Flow sorting of marine bacterioplankton after fluorescence in situ hybridization

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.     

Winter-to-summer changes in the composition and single-cell activity of near-surface Arctic prokaryotes

We collected surface samples in Franklin Bay (Western Arctic) from ice-covered to ice-free conditions, to determine seasonal changes in the identity and in situ activity of the prokaryotic assemblages. Catalysed reported fluorescence in situ hybridization was used to quantify the abundance of different groups, and combined with microautoradiography to determine the fraction of active cells taking up three substrates: glucose, amino acids and ATP. In surface waters, Archaea accounted for 16% of the total cell count in winter, but decreased to almost undetectable levels in summer, when Bacteria made up 97% of the total cell count. Alphaproteobacteria were the most abundant group followed by Bacteroidetes (average of 34% and 14% of total cell counts respectively). Some bacterial groups appearing in low abundances (< 10% of total cell counts), such as Betaproteobacteria , Roseobacter and Gammaproteobacteria , showed a high percentage of active cells. By contrast, more abundant groups, such as SAR11 or Bacteroidetes , had a lower percentage of active cells in the uptake of the substrates tested. Archaea showed low heterotrophic activity throughout the year. In comparison with temperate oceans, the percentage of active Bacteria in the uptake of the substrates was relatively high, even during the winter season.     

The microbiome of North Sea copepods

Copepods can be associated with different kinds and different numbers of bacteria. This was already shown in the past with culture-dependent microbial methods or microscopy and more recently by using molecular tools. In our present study, we investigated the bacterial community of four frequently occurring copepod species, Acartia sp., Temora longicornis, Centropages sp. and Calanus helgolandicus from Helgoland Roads (North Sea) over a period of 2 years using DGGE (denaturing gradient gel electrophoresis) and subsequent sequencing of 16S-rDNA fragments. To complement the PCR-DGGE analyses, clone libraries of copepod samples from June 2007 to 208 were generated. Based on the DGGE banding patterns of the two years survey, we found no significant differences between the communities of distinct copepod species, nor did we find any seasonality. Overall, we identified 67 phylotypes (>97 % similarity) falling into the bacterial phyla of Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The most abundant phylotypes were affiliated to the Alphaproteobacteria. In comparison with PCR-DGGE and clone libraries, phylotypes of the Gammaproteobacteria dominated the clone libraries, whereas Alphaproteobacteria were most abundant in the PCR-DGGE analyses.     

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.     

Phylogenetic composition of bacterioplankton assemblages from the Arctic Ocean

We analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domain Bacteria: alpha(36%)-, gamma(32%)-, delta(14%)-, and epsilon(1%)-Proteobacteria; Cytophaga-Flexibacter-Bacteroides spp. (9%); Verrucomicrobium spp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 and Roseobacter groups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related to Cytophaga and Polaribacter species were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) with Alteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) with Oceanospirillum sp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to alpha-Proteobacteria (dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to gamma-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.     

Bacterioplankton composition of the coastal upwelling system of 'Ría de Vigo', NW Spain

Catalysed reported deposition-FISH and clone libraries indicated that Roseobacter , followed by Bacteroidetes , and some gammaproteobacterial groups such as SAR86, dominated the composition of bacterioplankton in Ría de Vigo, NW Spain, in detriment to SAR11 (almost absent in this upwelling ecosystem). Since we sampled four times during the year, we observed pronounced changes in the structure of each bacterioplankton component, particularly for the Roseobacter lineage. We suggest that such variations in the coastal upwelling ecosystem of Ría de Vigo were associated with the characteristic phytoplankton communities of the four different hydrographical situations: winter mixing, spring bloom, summer stratification, and autumn upwelling. We retrieved new sequences among the major marine bacterial lineages, particularly among Roseobacter , SAR11, and especially SAR86. The spring community was dominated by two Roseobacter clades that had previously been related to phytoplankton blooms. In the other seasons, communities with higher diversity than the spring one were detected.     

Bacterial Diversity in the South Adriatic Sea during a Strong,Deep Winter Convection Year

The South Adriatic Sea is the deepest part of the Adriatic Sea and represents a key area for both the Adriatic Sea and the deep eastern Mediterranean. It has a role in dense water formation for the eastern Mediterranean deep circulation cell, and it represents an entry point for water masses originating from the Ionian Sea. The biodiversity and seasonality of bacterial picoplankton before, during, and after deep winter convection in the oligotrophic South Adriatic waters were assessed by combining comparative 16S rRNA sequence analysis and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The picoplankton communities reached their maximum abundance in the spring euphotic zone when the maximum value of the chlorophyll a in response to deep winter convection was recorded. The communities were dominated by Bacteria, while Archaea were a minor constituent. A seasonality of bacterial richness and diversity was observed, with minimum values occurring during the winter convection and spring postconvection periods and maximum values occurring under summer stratified conditions. The SAR11 clade was the main constituent of the bacterial communities and reached the maximum abundance in the euphotic zone in spring after the convection episode. Cyanobacteria were the second most abundant group, and their abundance strongly depended on the convection event, when minimal cyanobacterial abundance was observed. In spring and autumn, the euphotic zone was characterized by Bacteroidetes and Gammaproteobacteria. Bacteroidetes clades NS2b, NS4, and NS5 and the gammaproteobacterial SAR86 clade were detected to co-occur with phytoplankton blooms. The SAR324, SAR202, and SAR406 clades were present in the deep layer, exhibiting different seasonal variations in abundance. Overall, our data demonstrate that the abundances of particular bacterial clades and the overall bacterial richness and diversity are greatly impacted by strong winter convection.     

Bacterial diversity of water and sediment in the Changjiang estuary and coastal area of the East China Sea

The Changjiang estuary and the coastal area of the East China Sea (ECS) represent important interfaces of terrestrial and marine environments. This study included analyses of water and sediments collected during different seasons in these regions to determine the composition of microbial assemblages by means of 16S rRNA gene clone libraries. We retrieved 1946 sequences and 779 distinct operational taxonomic units from 36 clone libraries. Shannon–Weaver diversity index values and rarefaction analysis indicated that bacterial diversity in the sediment samples was much higher than in the water samples. Proteobacteria (72.9%) was the most abundant phylum, followed by Firmicutes (6.4%), Bacteroidetes (4.6%) and Actinobacteria (4.1%). In the water, clone sequences related to Alphaproteobacteria were the most abundant, whereas in the sediment samples, sequences affiliated with Gammaproteobacteria were predominant. Principal coordinate analysis showed that water samples collected from the Changjiang estuary and the ECS clustered separately. However, this spatial pattern could not be observed in sediment samples, which were mainly distinguished from one another by the season. Bacterial diversity in the Changjiang estuary was higher than that in the ECS, which may be the result of the mixing of bacterial communities from the Changjiang River, the estuary and the coastal ocean.     

Seasonal and spatial variability of sediment bacterial communities inhabiting the large shallow Lake Balaton

Seasonal studies of surface sediment bacterial communities, from two basins with differing trophic states within Lake Balaton (Hungary), were carried out using molecular (denaturing gradient gel electrophoresis, DGGE) and cultivation-based techniques. The presence of polyphosphate accumulates was tested using Neisser staining, and phosphatase activity was investigated on organic phosphorus (P) compound. Aerobic viable cell counts were significantly higher in the eutrophic than mesotrophic basin in each season. The lowest viable counts were observed in the autumn and the highest in spring and summer month in both basins. The DGGE fingerprints of the samples reflected that the composition of sediment bacterial communities in the two basins were distinct in spring and summer, and similar in autumn, but similarly diverse in all seasons. On the basis of partial 16S rDNA sequences, the 216 strains were affiliated with six major bacterial lineages: Firmicutes; Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Common species characterized from both basins constituted up to 66% of all identified phylotypes. Strains related to Bacillus sp. were dominant in all but one sample. Isolates affiliated with Aeromonas sp. prevailed in the sample taken from the mesotrophic basin in spring. The majority of the strains showed excess poly-P accumulation. Association of Neisser staining and phosphatase activity test results suggested that excess poly-P accumulation serves as P storage for sediment bacteria. Our study implied the importance of Firmicutes, Actinobacteria, Alphaproteobacteria, and Aeromonas species in benthic bacterial P retention.     

Molecular characterization of activated sludge from a seawater‐processing wastewater treatment plant

The prokaryotic community composition of activated sludge from a seawater‐processing wastewater treatment plant (Almeria, Spain) was investigated by using the rRNA approach, combining different molecular techniques such as denaturing gradient gel electrophoresis (DGGE), clone libraries and in situ hybridization (FISH and CARD‐FISH). Most of the sequences retrieved in the DGGE and the clone libraries were similar to uncultured members of different phyla. The most abundant sequence recovered from Bacteria in the clone library corresponded to a bacterium from the Deinococcus–Thermus cluster (almost 77% of the clones), and the library included members from other groups such as the Alpha, Gamma and Delta subclasses of Proteobacteria, the Bacteroidetes and Firmicutes. Concerning the archaeal clone library, we basically found sequences related to different orders of methanogenic Archaea, in correspondence with the recovered DGGE bands. Enumeration of DAPI (4′,6‐diamidino‐2‐phenylindole) stained cells from two different activated sludge samples after a mechanical flocculation disruption revealed a mean cell count of 1.6 × 10⁹ ml^?1. Around 94% of DAPI counts (mean value from both samples) hybridized with a Bacteria specific probe. Alphaproteobacteria were the dominant bacterial group (36% of DAPI counts), while Beta‐, Delta‐ and Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes contributed to lower proportions (between 0.5–5.7% of DAPI counts). Archaea accounted only for 6% of DAPI counts. In addition, specific primers for amplification of the amoA (ammonia monooxygenase) gene were used to detect the presence of Beta, Gamma and archaeal nitrifiers, yielding positive amplifications only for Betaproteobacteria. This, together with negative in situ hybridizations with probes for well‐known nitrifiying bacteria, suggests that nitrification is performed by still undetected microorganisms. In summary, the combination of the three approaches provided different and complementary pictures of the real assemblage composition and allowed to get closer to the main microorganisms involved in key processes of seawater‐processing activated sludge.     

Temporal variability of coastal Planctomycetes clades at Kabeltonne station, North Sea

Members of the bacterial phylum Planctomycetes are reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution of Planctomycetes in surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Generally Planctomycetes are more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed that Planctomycetes abundance was correlated to the Centrales diatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ~90% of the Planctomycetes reside in the >3-μm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, named Planctomyces-related group A, uncultured Planctomycetes group B, and Pirellula-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. Uncultured Planctomycetes group B was most abundant in autumn samples, while Planctomyces-related group A was present in high numbers only during late autumn and winter. The levels of Pirellula-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of the Planctomycetes is correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.     

Bacterial diversity in the sediment from polymetallic nodule fields of the Clarion-Clipperton Fracture Zone

The Clarion-Clipperton Fracture Zone (CCFZ) is located in the northeastern equatorial Pacific and contains abundant polymetallic nodules. To investigate its bacterial diversity, four libraries of 16S rRNA genes were constructed from sediments of four stations in different areas of the CCFZ. In total, 313 clones sequenced from the 4 libraries were assigned into 14 phylogenetic groups and 1 group of 28 unclassified bacteria. High bacterial diversity was predicted by the rarefaction analysis. The most dominant group overall was Proteobacteria, but there was variation in each library: Gammaproteobacteria was the most dominant group in two libraries, E2005-01 and ES0502, while Alphaproteobacteria and Deltaproteobacteria were the most dominant groups in libraries EP2005-03 and WS0505, respectively. Seven groups, including Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Betaproteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes, were common to all four libraries. The remaining minor groups were distributed in libraries with different patterns. Most clones sequenced in this study were clustered with uncultured bacteria obtained from the environment, such as the ocean crust and marine sediment, but only distantly related to isolates. Bacteria involved in the cycling of metals, sulfur and nitrogen were detected, and their relationship with their habitat was discussed. This study sheds light on the bacterial communities associated with polymetallic nodules in the CCFZ and provides primary data on the bacterial diversity of this area.     

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.     

Abundant proteorhodopsin genes in the North Atlantic Ocean

Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria, including Pelagibacter ubique , a member of the ubiquitous SAR11 clade. The goals of this study were to explore the diversity of PR genes and to estimate their abundance in the North Atlantic Ocean using quantitative polymerase chain reaction (QPCR). We found that PR genes in the western portion of the Sargasso Sea could be grouped into 27 clusters, but five clades had the most sequences. Sets of specific QPCR primers were designed to examine the abundance of PR genes in the following four of the five clades: SAR11 ( P. ubique and other SAR11 Alphaproteobacteria ), BACRED17H8 ( Alphaproteobacteria ), HOT2C01 ( Alphaproteobacteria ) and an uncultured subgroup of the Flavobacteria . Two groups (SAR11 and HOT2C01) dominated PR gene abundance in oligotrophic waters, but were significantly less abundant in nutrient- and chlorophyll-rich waters. The other two groups (BACRED17H8 and Flavobacteria subgroup NASB) were less abundant in all waters. Together, these four PR gene types were found in 50% of all bacteria in the Sargasso Sea. We found a significant negative correlation between total PR gene abundance and nutrients and chlorophyll but no significant correlation with light intensity for three of the four PR types in the depth profiles north of the Sargasso Sea. Our data suggest that PR is common in the North Atlantic Ocean, especially in SAR11 bacteria and another marine alphaproteobacterial group (HOT2C01), and that these PR-bearing bacteria are most abundant in oligotrophic waters.     

Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high Arctic

The springs at Gypsum Hill and Colour Peak on Axel Heiberg Island in the Canadian Arctic originate from deep salt aquifers and are among the few known examples of cold springs in thick permafrost on Earth. The springs discharge cold anoxic brines (7.5 to 15.8% salts), with a mean oxidoreduction potential of -325 mV, and contain high concentrations of sulfate and sulfide. We surveyed the microbial diversity in the sediments of seven springs by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers. Dendrogram analysis of the DGGE banding patterns divided the springs into two clusters based on their geographic origin. Bacterial 16S rRNA clone sequences from the Gypsum Hill library (spring GH-4) were classified into seven phyla (Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria, Spirochaetes, and Verrucomicrobia); Deltaproteobacteria and Gammaproteobacteria sequences represented half of the clone library. Sequences related to Proteobacteria (82%), Firmicutes (9%), and Bacteroidetes (6%) constituted 97% of the bacterial clone library from Colour Peak (spring CP-1). Most GH-4 archaeal clone sequences (79%) were related to the Crenarchaeota while half of the CP-1 sequences were related to orders Halobacteriales and Methanosarcinales of the Euryarchaeota. Sequences related to the sulfur-oxidizing bacterium Thiomicrospira psychrophila dominated both the GH-4 (19%) and CP-1 (45%) bacterial libraries, and 56 to 76% of the bacterial sequences were from potential sulfur-metabolizing bacteria. These results suggest that the utilization and cycling of sulfur compounds may play a major role in the energy production and maintenance of microbial communities in these unique, cold environments.     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.     

Seasonal changes in an alpine soil bacterial community in the colorado rocky mountains

The period when the snowpack melts in late spring is a dynamic time for alpine ecosystems. The large winter microbial community begins to turn over rapidly, releasing nutrients to plants. Past studies have shown that the soil microbial community in alpine dry meadows of the Colorado Rocky Mountains changes in biomass, function, broad-level structure, and fungal diversity between winter and early summer. However, little specific information exists on the diversity of the alpine bacterial community or how it changes during this ecologically important period. We constructed clone libraries of 16S ribosomal DNA from alpine soil collected in winter, spring, and summer. We also cultivated bacteria from the alpine soil and measured the seasonal abundance of selected cultured isolates in hybridization experiments. The uncultured bacterial communities changed between seasons in diversity and abundance within taxa. The Acidobacterium division was most abundant in the spring. The winter community had the highest proportion of Actinobacteria and members of the Cytophaga/Flexibacter/Bacteroides (CFB) division. The summer community had the highest proportion of the Verrucomicrobium division and of beta-PROTEOBACTERIA: As a whole, alpha-Proteobacteria were equally abundant in all seasons, although seasonal changes may have occurred within this group. A number of sequences from currently uncultivated divisions were found, including two novel candidate divisions. The cultured isolates belonged to the alpha-, beta-, and gamma-Proteobacteria, the Actinobacteria, and the CFB groups. The only uncultured sequences that were closely related to the isolates were from winter and spring libraries. Hybridization experiments showed that actinobacterial and beta-proteobacterial isolates were most abundant during winter, while the alpha- and gamma-proteobacterial isolates tested did not vary significantly. While the cultures and clone libraries produced generally distinct groups of organisms, the two approaches gave consistent accounts of seasonal changes in microbial diversity.     

Phylogenetic Composition of Bacterioplankton Assemblages from the Arctic Ocean

We analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domain Bacteria: α (36%)-, γ (32%)-, δ (14%)-, and (1%)-Proteobacteria; Cytophaga-Flexibacter-Bacteroides spp. (9%); Verrucomicrobium spp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 and Roseobacter groups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related to Cytophaga and Polaribacter species were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) with Alteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) with Oceanospirillum sp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to α-Proteobacteria (dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to γ-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.     

Major differences of bacterial diversity and activity inside and outside of a natural iron-fertilized phytoplankton bloom in the Southern Ocean

One of the first comparisons of a natural iron fertilized bloom with a high-nutrient low-chlorophyll (HNLC) site was undertaken during the Kerguelen ocean and plateau compared study (KEOPS) cruise. To understand better the bacteria–phytoplankton relationship in the context of natural iron fertilization, bacterial diversity and activity was investigated in the bloom and in the adjacent HNLC region by 16S rDNA clone libraries and by single strand conformation polymorphism (SSCP) analysis. Both libraries were dominated by Alphaproteobacteria , Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cluster analysis at 99% sequence similarity yielded several microdiverse clusters and revealed striking differences between the two libraries. In the bloom, the dominant operational taxonomic units (OTUs) were the Roseobacter NAC11-7 cluster, SAR92 and a Cytophaga-Flavobacteria-Bacteroides cluster related to the agg58 group, whereas in the HNLC region, SAR11, Roseobacter RCA and Polaribacter dominated. SSCP analysis of 16S rDNA and 16S rRNA revealed contrasting dynamics of three different Roseobacter OTUs. Roseobacter NAC11-7 and NAC11-6 had higher relative abundances and activities in the bloom compared with the HNLC site and NAC11-6 was only detected at the decline of the bloom concomitant with a shift in phytoplankton composi tion. In contrast, Roseobacter RCA was relatively abundant and active both inside and outside of the bloom. These results suggest that the different OTUs within the Roseobacter group represent functional groups that each play an important role in the cycling of carbon.