Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected ^1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) ³. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.Download video file.^{(80M, mov)}     

Pirellula and OM43 are among the dominant lineages identified in an Oregon coast diatom bloom

Although bacterioplankton and phytoplankton are generally perceived as closely linked in marine systems, specific interactions between discrete bacterioplankton and phytoplankton populations are largely unknown. However, measurements of bacterioplankton distributions during phytoplankton blooms may indicate specific microbial lineages that are responding to phytoplankton populations, and potentially controlling them by producing allelopathic compounds. Here we use a comprehensive molecular approach to identify, characterize and quantify bacterioplankton community responses to an Oregon coast diatom bloom. Total DAPI counts increased by nearly sevenfold in bloom samples, reaching 5.7 x 10(9) cells l(-1), and lineage-specific cell counts using fluorescence in situ hybridization (FISH) indicated that Bacteria accounted for approximately 89% of observed increases. Several dominant members of the bacterial community present outside the bloom (SAR11 and SAR86) did not contribute significantly to observed increases in bloom samples. Clone library and FISH data indicated that uncultured planctomycetes most closely related to Pirellula, and members of the OM43 clade of beta proteobacteria, reached 0.5 x 10(8) and 1.2 x 10(8) cells l(-1), respectively, and were among the dominant lineages in bloom samples.     

Tracking differential incorporation of dissolved organic carbon types among diverse lineages of Sargasso Sea bacterioplankton

Bacterioplankton are the primary trophic conduit for dissolved organic carbon (DOC) and linking community structure with DOC utilization is central to understanding global carbon cycling. We coupled stable isotope probing (SIP) with 16S rRNA pyrosequencing in dark seawater culture experiments on euphotic and mesopelagic communities from the Sargasso Sea. Parallel cultures were amended with equimolar quantities of four DO¹³C substrates to simultaneously evaluate community utilization and population‐specific incorporation. Of the substrates tested – two cyanobacterial products (exudates or lysates from a culture of Synechococcus) and two defined monosaccharides (glucose or gluconic acid) – the cyanobacterial exudates were incorporated by the greatest diversity of oligotrophic bacterioplankton populations in surface waters, including taxa from > 10 major subclades within the Flavobacteria, Actinobacteria, Verrucomicrobia and Proteobacteria (including SAR11). In contrast, the monosaccharide glucose was not incorporated by any taxa belonging to extant oligotrophic oceanic clades. Conversely, proteobacterial copiotrophs, which were rare in the ambient water (< 0.1% of sequences), grew rapidly on all DOC amendments at both depths, but with different substrate preferences among lineages. We present a new analytical framework for using SIP to detect DOC incorporation across diverse oligotrophic bacterioplankton and discuss implications for the ecology of bacterial–DOC interactions among populations of diverging trophic strategies.     

Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 micro m) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.     

Depleted dissolved organic carbon and distinct bacterial communities in the water column of a rapid-flushing coral reef ecosystem

Coral reefs are highly productive ecosystems bathed in unproductive, low-nutrient oceanic waters, where microbially dominated food webs are supported largely by bacterioplankton recycling of dissolved compounds. Despite evidence that benthic reef organisms efficiently scavenge particulate organic matter and inorganic nutrients from advected oceanic waters, our understanding of the role of bacterioplankton and dissolved organic matter (DOM) in the interaction between reefs and the surrounding ocean remains limited. In this study, we present the results of a 4-year study conducted in a well-characterized coral reef ecosystem (Paopao Bay, Moorea, French Polynesia) where changes in bacterioplankton abundance and dissolved organic carbon (DOC) concentrations were quantified and bacterial community structure variation was examined along spatial gradients of the reef:ocean interface. Our results illustrate that the reef is consistently depleted in concentrations of both DOC and bacterioplankton relative to offshore waters (averaging 79 μmol l⁻¹ DOC and 5.5 × 10⁸ cells l⁻¹ offshore and 68 μmol l⁻¹ DOC and 3.1 × 10⁸ cells l⁻¹ over the reef, respectively) across a 4-year time period. In addition, using a suite of culture-independent measures of bacterial community structure, we found consistent differentiation of reef bacterioplankton communities from those offshore or in a nearby embayment across all taxonomic levels. Reef habitats were enriched in Gamma-, Delta-, and Betaproteobacteria, Bacteriodetes, Actinobacteria and Firmicutes. Specific bacterial phylotypes, including members of the SAR11, SAR116, Flavobacteria, and Synechococcus clades, exhibited clear gradients in relative abundance among nearshore habitats. Our observations indicate that this reef system removes oceanic DOC and exerts selective pressures on bacterioplankton community structure on timescales approximating reef water residence times, observations which are notable both because fringing reefs do not exhibit long residence times (unlike those characteristic of atoll lagoons) and because oceanic DOC is generally recalcitrant to degradation by ambient microbial assemblages. Our findings thus have interesting implications for the role of oceanic DOM and bacterioplankton in the ecology and metabolism of reef ecosystems.     

Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 μm) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.     

Transporter genes expressed by coastal bacterioplankton in response to dissolved organic carbon

Coastal ocean bacterioplankton control the flow of dissolved organic carbon (DOC) from terrestrial and oceanic sources into the marine food web, and regulate the release of inorganic carbon to atmospheric and offshore reservoirs. While the fate of the chemically complex coastal DOC reservoir has long been recognized as a critical feature of the global carbon budget, it has been problematic to identify both the compounds that serve as major conduits for carbon flux and the roles of individual bacterioplankton taxa in mediating that flux. Here we analyse random libraries of expressed genes from a coastal bacterial community to identify sequences representing DOC‐transporting proteins. Predicted substrates of expressed transporter genes indicated that carboxylic acids, compatible solutes, polyamines and lipids may be key components of the biologically labile DOC pool in coastal waters, in addition to canonical bacterial substrates such as amino acids, oligopeptides and carbohydrates. Half of the expressed DOC transporter sequences in this coastal ocean appeared to originate from just eight taxa: Roseobacter, SAR11, Flavobacteriales and five orders of γ‐Proteobacteria. While all major taxa expressed transporter genes for some DOC components (e.g. amino acids), there were indications of specialization within the bacterioplankton community for others (e.g. carbohydrates, carboxylic acids and polyamines). Experimental manipulations of the natural DOC pool that increased the concentration of phytoplankton‐ or vascular plant‐derived compounds invoked a readily measured response in bacterial transporter gene expression. This highly resolved view of the potential for carbon flux into heterotrophic bacterioplankton cells identifies possible bioreactive components of the coastal DOC pool and highlights differing ecological roles in carbon turnover for the resident bacterial taxa.     

Community differentiation and population enrichment of Sargasso Sea bacterioplankton in the euphotic zone of a mesoscale mode‐water eddy

Eddies are mesoscale oceanographic features (～ 200 km diameter) that can cause transient blooms of phytoplankton by shifting density isoclines in relation to light and nutrient resources. To better understand how bacterioplankton respond to eddies, we examined depth‐resolved distributions of bacterial populations across an anticyclonic mode‐water eddy in the Sargasso Sea. Previous work on this eddy has documented elevated phytoplankton productivity and diatom abundance within the eddy centre with coincident bacterial productivity and biomass maxima. We illustrate bacterial community shifts within the eddy centre, differentiating populations uplifted along isopycnals from those enriched or depleted at horizons of enhanced bacterial and primary productivity. Phylotypes belonging to the Roseobacter, OCS116 and marine Actinobacteria clades were enriched in the eddy core and were highly correlated with pigment‐based indicators of diatom abundance, supporting developing hypotheses that members of these clades associate with phytoplankton blooms. Typical mesopelagic clades (SAR202, SAR324, SAR406 and SAR11 IIb) were uplifted within the eddy centre, increasing bacterial diversity in the lower euphotic zone. Typical surface oligotrophic clades (SAR116, OM75, Prochlorococcus and SAR11 Ia) were relatively depleted in the eddy centre. The biogeochemical context of a bloom‐inducing eddy provides insight into the ecology of the diverse uncultured bacterioplankton dominating the oligotrophic oceans.     

Basin-scale patterns in the abundance of SAR11 subclades, marine Actinobacteria (OM1), members of the Roseobacter clade and OCS116 in the South Atlantic

Bacterioplankton are major biogeochemical agents responsible for mediating the flux of dissolved organic matter (DOM) and subsequent cycling of nutrients in the oceans. Most information about the composition of bacterioplankton communities has come from studies along well-defined biogeochemical gradients in the northern hemisphere. This study extends observations of spatial and temporal dynamics for SAR11, Actinobacteria and OCS116 in the North Atlantic by demonstrating distinct spatial variability in the abundance and distribution of these and other lineages across the South Atlantic gyre and in the Benguela upwelling system. We identified shifts in SAR11, Actinobacteria, OCS116, SAR86, SAR116 and members of the Roseobacter clade along basin-scale gradients in nutrients, chlorophyll and dissolved organic carbon (DOC). Distinct SAR11 subclades dominated the western and eastern regions of the gyre, and Actinobacteria, OCS116 and members of the Roseobacter lineages were most abundant at the deep chlorophyll maxima. SAR86 and SAR116 accounted for a significant fraction of coastal and open ocean communities, respectively, and members of the gamma sulfur oxidizer (GSO) clade persisted in the Benguela upwelling system. These data suggest that distinct communities are partitioned along basin-scale biogeochemical gradients, that SAR11 community structure varies across the gyre and that Actinobacteria, OCS116, and members of the Roseobacter clade are closely associated with phytoplankton in the gyre.     

Bromodeoxyuridine labelling and fluorescence‐activated cell sorting of polyamine‐transforming bacterioplankton in coastal seawater

Polyamines (PAs) are a group of nitrogen‐rich dissolved organic nitrogen (DON) compounds that are ubiquitously distributed in marine environments. To identify bacteria that are involved in PA transformations, coastal bacterioplankton microcosms were amended with a single PA model compound, i.e. putrescine (PUT) or spermidine (SPD), or with no addition as controls (CTRs). Bromodeoxyuridine (BrdU) was added to all the microcosms to label newly synthesized DNAs. Fluorescence‐activated cell sorting (FACS) analysis indicated significant increases in numbers of total cells and cells with both high and low levels of BrdU incorporation in the PUT and SPD microcosms, but not in the CTRs. 16S rDNA pyrotag sequencing of FACS‐sorted cells indicated that PUT‐ and SPD‐transforming bacteria were composed similarly of a diverse group of taxa affiliated with Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria (especially Roseobacter of its alpha lineage). Broad taxonomic distribution of PA‐transforming bacteria was also indicated by the abundance and distribution of PA transporter gene homologues in a survey of sequenced marine bacterial genomes. Our results suggest that PAs may be common DON substrates for marine bacterioplankton, in line with the hypothesis that bacterially mediated PA transformation accounts for an important proportion of marine DON flux.     

Bacterioplankton nutrient metabolism in the Eastern Tropical North Pacific

Bacterioplankton nutrient metabolism in the Eastern Tropical North Pacific (ETNP) was assessed using specific activities of intracellular nitrogen (N) assimilation enzymes and hydrolytic ectoenzymes during amendment experiments, mesocosms, and diel studies of in situ rates. Glutamine synthetase (GS) and assimilatory nitrate reductase (ANR) were used to investigate N bioavailability, alkaline phosphatase (AP) to assess phosphorous (P) bioavailability and β-glucosidase (β-Glu) to detect shifts in the use of labile dissolved organic carbon (DOC). Conditions regulating activity of each enzyme were tested using incubations of < 0.6 mm size-fractionated seawater amended with different combinations of N, P, and DOC as glucose. Overall, N-deficiency was indicated by pronounced growth stimulation and repression of GS and ANR activity in incubations amended with dissolved free amino acid and ammonium. Phosphate and glucose amendments produced little or no growth stimulation, but did influence activity of all enzymes measured. Enzyme activities of bacterioplankton in mesocosms of whole plankton indicated enhanced N-deficiency and glucoside hydrolysis when the plankton community was released from any P-deficiency. Spatially, enzyme activity of bacterioplankton during two diel studies (at one slope and one open-ocean station) suggested greater N-deficiency at surface depths than within the chlorophyll maximum where activity of AP and b-Glu was often greatest. There was also greater GS and ANR activity at the open-ocean station, which had lower concentrations of dissolved inorganic N (DIN) relative to soluble reactive P (SRP), than along the continental slope of Mexico. These data suggest that bacterioplankton in surface waters of the ETNP require a large flux of DOC to drive N-deficiency; whereas, bacterioplankton deeper in the chlorophyll maximum depend on hydrolysis of complex DOC and DOP to meet their carbon demand in the presence of elevated nutrients with a low DIN:SRP ratio.     

Similar composition but differential stability of mineral retained organic matter across four classes of clay minerals

Adsorption of dissolved organic compounds onto mineral surfaces is increasingly recognized as a significant, if not dominant, carbon stabilisation mechanism in many soils. By utilising carbon-13 enriched dissolved organic carbon (DOC) source materials in a repeated leaching-sorption-incubation study, we show here that the biochemical composition of mineral-retained organic matter (OM) is similar across four different classes of clay minerals but the quantity and stability of this OM is both a function of source material and clay mineralogy. Three to eight times as much carbon was retained on a mass basis when the same amount of DOC derived from eucalyptus versus wheat litter was applied, and the retained wheat-derived OM was up to 2.4 times more degradable than that of the eucalyptus source. For both litter types, carbon retention across the clay types was not significantly different; whereas, the stability of the retained OM was different but depended on which litter extract had been applied. The wheat-derived DOC was more stable when retained by allophane and oxides than by illite and smectite. Solid-state ¹³C NMR spectroscopic results indicated that despite large compositional differences in both source litter and resultant DOC, the composition of the mineral-retained OM was similar across clay classes with lignin-derived aromatic and carboxylic compounds dominating. Differences in the amount of carbon retained were related to differences in the proportions of aromatic, phenolic and carboxylic C in the DOC produced from the two litter sources. Differences in the stability across the clay classes were correlated with the abundance of metals and short-range ordered minerals. These results suggest that whenever reactive mineral surfaces and metals are present in a soil, a similar form of relatively unaltered litter derived OM can be adsorbed but that the longer term stability of sorbed OM, and thus in situ composition, will be a function of the mineralogy (reactivity) of the specific minerals involved in the binding process.     

The use of antibiotics to reduce bacterioplankton uptake of phytoplankton extracellular organic carbon (EOC) in the Potomac River estuary

Phytoplankton production and accumulation of extracellular organic carbon (EOC) was tracked during diel intervals in microcosms by inhibiting bacterioplankton assimilation of EOC with streptomycin and kanamycin. Bacterioplankton production (³H-thymidine incorporation) and metabolism (¹⁴C-glucose incorporation) were monitored in samples collected from the Potomac River estuary to determine the effect of the antibiotics. Particulate (i.e., raw water) primary production and EOC (i.e., water passing through 1.0 μm glass fiber filter) production rates were monitored to determine the impact of antibiotics on phytoplankton. In preliminary experiments, neither streptomycin nor kanamycin alone significantly inhibited bacterioplankton activity compared to controls, but when both were present secondary production and metabolism were reduced up to 90%, and remained as such for 45 h. During field evaluations using a streptomycin and kanamycin mixture (50 μM each) particulate primary production and EOC production were not statistically different in control and antibiotic treated samples indicating that the antibiotics did not negatively influence phytoplankton production rates. In the presence of antibiotics dissolved free amino acids (DFAA) and, to a lesser extent, monosaccharides were significantly elevated compared to controls. This study demonstrates that streptomycin and kanamycin are capable of inhibiting bacterioplankton metabolism and uptake of dissolved organic carbon (DOC) in the samples tested so that the contribution of EOC to the DOC pool and to bacterioplankton metabolism could be measured and assessed.     

Limitation of lowland riverine bacterioplankton by dissolved organic carbon and inorganic nutrients

Flow regulation in lowland rivers has reduced the amount of allochthonous dissolved organic carbon (DOC) entering main channels through less frequent wetting of benches, flood runners and floodplains. The hypothesis tested was that lowland riverine bacterioplankton are DOC limited when flow events are absent and simulating an increase in assimilable DOC similar to that expected during an environmental flow will lead to heterotrophic dominance. Experiments took place in the Namoi River, a highly regulated lowland river in Australia. Specifically, in situ microcosms were used to examine the responses of bacterioplankton and phytoplankton to various additions of DOC as glucose or leaf leachate, with and without additions of inorganic nutrients. The results indicated that ambient DOC availability limited the bacterioplankton for the three seasons over which we conducted the experiments. When DOC was added alone, dissolved oxygen concentrations decreased primarily because of increased bacterial respiration and bacterioplankton growth generally increased relative to controls. Additions of DOC alone led to a pattern of decreased chlorophyll a concentration relative to controls, except for willow leachate. Additions of inorganic nutrients alone increased chlorophyll a concentrations above controls, indicating limitation of phytoplankton. These findings support our hypothesis. Based on the present results, environmental flows should increase the duration of allochthonously driven heterotrophic dominance, thus shifting regulated lowland rivers to more natural (pre-regulation) conditions for greater periods.     

Influence of the Hydrological Cycle on the Bacterioplankton of an Impacted Clear Water Amazonian Lake

Abstract Free-living and attached bacterial population sizes were determined fortnightly from December 1991 to December 1992 in natural and disturbed areas of an Amazonian clear water lake (Batata Lake, Pará, Brazil) impacted by bauxite tailings. The bacterioplankton showed distinct patterns during different phases of the hydrological cycle. Total bacterial population size and rates of thymidine incorporation (measured during high and low water phases) were high during low water, with values ranging from 3.3 × 10⁵ to 1.1 × 10⁶ cells ml⁻¹, and from 0.28 to 4.01 μg C l⁻¹ h⁻¹, respectively. The population size of free-living bacteria was larger at the natural station, while no differences were observed between attached bacterial populations at both stations. However, production and turnover rate of attached bacteria were high at the disturbed area. During low water, bacterial growth appeared to be driven mainly by the input of dissolved organic carbon (DOC) from phytoplankton origin. During high water, bacterial abundance was reduced, probably as the result of dilution and the input of less labile DOC from floodplains. The presence of bauxite tailings seems to influence bacterial dynamics in an indirect way, probably due to shading of phytoplankton cells and, hence, reducing the DOC supply for bacterial growth. This study, the first on the microbial ecology of an Amazonian clear water lake, demonstrated that water level variations exert a strong influence on the bacterioplankton dynamics. Received: 9 January 1996; Accepted 6 November 1996     

Ammonia-oxidizing archaea release a suite of organic compounds potentially fueling prokaryotic heterotrophy in the ocean

Ammonia-oxidizing archaea (AOA) constitute a considerable fraction of microbial biomass in the global ocean, comprising 20%–40% of the ocean's prokaryotic plankton. However, it remains enigmatic to what extent these chemolithoautotrophic archaea release dissolved organic carbon (DOC). A combination of targeted and untargeted metabolomics was used to characterize the exometabolomes of three model AOA strains of the Nitrosopumilus genus. Our results indicate that marine AOA exude a suite of organic compounds with potentially varying reactivities, dominated by nitrogen-containing compounds. A significant fraction of the released dissolved organic matter (DOM) consists of labile compounds, which typically limit prokaryotic heterotrophic activity in open ocean waters, including amino acids, thymidine and B vitamins. Amino acid release rates corresponded with ammonia oxidation activity and the three Nitrosopumilus strains predominantly released hydrophobic amino acids, potentially as a result of passive diffusion. Despite the low contribution of DOC released by AOA (~0.08%–1.05%) to the heterotrophic prokaryotic carbon demand, the release of physiologically relevant metabolites could be crucial for microbes that are auxotrophic for some of these compounds, including members of the globally abundant and ubiquitous SAR11 clade.     

Changes in dimethylsulfoniopropionate demethylase gene assemblages in response to an induced phytoplankton bloom

Over half of the bacterioplankton cells in ocean surface waters are capable of carrying out a demethylation of the phytoplankton metabolite dimethylsulfoniopropionate (DMSP) that routes the sulfur moiety away from the climatically active gas dimethylsulfide (DMS). In this study, we tracked changes in dmdA, the gene responsible for DMSP demethylation, over the course of an induced phytoplankton bloom in Gulf of Mexico seawater microcosms. Analysis of >91,000 amplicon sequences indicated 578 different dmdA sequence clusters at a conservative clustering criterion of ≥90% nucleotide sequence identity over the 6-day study. The representation of the major clades of dmdA, several of which are linked to specific taxa through genomes of cultured marine bacterioplankton, remained fairly constant. However, the representation of clusters within these major clades shifted significantly in response to the bloom, including two Roseobacter-like clusters and a SAR11-like cluster, and the best correlate with shifts of the dominant dmdA clades was chlorophyll a concentration. Concurrent 16S rRNA amplification and sequencing indicated the presence of Roseobacter, SAR11, OM60, and marine Rhodospirillales populations, all of which are known to harbor dmdA genes, although the largest taxonomic change was an increase in Flavobacteriaceae, a group not yet demonstrated to have DMSP-demethylating capabilities. Sequence heterogeneity in dmdA and other functional gene populations is becoming increasingly evident with the advent of high-throughput sequencing technologies, and understanding the ecological implications of this heterogeneity is a major challenge for marine microbial ecology.     

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.     

Improved culturability of SAR11 strains in dilution-to-extinction culturing from the East Sea, West Pacific Ocean

Although the SAR11 clade of the Alphaproteobacteria represents the most abundant and ubiquitous bacterioplankton in the ocean, very few laboratories have successfully cultured SAR11 cells. All of the SAR11 strains isolated thus far have been retrieved from the Oregon coast and the Sargasso Sea. In this study, a modified dilution-to-extinction culturing with prolonged incubation at low temperature was applied in an effort to cultivate major bacterioplankton lineages in the East Sea, Western Pacific Ocean. Five to 10 cells were inoculated into each well of 48-well plates, followed by the incubation of the plates at 10 °C for 4, 8, 20, and 24 weeks. Among a total of 35 isolated strains, 18 strains assigned to the SAR11 clade were isolated after 8, 20, and 24 weeks of incubation, whereas no SAR11 cells were detected in the samples after 4 weeks of incubation. The SAR11 isolates, noticeably, comprised 64–82% of the total isolates from the plates incubated for 20 and 24 weeks. Extinction cultures belonging to the Roseobacter , OM43, and SAR92 clades were also cultivated. The results of this study suggest that long-term incubation at low temperatures might prove an alternative for the efficient cultivation of new variants of the members of the SAR11 clade.