Flavobacteria blooms in four eutrophic lakes: linking population dynamics of freshwater bacterioplankton to resource availability

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.     

Do Patterns of Bacterial Diversity along Salinity Gradients Differ from Those Observed for Macroorganisms?

It is widely accepted that biodiversity is lower in more extreme environments. In this study, we sought to determine whether this trend, well documented for macroorganisms, also holds at the microbial level for bacteria. We used denaturing gradient gel electrophoresis (DGGE) with phylum-specific primers to quantify the taxon richness (i.e., the DGGE band numbers) of the bacterioplankton communities of 32 pristine Tibetan lakes that represent a broad salinity range (freshwater to hypersaline). For the lakes investigated, salinity was found to be the environmental variable with the strongest influence on the bacterial community composition. We found that the bacterial taxon richness in freshwater habitats increased with increasing salinity up to a value of 1‰. In saline systems (systems with >1‰ salinity), the expected decrease of taxon richness along a gradient of further increasing salinity was not observed. These patterns were consistently observed for two sets of samples taken in two different years. A comparison of 16S rRNA gene clone libraries revealed that the bacterial community of the lake with the highest salinity was characterized by a higher recent accelerated diversification than the community of a freshwater lake, whereas the phylogenetic diversity in the hypersaline lake was lower than that in the freshwater lake. These results suggest that different evolutionary forces may act on bacterial populations in freshwater and hypersaline lakes on the Tibetan Plateau, potentially resulting in different community structures and diversity patterns.     

太湖夏季浮游细菌群落多样性的空间格局

为了研究太湖夏季浮游细菌群落多样性与水体营养盐的关系,在太湖全湖范围内开展了一次大规模浮游细菌采样调查,分析了太湖不同湖区浮游细菌丰度和多样性组成。研究发现,浮游细菌丰度在不同湖区中存在明显的空间差异,从北部和西部湖区沿湖流向东南方向至湖心和南部沿岸再到东部湖区呈下降趋势,这与太湖水体营养水平从高到低变化趋势一致。浮游细菌丰度与营养盐浓度回归分析结果显示,总磷（TP）与细菌丰度存在较好的正相关（R2=0.6392,n=29,P0.01）,而总氮（TN）与细菌丰度无显著相关（R2=0.0663,n=29,P0.05）。因此,磷是太湖夏季浮游细菌生长的限制因子。不同湖区营养盐与浮游细菌群落多样性也具有显著的正相关,随着营养水平的升高,浮游细菌多样性增加。此外,细菌群落的组成在不同湖区间亦具有明显的空间异质性,与不同湖区营养水平空间变化一致。研究结果将有助于人们更好地理解淡水湖泊中微生物循环和生态系统功能。       

A fast,highly sensitive double‐nested PCR‐based method to screen fish immunobiomes

Efficient methods for constructing 16S tag amplicon libraries for pyrosequencing are needed for the rapid and thorough screening of infectious bacterial diversity from host tissue samples. Here we have developed a double‐nested PCR methodology that generates 16S tag amplicon libraries from very small amounts of bacteria/host samples. This methodology was tested for 133 kidney samples from the lake whitefish Coregonus clupeaformis (Salmonidae) sampled in five different lake populations. The double‐nested PCR efficiency was compared with two other PCR strategies: single primer pair amplification and simple nested PCR. The double‐nested PCR was the only amplification strategy to provide highly specific amplification of bacterial DNA. The resulting 16S amplicon libraries were synthesized and pyrosequenced using 454 FLX technology to analyse the variation of pathogenic bacteria abundance. The proportion of the community sequenced was very high (Good’s coverage estimator; mean = 95.4%). Furthermore, there were no significant differences of sequence coverage among samples. Finally, the occurrence of chimeric amplicons was very low. Therefore, the double‐nested PCR approach provides a rapid, informative and cost‐effective method for screening fish immunobiomes and most likely applicable to other low‐density microbiomes as well.     

Comparative analysis of bacterioplankton assemblages from maritime Antarctic freshwater lakes with contrasting trophic status

The bacterioplankton assemblages of eight maritime Antarctic lakes with a wide range of trophic status and geographic span (six lakes from Hope Bay, Antarctic Peninsula and two from Potter Peninsula, King George Island) were described using denaturing gradient gel electrophoresis and band sequencing during two consecutive austral summers (2003–2004). Analyses of the gels identified a total of 230 bands spread across 57 different positions. Among those bands, 14 were shared between lakes from Hope Bay and Potter Peninsula, 17 were observed only in particular lakes, and 17 were registered both years in the same lake. We successfully reamplified and sequenced 43 bands located in 36 different positions belonging to Bacteroidetes, Actinobacteria, Betaproteobacteria and Cyanobacteria. The closest matches for 63% of the sequenced bands were from Antarctic or from other cold environment clones and sequences already in the databases, suggesting the widespread dominance of microbial communities adapted to cold habitats. The results of the multivariate analyses (Cluster Analysis and CCA) indicated that the nutrient status of the lake influences the bacterioplankton assemblages. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seasonal variations in bacterioplankton community structures in two small rivers in the Himi region of central Japan and their relationships with environmental factors

The aim of this study was to improve our understanding of seasonal variations and the effects of physicochemical conditions on the bacterioplankton communities in two small rivers, the Moo and Nakayachi Rivers in the Himi region of central Japan. These rivers are inhabited by unionid freshwater mussels, which are used for oviposition by the endangered Itasenpara bitterling (Acheilognathus longipinnis). Water samples were collected every month between March 2011 and February 2012. Changes in bacterioplankton community structures were analysed using an approach that did not require cultivating the bacteria and involved PCR and denaturing gradient gel electrophoresis. The bacterioplankton community structures in the two rivers were similar in all seasons except winter. The bacterial sequences identified were dominated by typical freshwater Actinobacteria, Bacteroidetes, Cyanobacteria, α-Proteobacteria, and β-Proteobacteria bacterioplankton. Many β-Proteobacteria species were detected in all seasons, but Bacteroidetes species were dominant in the winter. The bacterioplankton community structures were affected by biochemical oxygen demand, chemical oxygen demand, chlorophyll-a concentration, water depth, and water temperature. These results provide a foundation for a more detailed understanding of the conditions that provide a suitable unionid habitat.     

pH Influences the Importance of Niche-Related and Neutral Processes in Lacustrine Bacterioplankton Assembly

pH is an important factor that shapes the structure of bacterial communities. However, we have very limited information about the patterns and processes by which overall bacterioplankton communities assemble across wide pH gradients in natural freshwater lakes. Here, we used pyrosequencing to analyze the bacterioplankton communities in 25 discrete freshwater lakes in Denmark with pH levels ranging from 3.8 to 8.8. We found that pH was the key factor impacting lacustrine bacterioplankton community assembly. More acidic lakes imposed stronger environmental filtering, which decreased the richness and evenness of bacterioplankton operational taxonomic units (OTUs) and largely shifted community composition. Although environmental filtering was determined to be the most important determinant of bacterioplankton community assembly, the importance of neutral assembly processes must also be considered, notably in acidic lakes, where the species (OTU) diversity was low. We observed that the strong effect of environmental filtering in more acidic lakes was weakened by the enhanced relative importance of neutral community assembly, and bacterioplankton communities tended to be less phylogenetically clustered in more acidic lakes. In summary, we propose that pH is a major environmental determinant in freshwater lakes, regulating the relative importance and interplay between niche-related and neutral processes and shaping the patterns of freshwater lake bacterioplankton biodiversity.     

Bacterial community structure in a latitudinal gradient of lakes: the roles of spatial versus environmental factors

1. We analysed the latitudinal variation of bacterioplankton in 45 freshwater environments (lakes, shallow lakes and ponds) across a transect of more than 2100 km stretching from Argentinean Patagonia (45°S) to Maritime Antarctica (63°S), to determine the factors that mainly determine bacterioplankton community structure. 2. Bacterioplankton community composition (BCC) was assessed by a fingerprinting method (denaturing gradient gel electrophoresis) followed by band sequencing, whereas the abundances of total bacteria and picocyanobacteria were estimated by epifluorescence microscopy. 3. Bacterioplankton community composition was controlled by a combination of spatial (latitude and longitude) and environmental [e.g. phosphate, light diffuse attenuation coefficient (K_d) and dissolved organic carbon] factors. Total bacterioplankton abundance declined with latitude. A multiple regression analysis showed that phosphate, K_d and latitude had significant effects on total bacterioplankton abundance. 4. Of 76 operational taxonomic units identified in the studied lakes, 45 were shared between Patagonian and Antarctic water bodies, 28 were present only in Patagonian lakes and three were restricted to the Antarctic lakes. Significant differences were found in BCC between Patagonia and Antarctica. Among the sequences, 54% were similar (>97% sequence similarity) to others reported from cold habitats elsewhere on the planet (glaciers, high mountain lakes, Arctic). 5. Our results provide new evidence that supports the hypotheses of biogeographic patterns of bacterial assemblages and suggest that both spatial and environmental factors control bacterioplankton community structure.     

Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes

We characterized the bacterioplankton community and its seasonal dynamics in two neighbouring hypertrophic lakes by denaturing gradient gel electrophoresis (DGGE) analysis of short (193 bp) 16S ribosomal DNA polymerase chain reaction (PCR) products obtained with primers specific for the domain Bacteria. Lake Blankaart is turbid and has a high phytoplankton biomass and episodic cyanobacterial blooms, whereas biomanipulated Lake Visvijver is characterized by clearwater conditions and the establishment of a dense charophyte vegetation. Both lakes were dominated by bacterial groups commonly found in freshwater habitats (e.g. ACK4 cluster of Actinomycetes; ACK stands for clones isolated from the Adirondack mountain lakes) . Yet, cluster analysis and principal components analysis (PCA) revealed that taxon composition of the bacterioplankton community of the two lakes differs substantially and consistently throughout the season. During the study year (1998), the bacterioplankton community of both lakes showed a distinct seasonal pattern. Lake Blankaart showed a clear differentiation between winter, spring, summer and autumn. In Lake Visvijver, summer samples differed greatly from spring, autumn and winter samples. We hypothesize that the contrasting bacterioplankton in the two neighbouring shallow lakes is determined largely by the presence or absence of macrophytes.     

Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes

The diversity of freshwater bacterioplankton communities has not been extensively studied despite their key role in foodwebs and the cycling of carbon and associated major elements. In order to explore and characterize the composition of bacterioplankton associated with cyanobacterial blooms, large 16S rRNA clone libraries from four lakes experiencing such blooms were analysed. The four libraries contained 1461 clones, of which 559 were prokaryotic sequences of non-cyanobacterial origin. These clones were classified into 158 operational taxonomic units affiliated mainly with bacterial divisions commonly found in freshwater systems, e.g. Proteobacteria, Bacteriodetes, Actinobacteria, Verrucomicrobia and Planctomycetes. Richness and evenness of non-cyanobacterial clones were similar to other clone libraries obtained for freshwater bacterioplankton, suggesting that bacterial communities accompanying cyanobacterial blooms are as diverse as non-bloom communities. Many of the identified operational taxonomic units grouped with known freshwater clusters but the libraries also contained novel clusters of bacterial sequences that may be characteristic for cyanobacterial blooms. About 25% of the operational taxonomic units were detected in more than one lake. Even so, 16S rRNA heterogeneity analysis demonstrated large differences in community composition between lakes regardless of their similar characteristics and close proximity. Hence even the similar environmental conditions created by different cyanobacterial blooms may foster very dissimilar bacterial communities, which could indicate that the genetic diversity in lake bacteria have been underestimated in the past.     

The structure and stability of the bacterioplankton community in Antarctic freshwater lakes, subject to extremely rapid environmental change

In this study, variation in the bacterioplankton community structure of three Antarctic lakes of different nutrient status, was determined in relation to physical and chemical gradients at depth and at time intervals, across the seasonal transition from winter ice-cover to the summer ice-free period. The three lakes studied were: Moss Lake (low nutrient, with typical nutrient concentrations of 80 microg l(-1) nitrate and 10 microg l(-1) dissolved reactive phosphate), Sombre Lake (low nutrient, but becoming progressively enriched, with typical nutrient concentrations of 185 microg l(-1) nitrate and 7 microg l(-1) dissolved reactive phosphate) and Heywood Lake (enriched, with typical nutrient concentrations of 1180 microg l(-1) nitrate and 124 microg l(-1) dissolved reactive phosphate). Bacterioplankton community structure was determined using a combination of PCR amplification of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE). Results indicated marked changes in this bacterioplankton community structure, which were particularly associated with the transition period. However, significant changes also occurred during the period of holomixis. Comparison of the results from lakes of different nutrient status suggest that increased levels of nutrient input, and in the timing and duration of ice cover will lead to marked changes in the structure and stability of the bacterioplankton community at existing levels of environmental change.     

Evaluation of Extracellular,High-affinity β-N-acetylglucosaminidase Measurements from Freshwater Lakes: An Enzyme Assay to Estimate Protistan Grazing on Bacteria and Picocyanobacteria

Protistan community grazing rates upon both bacterioplankton and autotrophic picoplankton were estimated using fluorescently-labeled prey and by measurement of extracellular hydrolysis of 4-methylumbelliferyl (MUF) beta-N-acetylglucosaminide in a eutrophic reservoir and an oligo-mesotrophic lake during phytoplankton blooms. In addition, enzyme methods were optimized in bacterivorous flagellate cultures by two enzyme assays, based on fluorometric detection of protistan digestive activity, which were compared and calibrated independently against flagellate bacterivory. Enzymatic hydrolyses of MUF beta-N,N',N'-triacetylchitotriose and MUF beta-N-acetylglucosaminide were measured in cell-free (sonicated) and whole-cell (unsonicated) samples. The hydrolysis of both substrates, using the whole-cell enzyme assay at in situ pH, was correlated significantly with total grazing rate of Bodo saltans. Thus the whole-cell enzyme assay with MUF beta-N-acetylglucosaminide was used for freshwater samples. High-affinity (Km < 1 μmol l-1) and low-affinity (Km > 100μmol l-1) enzymes were distinguished kinetically in most samples from both systems studied. Activities (Vmax) of the high-affinity enzyme varied from 0.24 to 1.43 nmol l-1 h-1. Protistan community grazing on bacterioplankton was in the range of 0.15-1.36 μg C l-1 h-1 both for lake and reservoir, the differences being observed in grazing on picocyanobacteria (lake, 0.03-0.22 μg C l-1 h-1; reservoir, 0.35-1.56 μg C l-1 h-1). The enzyme activities were correlated significantly with the protistan grazing both on bacterioplakton (rs=0.62, P<0.001) and total procaryotic picoplankton (the sum of organic carbon grazed from bacteria and picocyanobacteria, rs=0.73, P<0.001) in the eutrophic reservoir. Weaker relationships (rs=0.42) with a lower slope were found for the oligo-mesotrophic lake. Ingestion rate studies are time-consuming and the digestive enzyme assay with MUF beta-N-acetylglucosaminide presents a rapid alternative for estimating total protistan prokaryotic picoplanktivory in freshwaters.     

Genome-resolved viral and cellular metagenomes revealed potential key virus-host interactions in a deep freshwater lake

Metagenomics has dramatically expanded the known virosphere, but freshwater viral diversity and their ecological interaction with hosts remain poorly understood. Here, we conducted a metagenomic exploration of planktonic dsDNA prokaryotic viruses by sequencing both virion (<0.22 μm) and cellular (0.22–5.0 μm) fractions collected spatiotemporally from a deep freshwater lake (Lake Biwa, Japan). This simultaneously reconstructed 183 complete (i.e., circular) viral genomes and 57 bacterioplankton metagenome-assembled genomes. Analysis of metagenomic read coverage revealed vertical partitioning of the viral community analogous to the vertically stratified bacterioplankton community. The hypolimnetic community was generally stable during stratification, but occasionally shifted abruptly, presumably due to lysogenic induction. Genes involved in assimilatory sulfate reduction were encoded in 20 (10.9%) viral genomes, including those of dominant viruses, and may aid viral propagation in sulfur-limited freshwater systems. Hosts were predicted for 40 (21.9%) viral genomes, encompassing 10 phyla (or classes of Proteobacteria) including ubiquitous freshwater bacterioplankton lineages (e.g., Ca. Fonsibacter and Ca. Nitrosoarchaeum). Comparison with viral genomes derived from published metagenomes revealed viral phylogeographic connectivity in geographically isolated habitats. Notably, analogous to their hosts, actinobacterial viruses were among the most diverse, ubiquitous and abundant viral groups in freshwater systems, with potential high lytic activity in surface waters.     

Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.     

Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time

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 ([¹⁴C]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.     

Nearly a decade‐long repeatable seasonal diversity patterns of bacterioplankton communities in the eutrophic Lake Donghu (Wuhan,China)

Uncovering which environmental factors govern community diversity patterns and how ecological processes drive community turnover are key questions related to understand the community assembly. However, the ecological mechanisms regulating long‐term variations of bacterioplankton communities in lake ecosystems remain poorly understood. Here we present nearly a decade‐long study of bacterioplankton communities from the eutrophic Lake Donghu (Wuhan, China) using 16S rRNA gene amplicon sequencing with MiSeq platform. We found strong repeatable seasonal diversity patterns in terms of both common (detected in more than 50% samples) and dominant (relative abundance >1%) bacterial taxa turnover. Moreover, community composition tracked the seasonal temperature gradient, indicating that temperature is a key environmental factor controlling observed diversity patterns. Total phosphorus also contributed significantly to the seasonal shifts in bacterioplankton composition. However, any spatial pattern of bacterioplankton communities across the main lake areas within season was overwhelmed by their temporal variabilities. Phylogenetic analysis further indicated that 75%–82% of community turnover was governed by homogeneous selection due to consistent environmental conditions within seasons, suggesting that the microbial communities in Lake Donghu are mainly controlled by niche‐based processes. Therefore, dominant niches available within seasons might be occupied by similar combinations of bacterial taxa with modest dispersal rates throughout different lake areas.     

Bacterioplankton communities in a high-altitude freshwater wetland

Microbial communities play a crucial role in various biogeochemical processes in aquatic ecosystems. However, existing knowledge on microbial communities in the waters of wetlands is still very scant. The objective of the present study was to investigate the bacterioplankton community in the Luoshijiang Wetland, a high-altitude freshwater wetland in the Yunnan-Kweichow Plateau. Water samples were collected from different sites. The bacterioplankton community was characterized using 16S rRNA gene clone library analysis. A spatial variation of the diversity and composition of the bacterioplankton community was observed. Verrucomicrobia and Proteobacteria were the most abundant components. Proteobacteria might play an important role in water self-purification, but the significance of Verrucomicrobia remained unclear. Moreover, Pearson’s correlation analysis showed that Actinobacteria and Gemmatimonadetes were positively correlated with nitrite nitrogen in waters, while Alphaproteobacteria with dissolved phosphorous.     

Effects of Resources and Trophic Interactions on Freshwater Bacterioplankton Diversity

Abstract In a study of bacterioplankton in an oligotrophic lake in northern Wisconsin, a community fingerprinting technique, automated ribosomal intergenic spacer analysis (ARISA), was used to determine the effect of resources and trophic interactions on bacterioplankton diversity. Inorganic nitrogen and phosphorus (NP), carbon in the form of glucose (G) or dissolved organic matter extracted from peat (DOM), and carbon and NP in combination were added to two types of experimental systems. Ten-liter mesocosms contained all components of the original aquatic community except for large zooplankton. One-liter dilution cultures were prepared so that the effects of grazers and phytoplankton were removed. During a 3-day incubation, bacterial production showed the greatest response to the carbon plus NP treatment in both experimental systems, but bacterial diversity was strikingly different between them. In the mesocosms, the number of ARISA-PCR fragments averaged 41 per profile, whereas the dilution culture communities were highly reduced in complexity, dominated in most cases by a single PCR fragment. Further analysis of the mesocosm data suggested that whereas the NPDOM addition caused the greatest aggregate bacterial growth response, the addition of NP alone caused the largest shifts in community composition. These results suggest that the measurement of aggregate responses, such as bacterial production, alone in studies of freshwater bacterial communities may mask the effects of resources on bacterioplankton. Received: 24 January 2000; Accepted: 10 May 2000; Online Publication: 28 August 2000