High-resolution metagenomic reconstruction of the freshwater spring bloom

Background The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. Results We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. Conclusion We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom

The Passive Yet Successful Way of Planktonic Life: Genomic and Experimental Analysis of the Ecology of a Free-Living Polynucleobacter Population

Background: The bacterial taxon Polynucleobacter necessarius subspecies asymbioticus represents a group of planktonic freshwater bacteria with cosmopolitan and ubiquitous distribution in standing freshwater habitats. These bacteria comprise,1 % to 70 % (on average about 20%) of total bacterioplankton cells in various freshwater habitats. The ubiquity of this taxon was recently explained by intra-taxon ecological diversification, i.e. specialization of lineages to specific environmental conditions; however, details on specific adaptations are not known. Here we investigated by means of genomic and experimental analyses the ecological adaptation of a persistent population dwelling in a small acidic pond. Findings: The investigated population (F10 lineage) contributed on average 11 % to total bacterioplankton in the pond during the vegetation periods (ice-free period, usually May to November). Only a low degree of genetic diversification of the population could be revealed. These bacteria are characterized by a small genome size (2.1 Mb), a relatively small number of genes involved in transduction of environmental signals, and the lack of motility and quorum sensing. Experiments indicated that these bacteria live as chemoorganotrophs by mainly utilizing low-molecular-weight substrates derived from photooxidation of humic substances. Conclusions: Evolutionary genome streamlining resulted in a highly passive lifestyle so far only known among free-living bacteria from pelagic marine taxa dwelling in environmentally stable nutrient-poor off-shore systems. Surprisingly, such a lifestyle is also successful in a highly dynamic and nutrient-richer environment such as the water column of the investigate

Genetic methods for determination of the cyanotixin producers

This work summarises and introduces modern methods of molecular biology, which are frequently used to both cyanobacterial identification and detection of cyanotoxin production

Insights into variability of actinorhodopsin genes of the LG1 cluster in two different freshwater habitats.

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

Maximum Likelihood tree of LG1 actinorhodopsin amino acid sequences.

<p>ML best tree of LG1 actinorhodopsin amino acid sequences (97 aa). Rooted with LG2 sequences (GS020_39, GS012_40, GS012_3). Bootstrap values from Maximum likelihood/Bayesian inference/Neighbor joining methods are depicted. Clones from Lake Mondsee are highlighted in blue and clones from Jiřická reservoir in red.</p

Amino acid sequence similarity.

<p>Amino acid sequence similarity (97 aa) calculated as mean distance for the presented clones and for all 156 sequences in the analyses.</p

Venn diagram depicting numbers of LG1 actinorodopsin clusters.

<p>Venn diagram depicting numbers of LG1 actinorodopsin clusters defined in Fig. 1 shared by the two investigated and other habitats. For instance, Jiřická reservoir contained sequences representing three clusters not found in Lake Mondsee or one of the other habitats represented by reference sequences, while Lake Mondsee shared exclusively with Jiřická reservoir zero clusters, but four clusters with other habitats. Only three out of thirteen clusters contained sequences from all three habitat groups.</p

Maximum Likelihood tree of LG1 actinorhodopsin nucleotide sequences.

<p>ML best tree of LG1 actinorhodopsin nucleotide sequences (291 bp). Rooted with LG2 sequences (GS020_39, GS012_40, GS012_3). Bootstrap values from Maximum likelihood/Bayesian inference/Neighbor joining methods are depicted. Clones from Lake Mondsee are highlighted in blue and clones from Jiřická reservoir in red. Star - reference sequence (16S rRNA gene sequence is available). Numbers on the side symbolize cluster numbers, color the group affiliation (yellow = LG1-A, rosa = LG1-C, green = LG1-B). Bold frames – new clusters.</p