Nejvíce citovaný článek - PubMed ID 15691929
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
- Klíčová slova
- Freshwater bacterioplankton, Long-read amplicon sequencing, Microdiversity, PacBio, Phylogeography, Ribosomal internal transcribed spacers,
- MeSH
- biodiverzita * MeSH
- fylogeneze MeSH
- fylogeografie * MeSH
- plankton klasifikace genetika izolace a purifikace MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA metody MeSH
- sladká voda * MeSH
- vodní organismy klasifikace genetika izolace a purifikace MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Japonsko MeSH
- Názvy látek
- RNA ribozomální 16S MeSH
Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator-prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Římov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.
- MeSH
- Bacteria klasifikace genetika izolace a purifikace metabolismus MeSH
- Cryptophyta mikrobiologie MeSH
- fylogeneze MeSH
- heterotrofní procesy MeSH
- hybridizace in situ fluorescenční MeSH
- plankton mikrobiologie MeSH
- potravní řetězec MeSH
- roční období MeSH
- sladká voda mikrobiologie parazitologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences.
- MeSH
- Burkholderiaceae genetika izolace a purifikace MeSH
- DNA bakterií genetika MeSH
- ekologie MeSH
- ekosystém MeSH
- fylogeneze MeSH
- genomika * MeSH
- plankton genetika izolace a purifikace MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- sladká voda mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA bakterií MeSH
- RNA ribozomální 16S MeSH
Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.
- MeSH
- Actinobacteria fyziologie MeSH
- Burkholderiaceae fyziologie MeSH
- časové faktory MeSH
- Comamonadaceae fyziologie MeSH
- Eukaryota růst a vývoj metabolismus fyziologie MeSH
- fyziologie bakterií MeSH
- geny rRNA genetika MeSH
- heterotrofní procesy MeSH
- potravní řetězec MeSH
- sladká voda mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Actinorhodopsins (ActRs) are recently discovered proteorhodopsins present in Actinobacteria, enabling them to adapt to a wider spectrum of environmental conditions. Frequently, a large fraction of freshwater bacterioplankton belongs to the acI lineage of Actinobacteria and codes the LG1 type of ActRs. In this paper we studied the genotype variability of the LG1 ActRs. We have constructed two clone libraries originating from two environmentally different habitats located in Central Europe; the large alkaline lake Mondsee (Austria) and the small humic reservoir Jiřická (the Czech Republic). The 75 yielded clones were phylogenetically analyzed together with all ActR sequences currently available in public databases. Altogether 156 sequences were analyzed and 13 clusters of ActRs were distinguished. Newly obtained clones are distributed over all three LG1 subgroups--LG1-A, B and C. Eighty percent of the sequences belonged to the acI lineage (LG1-A ActR gene bearers) further divided into LG1-A1 and LG1-A2 subgroups. Interestingly, the two habitats markedly differed in genotype composition with no identical sequence found in both samples of clones. Moreover, Jiřická reservoir contained three so far not reported clusters, one of them LG1-C related, presenting thus completely new, so far undescribed, genotypes of Actinobacteria in freshwaters.
- MeSH
- Actinobacteria klasifikace genetika růst a vývoj MeSH
- bakteriální proteiny klasifikace genetika MeSH
- DNA bakterií chemie genetika MeSH
- ekosystém * MeSH
- fylogeneze MeSH
- genetická variace MeSH
- hybridizace in situ fluorescenční MeSH
- molekulární sekvence - údaje MeSH
- multigenová rodina genetika MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza DNA MeSH
- sladká voda mikrobiologie MeSH
- zeměpis MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Rakousko MeSH
- Názvy látek
- bakteriální proteiny MeSH
- DNA bakterií MeSH
Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S-23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria. Instead, our observations suggest that the genus Limnohabitans, as well as its R-BT subgroup, represent ecologically heterogeneous taxa, which underwent pronounced ecological diversification.
- MeSH
- bakteriální RNA genetika MeSH
- biodiverzita * MeSH
- Comamonadaceae fyziologie MeSH
- fyziologická adaptace fyziologie MeSH
- hybridizace in situ fluorescenční MeSH
- koncentrace vodíkových iontů MeSH
- mikrobiologie vody * MeSH
- RNA ribozomální 16S genetika MeSH
- sladká voda mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální RNA MeSH
- RNA ribozomální 16S MeSH
A chemo-organotrophic, aerobic, non-motile strain, MWH-BRAZ-DAM2D(T), isolated from a freshwater pond in Brazil, was characterized phenotypically, phylogenetically and chemotaxonomically. Phylogenetic analysis of 16S rRNA gene sequences indicated affiliation of the strain with the genus Limnohabitans (Comamonadaceae, Betaproteobacteria). 16S rRNA gene sequence similarities between the isolate and Limnohabitans curvus MWH-C5(T), representing the type species of the genus, and the type strains of Limnohabitans parvus and Limnohabitans planktonicus were 98.2, 96.5 and 97.0 %, respectively. DNA-DNA reassociation analyses with DNA of the type strains of all three previously described Limnohabitans species revealed similarity values in the range 26.2-44.6 %. The predominant fatty acids of the isolate were C(16 : 1)ω7c/ω6c, C(16 : 0), C(12 : 0) and C(8 : 0) 3-OH, the major quinone was ubiquinone Q-8 and the DNA G+C content was 55.8 mol%. The isolate could be discriminated from the type strains of the three Limnohabitans species by several phenotypic traits including differences in the utilization of several carbon sources. Based on the phylogeny of the isolate and its differences from the three most closely related species, the isolate represents a novel species for which the name Limnohabitans australis sp. nov. is proposed. The type strain is MWH-BRAZ-DAM2D(T) (=DSM 21646(T)=CCUG 56719(T)).
- MeSH
- Comamonadaceae klasifikace genetika izolace a purifikace MeSH
- DNA bakterií genetika MeSH
- fylogeneze * MeSH
- hybridizace nukleových kyselin MeSH
- mastné kyseliny chemie MeSH
- molekulární sekvence - údaje MeSH
- RNA ribozomální 16S genetika MeSH
- rybníky mikrobiologie MeSH
- sekvenční analýza DNA MeSH
- sladká voda mikrobiologie MeSH
- techniky typizace bakterií MeSH
- ubichinon chemie MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Brazílie MeSH
- Názvy látek
- DNA bakterií MeSH
- mastné kyseliny MeSH
- RNA ribozomální 16S MeSH
- ubichinon MeSH
- ubiquinone 8 MeSH Prohlížeč
We investigated potential niche separation in two closely related (99.1% 16S rRNA gene sequence similarity) syntopic bacterial strains affiliated with the R-BT065 cluster, which represents a subgroup of the genus Limnohabitans. The two strains, designated B4 and D5, were isolated concurrently from a freshwater reservoir. Differences between the strains were examined through monitoring interactions with a bacterial competitor, Flectobacillus sp. (FL), and virus- and predator-induced mortality. Batch-type cocultures, designated B4+FL and D5+FL, were initiated with a similar biomass ratio among the strains. The proportion of each cell type present in the cocultures was monitored based on clear differences in cell sizes. Following exponential growth for 28 h, the cocultures were amended by the addition of two different concentrations of live or heat-inactivated viruses concentrated from the reservoir. Half of virus-amended treatments were inoculated immediately with an axenic flagellate predator, Poterioochromonas sp. The presence of the predator, of live viruses, and of competition between the strains significantly affected their population dynamics in the experimentally manipulated treatments. While strains B4 and FL appeared vulnerable to environmental viruses, strain D5 did not. Predator-induced mortality had the greatest impact on FL, followed by that on D5 and then B4. The virus-vulnerable B4 strain had smaller cells and lower biomass yield, but it was less subject to grazing. In contrast, the seemingly virus-resistant D5, with slightly larger grazing-vulnerable cells, was competitive with FL. Overall, our data suggest contrasting ecophysiological capabilities and partial niche separation in two coexisting Limnohabitans strains.
