 from two stations representing estuarine and ocean-influenced coastal waters, respectively. Synechococcus abundance in coastal waters gradually increased from 3.6 × 10³ cells ml⁻¹ in March, reaching a peak value of 5.7 × 10⁵ cells ml⁻¹ in July, and then gradually decreased to 9.3 × 10³ cells ml⁻¹ in December. The changes in Synechococcus abundance in estuarine waters followed a pattern similar to that in coastal waters, whereas its composition shifted from being dominated by phycoerythrin-rich (PE-type) strains in winter to phycocyanin-only (PC-type) strains in summer owing to the increase in freshwater discharge from the Pearl River and higher water temperature. The high abundance of PC-type Synechococcus was composed of subcluster 5.2 marine Synechococcus, freshwater Synechococcus (F-PC), and Cyanobium. The Synechococcus assemblage in the coastal waters, on the other hand, was dominated by marine PE-type Synechococcus, with subcluster 5.1 clades II and VI as the major lineages from April to September, when the summer monsoon prevailed. Besides these two clades, clade III cooccurred with clade V at relatively high abundance in summer. During winter, the Synechococcus assemblage compositions at the two sites were similar and were dominated by subcluster 5.1 clades II and IX and an undescribed clade (represented by Synechococcus sp. strain miyav). Clade IX Synechococcus was a relatively ubiquitous PE-type Synechococcus found at both sites, and our study demonstrates that some strains of the clade have the ability to deal with large variation of salinity in subtropical estuarine environments. Our study suggests that changes in seawater temperature and salinity caused by the seasonal variation of monsoonal forcing are two major determinants of the community composition and abundance of Synechococcus assemblages in Hong Kong waters.     

A novel clade of Prochlorococcus found in high nutrient low chlorophyll waters in the South and Equatorial Pacific Ocean

A novel high-light (HL)-adapted Prochlorococcus clade was discovered in high nutrient and low chlorophyll (HNLC) waters in the South Pacific Ocean by phylogenetic analyses of 16S ribosomal RNA (rRNA) and 16S–23S internal transcribed spacer (ITS) sequences. This clade, named HNLC fell within the HL-adapted Prochlorococcus clade with sequences above 99% similarity to one another, and was divided into two subclades, HNLC1 and HNLC2. The distribution of the whole HNLC clade in a northwest to southeast transect in the South Pacific (HNLC-to-gyre) and two 8°N to 8°S transects in the Equatorial Pacific was determined by quantitative PCR using specific primers targeting ITS regions. HNLC was the dominant HL Prochlorococcus clade (2–9% of bacterial 16S rRNA genes) at the three westernmost stations in the South Pacific but decreased to less than 0.1% at the other stations being replaced by the eMIT9312 ecotype in the hyperoligotrophic gyre. The highest contributions of HNLC Prochlorococcus in both Equatorial Pacific transects along the latitudinal lines of 170°W and 155°W were observed at the southernmost stations, reaching 16 and 6% of bacterial 16S rRNA genes, respectively, whereas eMIT9312 dominated near the Equator. Spearman Rank Order correlation analysis indicated that although both the HNLC clade and eMIT9312 were correlated with temperature, they showed different correlations with regard to nutrients. HNLC only showed significant correlations to ammonium uptake and regeneration rates, whereas eMIT9312 was negatively correlated with inorganic nutrients.