Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period.
ABSTRACT: Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3-20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.
Project description:The oxygenated hypolimnion accounts for a volumetrically significant part of the global freshwater systems. Previous studies have proposed the presence of hypolimnion-specific bacterioplankton lineages that are distinct from those inhabiting the epilimnion. To date, however, no consensus exists regarding their ubiquity and abundance, which is necessary to evaluate their ecological importance. The present study investigated the bacterioplankton community in the oxygenated hypolimnia of 10 deep freshwater lakes. Despite the broad geochemical characteristics of the lakes, 16S rRNA gene sequencing demonstrated that the communities in the oxygenated hypolimnia were distinct from those in the epilimnia and identified several predominant lineages inhabiting multiple lakes. Catalyzed reporter deposition fluorescence in situ hybridization revealed that abundant hypolimnion-specific lineages, CL500-11 (Chloroflexi), CL500-3, CL500-37, CL500-15 (Planctomycetes) and Marine Group I (Thaumarchaeota), together accounted for 1.5-32.9% of all bacterioplankton in the hypolimnion of the lakes. Furthermore, an analysis of single-nucleotide variation in the partial 16S rRNA gene sequence (oligotyping) suggested the presence of different sub-populations between lakes and water layers among the lineages occurring in the entire water layer (for example, acI-B1 and acI-A7). Collectively, these results provide the first comprehensive overview of the bacterioplankton community in the oxygenated hypolimnion of deep freshwater lakes.
Project description:<h4>Background</h4>Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on >?97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe.<h4>Results</h4>Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7-101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion.<h4>Conclusions</h4>Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future. Video abstract.
Project description:Bacteriophages of freshwater environments have not been well studied despite their numerical dominance and ecological importance. Currently, very few phages have been isolated for many abundant freshwater bacterial groups, especially for the family Comamonadaceae that is found ubiquitously in freshwater habitats. In this study, we report two novel phages, P26059A and P26059B, that were isolated from Lake Soyang in South Korea, and lytically infected bacterial strain IMCC26059, a member of the family Comamonadaceae. Morphological observations revealed that phages P26059A and P26059B belonged to the family Siphoviridae and Podoviridae, respectively. Of 12 bacterial strains tested, the two phages infected strain IMCC26059 only, showing a very narrow host range. The genomes of the two phages were different in length and highly distinct from each other with little sequence similarity. A comparison of the phage genome sequences and freshwater viral metagenomes showed that the phage populations represented by P26059A and P26059B exist in the environment with different distribution patterns. Presence of the phages in Lake Soyang and Lake Michigan also indicated a consistent lytic infection of the Comamonadaceae bacterium, which might control the population size of this bacterial group. Taken together, although the two phages shared a host strain, they showed completely distinctive characteristics from each other in morphological, genomic, and ecological analyses. Considering the abundance of the family Comamonadaceae in freshwater habitats and the rarity of phage isolates infecting this family, the two phages and their genomes in this study would be valuable resources for freshwater virus research.
Project description:The bacterioplankton diversity in large rivers has thus far been under-sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA-gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free-living and particle-associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the Polynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater-affiliated bacteria. Based on views of the meta-community and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity.
Project description:High-throughput culturing (HTC) methods that rely on dilution to extinction in very-low-nutrient media were used to obtain bacterial isolates from Crater Lake, Oregon. 16S rRNA sequence determination and phylogenetic reconstruction were used to determine the potential ecological significance of isolated bacteria, both in Crater Lake and globally. Fifty-five Crater Lake isolates yielded 16 different 16S rRNA gene sequences. Thirty of 55 (55%) Crater Lake isolates had 16S rRNA gene sequences with 97% or greater similarity to sequences recovered previously from Crater Lake 16S rRNA gene clone libraries. Furthermore, 36 of 55 (65%) Crater Lake isolates were found to be members of widely distributed freshwater groups. These results confirm that HTC is a significant improvement over traditional isolation techniques that tend to enrich for microorganisms that do not predominate in their environment and rarely correlate with 16S rRNA gene clone library sequences. Although all isolates were obtained under dark, heterotrophic growth conditions, 2 of the 16 different groups showed evidence of photosynthetic capability as assessed by the presence of puf operon sequences, suggesting that photoheterotrophy may be a significant process in this oligotrophic, freshwater habitat.
Project description:To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single-cell genomes and a 5-year metagenomic time series. The single-cell genomes grouped into 15 monophyletic clusters (termed "tribes") that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI Actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.
Project description:The frequency of freshwater cyanobacterial blooms is at risk of increasing as a consequence of climate change and eutrophication of waterways. It is increasingly apparent that abiotic data are insufficient to explain variability within the cyanobacterial community, with biotic factors such as heterotrophic bacterioplankton, viruses and protists emerging as critical drivers. During the Australian summer of 2012-2013, a bloom that occurred in a shallow ephemeral lake over a 6-month period was comprised of 22 distinct cyanobacteria, including Microcystis, Dolichospermum, Oscillatoria and Sphaerospermopsis. Cyanobacterial cell densities, bacterial community composition and abiotic parameters were assessed over this period. Alpha-diversity indices and multivariate analysis were successful at differentiating three distinct bloom phases and the contribution of abiotic parameters to each. Network analysis, assessing correlations between biotic and abiotic variables, reproduced these phases and assessed the relative importance of both abiotic and biotic factors. Variables possessing elevated betweeness centrality included temperature, sodium and operational taxonomic units belonging to the phyla Verrucomicrobia, Planctomyces, Bacteroidetes and Actinobacteria. Species-specific associations between cyanobacteria and bacterioplankton, including the free-living Actinobacteria acI, Bacteroidetes, Betaproteobacteria and Verrucomicrobia, were also identified. We concluded that changes in the abundance and nature of freshwater cyanobacteria are associated with changes in the diversity and composition of lake bacterioplankton. Given this, an increase in the frequency of cyanobacteria blooms has the potential to alter nutrient cycling and contribute to long-term functional perturbation of freshwater systems.
Project description:While fastidious microbes can be abundant and ubiquitous in their natural communities, many fail to grow axenically in laboratories due to auxotrophies or other dependencies. To overcome auxotrophies, these microbes rely on their surrounding cohort. A cohort may consist of kin (ecotypes) or more distantly related organisms (community) with the cooperation being reciprocal or nonreciprocal and expensive (Black Queen hypothesis) or costless (by-product). These metabolic partnerships (whether at single species population or community level) enable dominance by and coexistence of these lineages in nature. Here we examine the relevance of these cooperation models to explain the abundance and ubiquity of the dominant fastidious bacterioplankton of a dimictic mesotrophic freshwater lake. Using both culture-dependent (dilution mixed cultures) and culture-independent (small subunit [SSU] rRNA gene time series and environmental metagenomics) methods, we independently identified the primary cohorts of actinobacterial genera "Candidatus Planktophila" (acI-A) and "Candidatus Nanopelagicus" (acI-B) and the proteobacterial genus "Candidatus Fonsibacter" (LD12). While "Ca Planktophila" and "Ca. Fonsibacter" had no correlation in their natural habitat, they have the potential to be complementary in laboratory settings. We also investigated the bifunctional catalase-peroxidase enzyme KatG (a common good which "Ca Planktophila" is dependent upon) and its most likely providers in the lake. Further, we found that while ecotype and community cooperation combined may explain "Ca Planktophila" population abundance, the success of "Ca. Nanopelagicus" and "Ca. Fonsibacter" is better explained as a community by-product. Ecotype differentiation of "Ca. Fonsibacter" as a means of escaping predation was supported but not for overcoming auxotrophies.IMPORTANCE This study examines evolutionary and ecological relationships of three of the most ubiquitous and abundant freshwater bacterial genera: "Ca Planktophila" (acI-A), "Ca. Nanopelagicus" (acI-B), and "Ca. Fonsibacter" (LD12). Due to high abundance, these genera might have a significant influence on nutrient cycling in freshwaters worldwide, and this study adds a layer of understanding to how seemingly competing clades of bacteria can coexist by having different cooperation strategies. Our synthesis ties together network and ecological theory with empirical evidence and lays out a framework for how the functioning of populations within complex microbial communities can be studied.
Project description:Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([(14)C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and ACTINOBACTERIA: Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.
Project description:Heterotrophic bacteria are major contributors to biogeochemical cycles and influence water quality. Still, the lack of representative isolates and the few quantitative surveys leave the ecological role and significance of single bacterial populations to be revealed. Here we analyzed the diversity and dynamics of freshwater Flavobacteria populations in four eutrophic temperate lakes. From each lake, clone libraries were constructed using primers specific for either the class Flavobacteria or Bacteria. Sequencing of 194 Flavobacteria clones from 8 libraries revealed a diverse freshwater Flavobacteria community and distinct differences among lakes. Abundance and seasonal dynamics of Flavobacteria were assessed by quantitative PCR with class-specific primers. In parallel, the dynamics of individual populations within the Flavobacteria community were assessed with terminal restriction fragment length polymorphism analysis using identical primers. The contribution of Flavobacteria to the total bacterioplankton community ranged from 0.4 to almost 100% (average, 24%). Blooms where Flavobacteria represented more than 30% of the bacterioplankton were observed at different times in the four lakes. In general, high proportions of Flavobacteria appeared during episodes of high bacterial production. Phylogenetic analyses combined with Flavobacteria community fingerprints suggested dominance of two Flavobacteria lineages. Both drastic alterations in total Flavobacteria and in community composition of this class significantly correlated with bacterial production, emphasizing that resource availability is an important driver of heterotrophic bacterial succession in eutrophic lakes.