Nejvíce citovaný článek - PubMed ID 11375187
Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir
Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.
Heterotrophic nanoflagellates (HNF) are considered as major planktonic bacterivores, however, larger HNF taxa can also be important predators of eukaryotes. To examine this trophic cascading, natural protistan communities from a freshwater reservoir were released from grazing pressure by zooplankton via filtration through 10- and 5-µm filters, yielding microbial food webs of different complexity. Protistan growth was stimulated by amendments of five Limnohabitans strains, thus yielding five prey-specific treatments distinctly modulating protistan communities in 10- versus 5-µm fractions. HNF dynamics was tracked by applying five eukaryotic fluorescence in situ hybridization probes covering 55-90% of total flagellates. During the first experimental part, mainly small bacterivorous Cryptophyceae prevailed, with significantly higher abundances in 5-µm treatments. Larger predatory flagellates affiliating with Katablepharidacea and one Cercozoan lineage (increasing to up to 28% of total HNF) proliferated towards the experimental endpoint, having obviously small phagocytized HNF in their food vacuoles. These predatory flagellates reached higher abundances in 10-µm treatments, where small ciliate predators and flagellate hunters also (Urotricha spp., Balanion planctonicum) dominated the ciliate assemblage. Overall, our study reports pronounced cascading effects from bacteria to bacterivorous HNF, predatory HNF and ciliates in highly treatment-specific fashions, defined by both prey-food characteristics and feeding modes of predominating protists.
- Klíčová slova
- Cercozoa, Cryptophyceae, Katablepharidacea, bacterivorous and predatory flagellates, ciliates, freshwater microbial food webs,
- MeSH
- Cercozoa * MeSH
- Cryptophyta MeSH
- hybridizace in situ fluorescenční MeSH
- potravní řetězec * MeSH
- sladká voda MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The persistent inertia in the ability to culture environmentally abundant microbes from aquatic ecosystems represents an obstacle in disentangling the complex web of ecological interactions spun by a diverse assortment of participants (pro- and eukaryotes and their viruses). In aquatic microbial communities, the numerically most abundant actors, the viruses, remain the most elusive, and especially in freshwaters their identities and ecology remain unknown. Here, using ultra-deep metagenomic sequencing from pelagic freshwater habitats, we recovered complete genomes of > 2000 phages, including small "miniphages" and large "megaphages" infecting iconic freshwater prokaryotic lineages. For instance, abundant freshwater Actinobacteria support infection by a very broad size range of phages (13-200 Kb). We describe many phages encoding genes that likely afford protection to their host from reactive oxygen species (ROS) in the aquatic environment and in the oxidative burst in protist phagolysosomes (phage-mediated ROS defense). Spatiotemporal abundance analyses of phage genomes revealed evanescence as the primary dynamic in upper water layers, where they displayed short-lived existences. In contrast, persistence was characteristic for the deeper layers where many identical phage genomes were recovered repeatedly. Phage and host abundances corresponded closely, with distinct populations displaying preferential distributions in different seasons and depths, closely mimicking overall stratification and mixis.
- MeSH
- Actinobacteria virologie MeSH
- bakteriofágy genetika izolace a purifikace MeSH
- ekologie MeSH
- ekosystém MeSH
- fylogeneze MeSH
- genom virový MeSH
- metagenom genetika MeSH
- metagenomika MeSH
- mikrobiologie vody * MeSH
- sladká voda mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia coli cells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir Římov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers' removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.
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
The genus Limnohabitans (Comamonadaceae, Betaproteobacteria) is a common and a highly active component of freshwater bacterioplanktonic communities. To date, the genus has been considered to contain only heterotrophic species. In this study, we detected the photosynthesis genes pufLM and bchY in 28 of 46 strains from three Limnohabitans lineages. The pufM sequences obtained are very closely related to environmental pufM sequences detected in various freshwater habitats, indicating the ubiquity and potential importance of photoheterotrophic Limnohabitans in nature. Additionally, we sequenced and analyzed the genomes of 5 potentially photoheterotrophic Limnohabitans strains, to gain further insights into their phototrophic capacity. The structure of the photosynthesis gene cluster turned out to be highly conserved within the genus Limnohabitans and also among all potentially photosynthetic Betaproteobacteria strains. The expression of photosynthetic complexes was detected in a culture of Limnohabitans planktonicus II-D5T using spectroscopic and pigment analyses. This was further verified by a novel combination of infrared microscopy and fluorescent in situ hybridization.IMPORTANCE The data presented document that the capacity to perform anoxygenic photosynthesis is common among the members of the genus Limnohabitans, indicating that they may have a novel role in freshwater habitats.
- Klíčová slova
- FISH, IR microscopy, Limnohabitans, bacteriochlorophyll, bchY, freshwater Betaproteobacteria, photosynthetic bacteria, pufM,
- MeSH
- aerobióza MeSH
- bakteriální geny fyziologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Comamonadaceae genetika metabolismus MeSH
- fotosyntéza genetika MeSH
- fylogeneze MeSH
- multigenová rodina fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
We studied the diversity of Limnohabitans using reverse line blot hybridization with Limnohabitans lineage-specific probes in the freshwater canyon-shaped Římov reservoir (Czech Republic). To examine the succession of distinct lineages, we performed (i) a study of an intensive spring sampling program at the lacustrine part of the Římov reservoir (from ice melt through a phytoplankton peak to the clear-water phase), and (ii) a seasonal study (April to November) when the occurrence of distinct Limnohabitans lineages was related to the inherent longitudinal heterogeneity of the reservoir. Significant spatiotemporal changes in the compositions of distinct Limnohabitans lineages allowed for the identification of "generalists" that were always present throughout the whole season as well as "specialists" that appeared in the reservoir only for limited periods of time or irregularly. Our results indicate that some phytoplankton groups, such as cryptophytes or cyanobacteria, and zooplankton composition were the major factors modulating the distribution and dynamics of distinct Limnohabitans lineages. The highest Limnohabitans diversity was observed during the spring algal bloom, whereas the lowest was during the summer cyanobacterial bloom. The microdiversity also markedly increased upstream in the reservoir, being highest at the inflow, and thus likely reflecting strong influences of the watershed.IMPORTANCE The genus Limnohabitans is a typical freshwater bacterioplankton and is believed to play a significant role in inland freshwater habitats. This work is unique in detecting and tracing different closely related lineages of this bacterial genus in its natural conditions using the semiquantitative reverse line blot hybridization method and in discovering the factors influencing the microdiversity, subtype alternations, and seasonality.
- Klíčová slova
- ITS, Limnohabitans, bacterial diversity, canyon-shaped freshwater reservoir, reverse line blot hybridization, subtypes,
- MeSH
- Comamonadaceae klasifikace genetika izolace a purifikace MeSH
- Cryptophyta růst a vývoj MeSH
- ekosystém MeSH
- eutrofizace MeSH
- fytoplankton růst a vývoj MeSH
- roční období MeSH
- sinice růst a vývoj MeSH
- sladká voda mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Because their large growth potential is counterbalanced with grazing by heterotrophic nanoflagellates (HNF), bacteria of the genus Limnohabitans, which are common in many freshwater habitats, represent a valuable model for examining bacterial carbon flow to the grazer food chain. We conducted experiments with natural HNF communities taken from two distinct habitats, the meso-eutrophic Římov Reservoir and the oligo-mesotrophic Lake Cep (South Bohemia). HNF communities from each habitat at distinct seasonal phases, a late April algal bloom and a late May clear water phase, were each fed 3 Limnohabitans strains of differing cell sizes. Water samples were prefiltered (5 μm) to release natural HNF communities from zooplankton control and then amended with the Limnohabitans strains L. planktonicus II-D5 (medium sized, rod shaped), Limnohabitans sp. strain T6-5 (thin, long, curved rod), and Limnohabitans sp. strain 2KL-3 (large solenoid). Using temporal sampling and prey treatment, we determined HNF growth parameters such as doubling time, growth efficiency, and length of lag phase prior starting to exponential growth. All three Limnohabitans strains supported HNF growth but in significant prey-, site-, and season-dependent fashions. For instance, addition of the moderately large T6-5 strain yielded very rapid HNF growth with a short lag phase. In contrast, the curved morphology and larger cell size of strain 2KL-3 made this prey somewhat protected against grazing by smaller HNF, resulting in slower HNF growth and longer lag phases. These trends were particularly pronounced during the late May clear-water phase, which was dominated by smaller HNF cells. This may indicate a longer "adaptation time" for the flagellate communities toward the large prey size offered.
- MeSH
- Comamonadaceae cytologie růst a vývoj MeSH
- heterotrofní procesy MeSH
- jezera mikrobiologie parazitologie MeSH
- potravní řetězec MeSH
- roční období MeSH
- sladká voda mikrobiologie parazitologie MeSH
- zooplankton růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika 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
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
