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MODIS satellite image on September 26, 2016 of the phytoplankton bloom occurring in Monterey Bay and extending into the Pacific. The red dot represents the sampling station M0, located at 36.835 N, 121.901 W.
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Metagenomic and metatranscriptomic time-series data covering a 52-day period in the fall of 2016 provide an inventory of bacterial and archaeal community genes, transcripts, and taxonomy during an intense dinoflagellate bloom in Monterey Bay, CA, USA. The dataset comprises 84 metagenomes (0.8 terabases), 82 metatranscriptomes (1.1 terabases), and 8...
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... Several studies have already tested the metabarcoding approach for phytoplankton, many of them using a combination of two different barcodes, one targeting the prokaryotic fraction, like the 16S rRNA gene, and the other targeting the eukaryotic fraction, like the 18S rRNA gene (e.g. Hug et al., 2016, Djurhuus et al., 2017, Filker et al., 2019, Nowinski et al., 2019, Yarimizu et al., 2020, Sildever et al., 2022. These two genetic markers, 18S rRNA and 16S rRNA are well represented in reference libraries like Silva and PR2 (Guillou et al., 2013). ...
DNA metabarcoding can be a promising alternative to microscopy for analysing phytoplankton, a key ecological indicator for freshwater ecosystems. The aim of this study was to evaluate the performance of different barcodes and associated primer pairs to assess microalgal diversity with DNA metabarcoding using a single barcode targeting all microalgae. We investigated barcodes in 16S and 23S rRNA genes, encoding for prokaryotic ribosomal sub-units, that are present in Cyanobacteria as well as in chloroplasts. In silico PCR tests were carried out on eight 16S and five 23S primer pairs using the Phytool reference library. Two and three pairs were selected for 16S and 23S, respectively, to perform an in vitro metabarcoding test based on a mock community made of DNA extracts of 10 microalgae strains. The 23S pairs enabled to detect all species, whereas 16S ones failed in the detection of some of them. One pair was selected for each genetic marker, based on its efficiency and specificity towards microalgae ( e.g. not heterotrophic bacteria). Another mock community covering a larger diversity (18 microalgae strains) was used to test the efficiency of the selected pairs and their ability to estimate relative abundances. The 23S pair performed better than the 16S one for detecting target species with also more accuracy to assess their relative abundances. We conclude that the 23S primer pair ECLA23S_F1/ECLA23S_R1 appears as a good candidate to decipher freshwater phytoplankton communities. As a next step, it will be necessary to confirm these results on a large diversity of natural communities.
... The community structure inferred from our study suggested that in the seawater of Heping Island, Alteromonadaceae, Litoricolaceae, and Cryomorphaceae formed a coexistence relationship with a group of bacteria, including AEGEAN_169_marine_group, Clade_I, Clade_II, Cyanobiaceae, Puniceicoccaceae, Porticoccaceae, Actinomarinaceae, SAR116_clade, and Kiritimatiellaceae, most of which were reported to be abundant in phytoplankton blooms [66,67]. Conversely, Rhodobacteraceae and Vibrionaceae showed higher abundances at Badouzi Fishing Port and formed mutual-exclusion relationships with the abovementioned bacteria, which maintained a relationship of coexistence. ...
Pollution in human-made fishing ports caused by petroleum from boats, dead fish, toxic chemicals, and effluent poses a challenge to the organisms in seawater. To decipher the impact of pollution on the microbiome, we collected surface water from a fishing port and a nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean. By employing 16S rRNA gene amplicon sequencing and whole-genome shotgun sequencing, we discovered that Rhodobacteraceae, Vibrionaceae, and Oceanospirillaceae emerged as the dominant species in the fishing port, where we found many genes harboring the functions of antibiotic resistance (ansamycin, nitroimidazole, and aminocoumarin), metal tolerance (copper, chromium, iron and multimetal), virulence factors (chemotaxis, flagella, T3SS1), carbohydrate metabolism (biofilm formation and remodeling of bacterial cell walls), nitrogen metabolism (denitrification, N2 fixation, and ammonium assimilation), and ABC transporters (phosphate, lipopolysaccharide, and branched-chain amino acids). The dominant bacteria at the nearby offshore island (Alteromonadaceae, Cryomorphaceae, Flavobacteriaceae, Litoricolaceae, and Rhodobacteraceae) were partly similar to those in the South China Sea and the East China Sea. Furthermore, we inferred that the microbial community network of the cooccurrence of dominant bacteria on the offshore island was connected to dominant bacteria in the fishing port by mutual exclusion. By examining the assembled microbial genomes collected from the coastal seawater of the fishing port, we revealed four genomic islands containing large gene-containing sequences, including phage integrase, DNA invertase, restriction enzyme, DNA gyrase inhibitor, and antitoxin HigA-1. In this study, we provided clues for the possibility of genomic islands as the units of horizontal transfer and as the tools of microbes for facilitating adaptation in a human-made port environment.
... pomeroyi transcripts averaged 38% of the bacterial reads in the metatranscriptome datasets [50]. Cells were collected by filtration after 90 min and processed for RNAseq analysis. ...
... Transporter expression reveals the metabolite landscape of a coastal phytoplankton bloom We used an R. pomeroyi gene expression dataset from a natural phytoplankton bloom in Fall 2016 in Monterey Bay, CA, USA [50] to assess the ecological relevance of the verified transporters. On 14 dates over 5 weeks during the decline of a bloom dominated by the dinoflagellate Akashiwo sanguinea, R. pomeroyi cells were introduced into samples from the natural community for 90 min [9]. ...
Metabolite exchange within marine microbial communities transfers carbon and other major elements through global cycles and forms the basis of microbial interactions. Yet lack of gene annotations and concern about the quality of existing ones remain major impediments to revealing currencies of carbon flux. We employed an arrayed mutant library of the marine bacterium Ruegeria pomeroyi DSS-3 to experimentally annotate substrates of organic compound transporter systems, using mutant growth and compound drawdown analyses to link transporters to their cognate substrates. Mutant experiments verified substrates for thirteen R. pomeroyi transporters. Four were previously hypothesized based on gene expression data (taurine, glucose/xylose, isethionate, and cadaverine/putrescine/spermidine); five were previously hypothesized based on homology to experimentally annotated transporters in other bacteria (citrate, glycerol, N -acetylglucosamine, fumarate/malate/succinate, and dimethylsulfoniopropionate); and four had no previous annotations (thymidine, carnitine, cysteate, and 3-hydroxybutyrate). These bring the total number of experimentally-verified organic carbon influx transporters to 18 of 126 in the R. pomeroyi genome. In a longitudinal study of a coastal phytoplankton bloom, expression patterns of the experimentally annotated transporters linked them to different stages of the bloom, and also led to the hypothesis that citrate and 3-hydroxybutyrate were among the most highly available bacterial substrates. Improved functional annotation of the gatekeepers of organic carbon uptake is critical for deciphering carbon flux and fate in microbial ecosystems.
... A bioinformatic search across publicly available metagenomes identified significant hits (e < 10 −15 ) of the Tritonibacter mobilis A3R06 phage-plasmid repressor in several natural microbial communities across the globe (Fig. S12). Hits were found mainly in nutrient rich marine environment, such as an shrimp aquaculture in the eastern coast of China 28 , particle-attached Mediterranean water column 29 , as well as an intense algal bloom in California 30 . Further studies are required to get more complete sequence information of phageplasmids and to capture and characterize their eco-evolutionary dynamics in natural environments. ...
Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in a population of marine Roseobacter. Recurrent loss-of-function mutations in the phage repressor that controls prophage induction leads to constitutively lytic phage-plasmids that spread rapidly throughout the population. The entire phage-plasmid genome is packaged into virions, which were horizontally transferred by re-infecting lysogenized cells, leading to an increase in phage-plasmid copy number and to heterozygosity in a phage repressor locus in re-infected cells. However, the uneven distribution of phage-plasmids after cell division (i.e., segregational drift) leads to the production of offspring carrying only the constitutively lytic phage-plasmid, thus restarting the lysis-reinfection-segregation life cycle. Mathematical models and experiments show that these dynamics lead to a continuous productive infection of the bacterial population, in which lytic and lysogenic phage-plasmids coexist. Furthermore, analyses of marine bacterial genome sequences indicate that the plasmid backbone here can carry different phages and disseminates trans-continentally. Our study highlights how the interplay between phage infection and plasmid genetics provides a unique eco-evolutionary strategy for phage-plasmids.
... During a phytoplankton bloom in Monterey Bay, CA, USA in the fall of 2016 [7], the in situ robotic Environmental Sample Processor (ESP) [8] collected and stored microbial cells for nucleic acid analysis at Station M0. 16S rRNA gene amplicon sequencing data from 41 dates during this longitudinal study [9] revealed that the most abundant taxon in the bacterial community was represented by an amplicon sequence variant (ASV) belonging to a marine Roseobacter (Rhodobacteraceae) from the NAC11-7 lineage. Roseobacter species vary dramatically in life history characteristics, some with large genomes (>4 Mb) that are amenable to culturing, and others with streamlined genomes (<2.3 Mb) that largely remain uncultured [10]. ...
... Microbial cells in the ≤5 μm to ≥0.22 μm size range were collected at Monterey Bay Station M0 over a 52 d period from September 26 through Sequencing and assembly DNA and RNA sequencing and assembly of libraries representing the microbial community were carried out as described previously [9,23,24]. Our protocol included addition of two genomic DNA internal standards from non-marine bacteria Thermus thermophilus and Blautia producta [25,26] and two mRNA internal standards (each~1000 bp) transcribed from custom templates [27]; these were added in known amounts to sample filters prior to processing. ...
... To obtain the pangenome of the two NAC11-7 lineage species, metagenomic and metatranscriptomic reads from Monterey Bay from 2016 [9] were mapped to the 31 NAC11-7 genomes (the original HTCC2255 isolate [33] along with 2 MAGs and 28 SAGs from this study) using Bowtie2. anvi'o v6.1 [34] was used to create a database of DNA, amino acid sequences, and read mapping profiles of the 31 genomes. ...
Identifying mechanisms by which bacterial species evolve and maintain genomic diversity is particularly challenging for the uncultured lineages that dominate the surface ocean. A longitudinal analysis of bacterial genes, genomes, and transcripts during a coastal phytoplankton bloom revealed two co-occurring, highly related Rhodobacteraceae species from the deeply branching and uncultured NAC11-7 lineage. These have identical 16S rRNA gene amplicon sequences, yet their genome contents assembled from metagenomes and single cells indicate species-level divergence. Moreover, shifts in relative dominance of the species during dynamic bloom conditions over 7 weeks confirmed the syntopic species’ divergent responses to the same microenvironment at the same time. Genes unique to each species and genes shared but divergent in per-cell inventories of mRNAs accounted for 5% of the species’ pangenome content. These analyses uncover physiological and ecological features that differentiate the species, including capacities for organic carbon utilization, attributes of the cell surface, metal requirements, and vitamin biosynthesis. Such insights into the coexistence of highly related and ecologically similar bacterial species in their shared natural habitat are rare.
... Using the HMM profiles of the Rep and Cap proteins from thirtythree reference microviruses, a total of 12,495 circular contigs and genomes similar to microvirus ones were identified in sequence data from GenBank, in IMG microbial metagenomes and metatranscriptomes (Nowinski et al. 2019), and in 2,946 assembled viral metagenomes. Although many contigs (1,314) were shorter than the smallest genome of a cultivated microvirus (4.248 kb), only a handful of contigs stood out as extremely small ( Supplementary Fig. S1). ...
Small circular single-stranded DNA viruses of the Microviridae family are both prevalent and diverse in all ecosystems. They usually harbor a genome between 4.3 and 6.3 kb, with a microvirus recently isolated from a marine Alphaproteobacteria being the smallest known genome of a DNA phage (4.248 kb). A subfamily, Amoyvirinae, has been proposed to classify this virus and other related small Alphaproteobacteria-infecting phages. Here, we report the discovery, in meta-omics data sets from various aquatic ecosystems, of sixteen complete microvirus genomes significantly smaller (2.991–3.692 kb) than known ones. Phylogenetic analysis reveals that these sixteen genomes represent two related, yet distinct and diverse, novel groups of microviruses—amoyviruses being their closest known relatives. We propose that these small microviruses are members of two tentatively named subfamilies Reekeekeevirinae and Roodoodoovirinae. As known microvirus genomes encode many overlapping and overprinted genes that are not identified by gene prediction software, we developed a new methodology to identify all genes based on protein conservation, amino acid composition, and selection pressure estimations. Surprisingly, only four to five genes could be identified per genome, with the number of overprinted genes lower than that in phiX174. These small genomes thus tend to have both a lower number of genes and a shorter length for each gene, leaving no place for variable gene regions that could harbor overprinted genes. Even more surprisingly, these two Microviridae groups had specific and different gene content, and major differences in their conserved protein sequences, highlighting that these two related groups of small genome microviruses use very different strategies to fulfill their lifecycle with such a small number of genes. The discovery of these genomes and the detailed prediction and annotation of their genome content expand our understanding of ssDNA phages in nature and are further evidence that these viruses have explored a wide range of possibilities during their long evolution.
... Primers targeting the 16S and 18S rRNA V4 gene region were used in many pro-and eukaryotic phytoplankton metabarcoding studies (e.g. Hu et al., 2016, Djurhuus et al., 2017, Filker et al., 2019, Nowinski et al,.2019, Yarimizu et al., 2020 and they are well represented in databases like Silva (Quast et al., 2013) and PR 2 (Guillou et al., 2013). ...
This document is a methodological guide for using a genomic ecosystem survey technique (eDNA metabarcoding) to supplement conventional phytoplankton monitoring of the Finnish marine monitoring program. The guidelines describe the detection of eukaryotic and prokaryotic phytoplankton with 18S and 16S rDNA gene primers, using high-throughput sequencing. The document includes information on sampling, sample processing, molecular biological work, quality control, and bioinformatics so that the method can be applied in addition to standardized light microscopy. The guidelines are based on a first pilot project testing the integration of eDNA metabarcoding in Finnish marine phytoplankton monitoring and will be developed further, according to evolving genetic methods and international guidelines and standards. Suggestions on steps towards introducing eDNA methodology in phytoplankton monitoring are included in the guidelines. Using eDNA metabarcoding to complement standardized light microscopy advances conventional monitoring and research of phytoplankton communities to assess biodiversity and the status of the marine environment.
ISBN 978-952-11-5524-6 (PDF)
ISSN 1796-1726 (online)
link http://urn.fi/URN:ISBN:978-952-11-5524-6
... We used the DADA2 algorithm to denoise and remove sequencing errors [27], together with R version 4.1.0 (Global (CDN)-Rstudio) [28]. We then imported high quality reads into QIIME2 for the next analyzes. ...
... All ASVs assigned to mitochondrial and chloroplast sequences were removed from the data set. Finally, all the data files generated with QIIME 2 were loaded into the R software [28], for subsequent statistical analysis. ...
... microbiomeanalyst.ca/MicrobiomeAnalyst/upload/OtuUploadView.xhtml, accessed on Microorganisms 2022, 10, 2191 5 of 16 28 March 2022 [13,31]). Due to a disparate number of samples collected in the two sites, comparative analyses between sites were carried out using all samples collected for each site and also by comparing just one month of data for each site. ...
Mangrove ecosystems are threatened worldwide by a wide range of factors including climate change, coastal development, and pollution. The effects of these factors on soil bacterial communities of Neotropical mangroves and their temporal dynamics is largely undocumented. Here we compared the diversity and taxonomic composition of bacterial communities in the soil of two mangrove forest sites of the Panama Bay: Juan Diaz (JD), an urban mangrove forest in Panama City surrounded by urban development, with occurrence of five mangrove species, and polluted with solid waste and sewage; and Bayano (B), a rural mangrove forest without urban development, without solid waste pollution, and with the presence of two mangrove species. Massive amplicon sequencing of the V4 region of the 16S rRNA gene and community analyses were implemented. In total, 20,691 bacterial amplicon sequence variants were identified, and the bacterial community was more diverse in the rural mangrove forest based on Faith’s phylogenetic diversity index. The three dominant phyla of bacteria found and shared between the two sites were Proteobacteria, Desulfobacterota, and Chloroflexi. The ammonia oxidizing archaea class Nitrosphaeria was found among the top 10 most abundant. Dominant genera of bacteria that occurred in the two mangrove sites were: BD2-11_terrestrial_group (Gemmatimonadota), EPR3968-O8a-Bc78 (Gammaproteobacteria), Salinimicrobium (Bacteroidetes), Sulfurovum (Campylobacteria), and Woeseia (Gammaproteobacteria) of which the first three and Methyloceanibacter had increased in relative abundance in the transition from rainy to dry to rainy season in the urban mangrove forest. Altogether, our study suggests that factors such as urban development, vegetation composition, pollution, and seasonal changes may cause shifts in bacterial diversity and relative abundance of specific taxa in mangrove soils. In particular, taxa with roles in biogeochemical cycles of carbon, nitrogen, sulfur, and phosphorus, and on rhizosphere taxa, could be important for mangrove plant resilience to environmental stress.
... Metagenome sequencing gained noticeable popularity in the past decade, as multiple projects shed light on microbial communities in various ecosystems (Poretsky et al., 2005;Nowinski et al., 2019) and eukaryotic microbiomes (Turnbaugh et al., 2007;Arumugam et al., 2011;Lloyd-Price et al., 2019). However, these studies required the development of novel software tools, as the previously designed methods for conventional sequencing data analysis appeared to be underperforming on large and complex metagenomic datasets. ...
While metagenome sequencing may provide insights on the genome sequences and composition of microbial communities, metatranscriptome analysis can be useful for studying the functional activity of a microbiome. RNA-Seq data provides the possibility to determine active genes in the community and how their expression levels depend on external conditions. Although the field of metatranscriptomics is relatively young, the number of projects related to metatranscriptome analysis increases every year and the scope of its applications expands. However, there are several problems that complicate metatranscriptome analysis: complexity of microbial communities, wide dynamic range of transcriptome expression and importantly, the lack of high-quality computational methods for assembling meta-RNA sequencing data. These factors deteriorate the contiguity and completeness of metatranscriptome assemblies, therefore affecting further downstream analysis.
Here we present MetaGT, a pipeline for de novo assembly of metatranscriptomes, which is based on the idea of combining both metatranscriptomic and metagenomic data sequenced from the same sample. MetaGT assembles metatranscriptomic contigs and fills in missing regions based on their alignments to metagenome assembly. This approach allows to overcome described complexities and obtain complete RNA sequences, and additionally estimate their abundances. Using various publicly available real and simulated datasets, we demonstrate that MetaGT yields significant improvement in coverage and completeness of metatranscriptome assemblies compared to existing methods that do not exploit metagenomic data. The pipeline is implemented in NextFlow and is freely available from https://github.com/ablab/metaGT .
... Completeness and contamination of genome bins were estimated by taxon-specific sets of single-copy marker genes through the lineage-specific workflow of CheckM v1.0.13 43 . After removal of genomes likely derived from an internal standard (n = 63; Thermus thermophilus and Blautias producta 44 ), 54,614 genome bins were obtained (Fig. 1c). ...
Marine microorganisms are immensely diverse and play fundamental roles in global geochemical cycling. Recent metagenome-assembled genome studies, with particular attention to large-scale projects such as Tara Oceans, have expanded the genomic repertoire of marine microorganisms. However, published marine metagenome data is still underexplored. We collected 2,057 marine metagenomes covering various marine environments and developed a new genome reconstruction pipeline. We reconstructed 52,325 qualified genomes composed of 8,466 prokaryotic species-level clusters spanning 59 phyla, including genomes from the deep-sea characterized as deeper than 1,000 m (n = 3,337), low-oxygen zones of <90 μmol O2 per kg water (n = 7,884), and polar regions (n = 7,752). Novelty evaluation using a genome taxonomy database shows that 6,256 species (73.9%) are novel and include genomes of high taxonomic novelty, such as new class candidates. These genomes collectively expanded the known phylogenetic diversity of marine prokaryotes by 34.2%, and the species representatives cover 26.5–42.0% of prokaryote-enriched metagenomes. Thoroughly leveraging accumulated metagenomic data, this genome resource, named the OceanDNA MAG catalog, illuminates uncharacterized marine microbial ‘dark matter’ lineages.
