Bacterioplankton community composition along a salinity gradient of sixteen high-mountain lakes located on the Tibetan Plateau, China

The influence of altitude and salinity on bacterioplankton community composition (BCC) in 16 high-mountain lakes located at altitudes of 2,817 to 5,134 m on the Eastern Qinghai-Xizang (Tibetan) Plateau, China, spanning a salinity gradient from 0.02% (freshwater) to 22.3% (hypersaline), was investigated. Three different methods, fluorescent in situ hybridization, denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing, and reverse line blot hybridization (RLB) with probes targeting 17 freshwater bacterial groups, were used for analysis of BCC. Furthermore, the salt tolerances of 47 strains affiliated with groups detected in or isolated from the Tibetan habitats were investigated. Altitude was not found to influence BCC significantly within the investigated range. Several groups of typical freshwater bacteria, e.g., the ACK-M1 cluster and the Polynucleobacter group, were detected in habitats located above 4,400 m. Salinity was found to be the dominating environmental factor controlling BCC in the investigated lakes, resulting in only small overlaps in the BCCs of freshwater and hypersaline lakes. The relative abundances of different classes of Proteobacteria showed a sharp succession along the salinity gradient. Both DGGE and RLB demonstrated that a few freshwater bacterial groups, e.g., GKS98 and LD2, appeared over wide salinity ranges. Six freshwater isolates affiliated with the GKS98 cluster grew in ecophysiological experiments at maximum salinities of 0.3% to 0.7% (oligosaline), while this group was detected in habitats with salinities up to 6.7% (hypersaline). This observation indicated ecologically significant differences in ecophysiological adaptations among members of this narrow phylogenetic group and suggested ecological significance of microdiversity.     

High predictability of the seasonal dynamics of a species-like Polynucleobacter population in a freshwater lake

One of the key questions in microbial ecology is if seasonal patterns of bacterial community composition (BCC) observed in one year repeat in the following years. We have investigated if the recorded annual dynamics of a species-like Polynucleobacter (subcluster PnecB) population allowed the prediction of the population dynamics in another year. The abundance of PnecB bacteria in the pelagic of temperate Lake Mondsee was investigated by fluorescence in situ hybridization (FISH) over three consecutive years. The PnecB bacteria formed a persistent population, and were present in the entire water body of the lake. Two of the three investigated years differed strongly in summer temperatures and precipitation, which resulted in markedly different growth conditions. But despite of these different environmental conditions, the PnecB population demonstrated remarkably similar seasonal dynamics in the three investigated years. Water temperature was the best predictor of the population dynamics during the first half of the annual cycles. Statistical analysis also indicated influences of phytoplankton and metazooplankton successions on the PnecB population dynamics. Furthermore, 65 lakes and ponds were investigated for the presence of PnecB bacteria. They were detected in the majority (78%) of circum-neutral and alkaline freshwater habitats, but not in any investigated acidic or saline habitat.     

Recurrent seasonal variations in abundance and composition of filamentous SOL cluster bacteria (Saprospiraceae, Bacteroidetes) in oligomesotrophic Lake Mondsee (Austria)

The spatial and temporal variation of SOL cluster bacteria was assessed in oligomesotrophic Lake Mondsee and adjacent lakes by fluorescence in situ hybridization over two annual cycles. The filamentous SOL bacteria were present in Lake Mondsee throughout the study period, and the seasonal dynamics of the SOL community were remarkably similar with respect to both abundance and composition in the two consecutive years. Only two of the three SOL subclusters were detected in Lake Mondsee and four connected lakes. These two populations significantly differed in size distribution and demonstrated pronounced but recurrent differences in seasonality and length of period of appearance in Lake Mondsee. Extensive sampling of the lakes in September 2003 revealed low horizontal variation in the composition of the SOL community within Lake Mondsee but marked variations with depth. Between connected habitats pronounced differences in the composition and abundance of the SOL community were detected. The interaction of SOL bacteria with bacterivorous protists, mesozooplankton, and phytoplankton was investigated in order to reveal variables controlling the structure and dynamics of SOL communities. No strong indication for a bottom-up influence of phytoplankton was found, while the estimated community grazing rates of mesozooplankton on SOL bacteria indicated a top-down control of SOL abundance during mesozooplankton peaks in spring and early autumn. Furthermore, species-specific differences in grazing of mesozooplankton on SOL bacteria were observed. In general, the overall composition of SOL communities was controlled by abiotic factors (water chemistry), while their dynamics seemed to be controlled by abiotic and biotic interactions.     

Recurrent Seasonal Variations in Abundance and Composition of Filamentous SOL Cluster Bacteria (Saprospiraceae, Bacteroidetes) in Oligomesotrophic Lake Mondsee (Austria)

The spatial and temporal variation of SOL cluster bacteria was assessed in oligomesotrophic Lake Mondsee and adjacent lakes by fluorescence in situ hybridization over two annual cycles. The filamentous SOL bacteria were present in Lake Mondsee throughout the study period, and the seasonal dynamics of the SOL community were remarkably similar with respect to both abundance and composition in the two consecutive years. Only two of the three SOL subclusters were detected in Lake Mondsee and four connected lakes. These two populations significantly differed in size distribution and demonstrated pronounced but recurrent differences in seasonality and length of period of appearance in Lake Mondsee. Extensive sampling of the lakes in September 2003 revealed low horizontal variation in the composition of the SOL community within Lake Mondsee but marked variations with depth. Between connected habitats pronounced differences in the composition and abundance of the SOL community were detected. The interaction of SOL bacteria with bacterivorous protists, mesozooplankton, and phytoplankton was investigated in order to reveal variables controlling the structure and dynamics of SOL communities. No strong indication for a bottom-up influence of phytoplankton was found, while the estimated community grazing rates of mesozooplankton on SOL bacteria indicated a top-down control of SOL abundance during mesozooplankton peaks in spring and early autumn. Furthermore, species-specific differences in grazing of mesozooplankton on SOL bacteria were observed. In general, the overall composition of SOL communities was controlled by abiotic factors (water chemistry), while their dynamics seemed to be controlled by abiotic and biotic interactions.     

Contrasting trends in distribution of four major planktonic betaproteobacterial groups along a pH gradient of epilimnia of 72 freshwater habitats

The distribution and abundance of Betaproteobacteria and three of its genera - Limnohabitans (R-BT065 lineage), Polynucleobacter (including subclusters Polynucleobacter necessarius and Polynucleobacter acidiphobus/Polynucleobacter difficilis), and Methylophilus - across the epilimnia of 72 limnologically diverse freshwater habitats were investigated using fluorescence in situ hybridization. Moreover, seasonal development of Betaproteobacteria subgroups along the longitudinal axis of a reservoir was followed. Betaproteobacteria comprised on average 29.1%, Polynucleobacter 11.6%, P.?necessarius 10.1%, P.?acidiphobus/difficilis 0.5%, Limnohabitans 8.9%, and Methylophilus 0.9% of total bacterioplankton cells in the investigated habitats. Polynucleobacter necessarius and Limnohabitans coexisted in the majority of habitats but showed contrasting abundance patterns along the pH gradient of habitats (pH, 3.8-8.5). The observed distribution patterns could theoretically be explained by different preferences for substrate sources, that is, substances of humic origin in acidic waters and algal-derived substances in alkaline waters. However, substrate utilization patterns observed in laboratory experiments indicate no coherent group-specific differences in substrate preferences. Interestingly, similar distribution patterns were revealed for Limnohabitans and P.?acidiphobus/difficilis, suggesting similar ecological adaptations of these distantly related taxa. Our findings further emphasize that at least two taxa of freshwater Betaproteobacteria represent ecologically diversified groups. Investigations at higher phylogenetic resolution are required for obtaining further insights into their ecology.     

Rhodoferax-related pufM gene cluster dominates the aerobic anoxygenic phototrophic communities in German freshwater lakes

The presence of aerobic anoxygenic phototrophs (AAPs) has been repeatedly reported from various marine environments, but their distribution in freshwater lakes was neglected until recently. We investigated the phylogenetic composition of AAP communities in 10 lakes in Northeastern Germany with different trophic status including oligotrophic Lake Stechlin and humic matter rich Lake Grosse Fuchskuhle. The AAP community was composed by members of Alpha- and Betaproteobacteria, but their contribution varied largely among the studied lakes. Our results show that AAP community composition in the studied lakes was affected mostly by pH and humic matter content. While alkaline lakes were mostly composed of Betaproteobacteria, the acidic and humic matter rich south-west (SW) basin of Lake Grosse Fuchskule was dominated (87%) by Alphaproteobacteria. The most frequent group within Betaproteobacteria was a cluster of pufM genes which was phylogenetically related to Rhodoferax representing 38.5% of all retrieved sequences. Alphaproteobacteria-related sequences had a broader phylogenetic diversity including six different taxa dominated by Sphingomonas- and Rhodobacter-like bacteria in lakes with alkaline to neutral pH. In the acidic and humic matter-rich SW basin of Lake Grosse Fuchskuhle, however, Methylobacterium-related sequences dominated the AAP community. We suggest that the variable AAP community structure might reflect the potential of these bacteria to cope with the contrasting conditions in freshwater environments.     

Low intraspecific diversity in a polynucleobacter subcluster population numerically dominating bacterioplankton of a freshwater pond

Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.     

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.     

Isolation of novel ultramicrobacteria classified as actinobacteria from five freshwater habitats in Europe and Asia

We describe the first freshwater members of the class Actinobacteria that have been isolated. Nine ultramicro-size (<0.1 microm(3)) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 microm(3) and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae. The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp. strain DS.     

Temporal Patterns in Glycolate-Utilizing Bacterial Community Composition Correlate with Phytoplankton Population Dynamics in Humic Lakes

Previous observations of correlated community dynamics between phytoplankton and bacteria in lakes indicate that phytoplankton populations may influence bacterial community structure. To investigate the possibility that bacterial use of phytoplankton exudates contributes to observed patterns of community change, we characterized the diversity and dynamics of heterotrophic bacterioplankton with genetic potential to use glycolate, a photorespiration-specific exudate, in five lakes over a 15-week period. Culture-independent approaches were used to track different bacterial phylotypes represented by DNA sequence variation in the functional gene glycolate oxidase subunit D (glcD). glcD gene sequences from freshwater bacteria exhibited broad phylogenetic diversity, including sequences representing the Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia. The majority of glcD gene sequences were betaproteobacterial, with 48% of the sequences clustering with the glcD gene from the cosmopolitan freshwater species Polynucleobacter necessarius. Terminal restriction fragment length polymorphism fingerprinting of the glcD gene revealed changes in glycolate-utilizing assemblages over time. An average of 39% of within-lake temporal variation in glycolate-utilizing assemblages across five lakes was explained by phytoplankton community composition and dynamics. The interaction between phytoplankton populations and the environment explained an additional 17% of variation on average. These observations offer new insight into the diversity and temporal dynamics of freshwater bacteria with genetic potential to use glycolate and support the hypothesis that algal exudates influence the structure of bacterial communities.     

Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria)

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approximately 98.5%, have been reported to occur as obligate endosymbionts of ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S-23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex life cycle. These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and obligately free-living bacteria ever revealed.     

Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria

In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenk?llesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia). The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin. Six of the clusters were affiliated with the alpha, four were affiliated with the beta, and one was affiliated with the gamma subclass of the Proteobacteria; four were affiliated with the Cytophaga-Flavobacterium-Bacteroides group; and one was affiliated with the class Actinobacteria (formerly known as the high-G+C gram-positive bacteria). The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments. Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 x 10(5) cells ml(-1) in Lake Gossenk?llesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated. Cell size measurements revealed that Actinobacteria in Lake Gossenk?llesee can account for up to 63% of the bacterioplankton biomass. A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class Actinobacteria in freshwater ecosystems.     

Multivariate analysis of microbial communities in the River Elbe (Germany) on different phylogenetic and spatial levels of resolution

The microbial communities of three different habitat types and from two sediment depths in the River Elbe were investigated by fluorescence in situ hybridization at various levels of complexity. Differences in the microbial community composition of free-flowing river water, water within the hyporheic interstitial and sediment-associated bacteria were quantitatively analyzed using domain- and group-specific oligonucleotide probes. Qualitative data on the presence/absence of specific bacterial taxa were gathered using genus- and species-specific probes. The complete data set was statistically processed by univariate statistical approaches, and two-dimensional ordinations of nonmetric multidimensional scaling. The analysis showed: (1) that the resolution of microbial community structures at microenvironments, habitats and locations can be regulated by targeted application of oligonucleotides on phylogenetic levels ranging from domains to species, and (2) that an extensive qualitative presence/absence analysis of multiparallel hybridization assays enables a fine-scale apportionment of spatial differences in microbial community structures that is robust against apparent limitations of fluorescence in situ hybridization such as false positive hybridization signals or inaccessibility of in situ oligonucleotide probes. A general model for the correlation of the phylogenetic depth of focus and the relative spatial resolution of microbial communities by fluorescence in situ hybridization is presented.     

Ubiquity of Polynucleobacter necessarius ssp. asymbioticus in lentic freshwater habitats of a heterogenous 2000 km2 area

We present a survey on the distribution and habitat range of Polynucleobacter necessarius ssp. asymbioticus (PnecC), a numerically and functionally important taxon in the plankton of freshwater systems. We systematically sampled stagnant freshwater habitats in a heterogeneous 2000 km² area, together with ecologically different habitats outside this area. In total, 137 lakes, ponds and puddles were investigated, which represent an enormous diversity of habitats differing, e.g. in depth (< 10 cm – 171 m) and pH (3.9–8.5). PnecC bacteria were detected by cultivation‐independent methods in all investigated habitats, and their presence was confirmed by cultivation of strains from selected habitats representing the whole studied ecological range. The determined relative abundance of the subspecies ranged from values close to the detection limit of FISH (0.2%) to 67% (average 14.5%), and the highest observed absolute abundance was 5.3 × 10⁶ cells ml^?1. Statistical analyses revealed that the abundance of PnecC bacteria was partially controlled by factors linked to concentrations of humic substances, which support the hypothesis that these bacteria utilize photodegradation products of humic substances. Based on the revealed statistical relationships, an average relative abundance of this subspecies of 20% in global freshwater habitats was extrapolated. Our study provides important implications for the current debate on ubiquity and biogeography in microorganisms.     

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.     

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.     

Euxinic freshwater hypolimnia promote bacterial endemicity in continental areas

Bacteria and archaea represent the vast majority of biodiversity on Earth. The ways that dynamic ecological and evolutionary processes interact in the microbial world are, however, poorly known. Here, we have explored community patterns of planktonic freshwater bacteria inhabiting stratified lakes with oxic/anoxic interfaces and euxinic (anoxic and sulfurous) water masses. The interface separates a well-oxygenated upper water mass (epilimnion) from a lower anoxic water compartment (hypolimnion). We assessed whether or not the vertical zonation of lakes promoted endemism in deeper layers by analyzing bacterial 16S rRNA gene sequences from the water column of worldwide distributed stratified lakes and applying a community ecology approach. Community similarity based on the phylogenetic relatedness showed that bacterial assemblages from the same water layer were more similar across lakes than to communities from different layer within lakes and that anoxic hypolimnia presented greater β-diversity than oxic epilimnia. Higher β-diversity values are attributable to low dispersal and small connectivity between community patches. In addition, surface waters had significant spatial but non-significant environmental components controlling phylogenetic β-diversity patterns, respectively. Conversely, the bottom layers were significantly correlated with environment but not with geographic distance. Thus, we observed different ecological mechanisms simultaneously acting on the same water body. Overall, bacterial endemicity is probably more common than previously thought, particularly in isolated and environmentally heterogeneous freshwater habitats. We argue for a microbial diversity conservation perspective still lacking in the global and local biodiversity preservation policies.     

Diversity and seasonal dynamics of Actinobacteria populations in four lakes in northeastern Germany

The phylogenetic diversity and seasonal dynamics of freshwater Actinobacteria populations in four limnologically different lakes of the Mecklenburg-Brandenburg Lake District (northeastern Germany) were investigated. Fluorescence in situ hybridization was used to determine the seasonal abundances and dynamics of total Actinobacteria (probe HGC69a) and the three actinobacterial subclusters acI, acI-A, and acI-B (probes AcI-852, AcI-840-1, and AcI-840-2). Seasonal means of total Actinobacteria abundances in the epilimnia of the lakes varied from 13 to 36%, with maximum values of 30 to 58%, of all DAPI (4',6'-diamidino-2-phenylindole)-stained cells. Around 80% of total Actinobacteria belonged to the acI cluster. The two subclusters acI-A and acI-B accounted for 60 to 91% of the acI cluster and showed seasonal means of 49% (acI-B) and 23% (acI-A) in relation to the acI cluster. Total Actinobacteria and members of the clusters acI and acI-B showed distinct seasonal changes in their absolute abundances, with maxima in late spring and fall/winter. In eight clone libraries constructed from the lakes, a total of 76 actinobacterial 16S rRNA gene sequences were identified from a total of 177 clones. The majority of the Actinobacteria sequences belonged to the acI and acIV cluster. Several new clusters and subclusters were found (acSTL, scB1-4, and acIVA-D). The majority of all obtained 16S rRNA gene sequences are distinct from those of already-cultured freshwater Actinobacteria.     

Intra-habitat Differences in the Composition of the Methanogenic Archaeal Community between the Microcystis-Dominated and the Macrophyte-Dominated Bays in Taihu Lake

A regime shift between a macrophyte-dominated clear state and a phytoplankton-dominated turbid state can have considerable impact on ecosystem structure and function of shallow lakes. However, very little is known about the response of the methanogenic archaeal community in the sediment during this regime shift. We investigated the methanogenic archaeal community at two sites in the large, shallow, eutrophic Taihu Lake over the course of one year. One site is located in Meiliang Bay and is dominated by Microcystis blooms, and the other site is located in East Taihu Bay and is dominated by aquatic macrophytes. Terminal restriction fragment length polymorphism (T-RFLP) and phylogenetic analyses of archaeal 16S rRNA genes were used to analyze the methanogenic community. Higher ratio of methanogens in Archaea was observed in East Taihu Bay than in Meiliang Bay. The methanogenic archaeal community was dominated by the Methanobacteriales and the LDS cluster in macrophytes-dominated East Taihu Bay, while it was dominated by the Methanosarcinaceae, Methanobacteriales, and the LDS cluster in Microcystis-dominated Meiliang Bay. Clustering analysis of all of the samples revealed differences in the composition of the methanogenic archaeal communities between the two sites that were independent of seasonal variations. Further statistical analysis indicated that the chlorophyll a (Chla) concentration had a profound impact on the composition of the methanogenic archaeal community in Meiliang Bay, whereas it was primarily influenced by total organic carbon (TOC) levels in East Taihu Bay. Overall, this investigation demonstrates that intra-habitat differences in the composition of methanogenic archaeal communities are likely driven by changes in the available organic materials.     

Assessing the diversity and specificity of two freshwater viral communities through metagenomics

Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale.To help identify the characteristics and potential specificities of freshwater viral communities, such communities from two lakes differing by their ecological parameters were studied through metagenomics. Both the cluster richness and the species richness of the Lake Bourget virome were significantly higher that those of the Lake Pavin, highlighting a trend similar to the one observed for microorganisms (i.e. the specie richness observed in mesotrophic lakes is greater than the one observed in oligotrophic lakes). Using 29 previously published viromes, the cluster richness was shown to vary between different environment types and appeared significantly higher in marine ecosystems than in other biomes. Furthermore, significant genetic similarity between viral communities of related environments was highlighted as freshwater, marine and hypersaline environments were separated from each other despite the vast geographical distances between sample locations within each of these biomes. An automated phylogeny procedure was then applied to marker genes of the major families of single-stranded (Microviridae, Circoviridae, Nanoviridae) and double-stranded (Caudovirales) DNA viruses. These phylogenetic analyses all spotlighted a very broad diversity and previously unknown clades undetectable by PCR analysis, clades that gathered sequences from the two lakes. Thus, the two freshwater viromes appear closely related, despite the significant ecological differences between the two lakes. Furthermore, freshwater viral communities appear genetically distinct from other aquatic ecosystems, demonstrating the specificity of freshwater viruses at a community scale for the first time.