The Archaea domain is ubiquitously distributed and extremely diverse, however, environmental factors that shape archaeal community structure are not well known. Aquatic environments, including the water column and sediments harbor many new uncultured archaeal species from which metabolic and ecological roles remain elusive. Some environments are especially neglected in terms of archaeal diversity, as is the case of pristine tropical areas. Here we investigate the archaeal composition in marine and freshwater systems from Ilha Grande, a South Atlantic tropical environment. All sampled habitats showed high archaeal diversity. No OTUs were shared between freshwater, marine and mangrove sediment samples, yet these environments are interconnected and geographically close, indicating environment-specific community structuring. Group II Euryarchaeota was the main clade in marine samples, while the new putative phylum Thaumarchaeota and LDS/RCV Euryarchaeota dominated freshwaters. Group III Euryarchaeota, a rare clade, was also retrieved in reasonable abundance in marine samples. The archaeal community from mangrove sediments was composed mainly by members of mesophilic Crenarchaeota and by a distinct clade forming a sister-group to Crenarchaeota and Thaumarchaeota. Our results show strong environment-specific community structuring in tropical aquatic Archaea, as previously seen for Bacteria.
Although numerous marine bacteria are known to produce antibiotics via hybrid NRPS-PKS gene clusters, none have been previously described in an Alteromonas species. In this study, we describe in detail a novel hybrid NRPS-PKS cluster identified in the plasmid of the Alteromonasmacleodii strain AltDE1 and analyze its relatedness to other similar gene clusters in a sequence-based characterization. This is a mobile cluster, flanked by transposase-like genes, that has even been found inserted into the chromosome of some Alteromonasmacleodii strains. The cluster contains separate genes for NRPS and PKS activity. The sole PKS gene appears to carry a novel acyltransferase domain, quite divergent from those currently characterized. The predicted specificities of the adenylation domains of the NRPS genes suggest that the final compound has a backbone very similar to bleomycin related compounds. However, the lack of genes involved in sugar biosynthesis indicates that the final product is not a glycopeptide. Even in the absence of these genes, the presence of the cluster appears to confer complete or partial resistance to phleomycin, which may be attributed to a bleomycin-resistance-like protein identified within the cluster. This also suggests that the compound still shares significant structural similarity to bleomycin. Moreover, transcriptomic evidence indicates that the NRPS-PKS cluster is expressed. Such sequence-based approaches will be crucial to fully explore and analyze the diversity and potential of secondary metabolite production, especially from increasingly important sources like marine microbes.
The current study describes the taxonomic and functional composition of metagenomic sequences obtained from a filamentous microbial mat isolated from the Comau fjord, located in the northernmost part of the Chilean Patagonia. The taxonomic composition of the microbial community showed a high proportion of members of the Gammaproteobacteria, including a high number of sequences that were recruited to the genomes of Moritella marina MP-1 and Colwelliapsycherythraea 34H, suggesting the presence of populations related to these two psychrophilic bacterial species. Functional analysis of the community indicated a high proportion of genes coding for the transport and metabolism of amino acids, as well as in energy production. Among the energy production functions, we found protein-coding genes for sulfate and nitrate reduction, both processes associated with Gammaproteobacteria-related sequences. This report provides the first examination of the taxonomic composition and genetic diversity associated with these conspicuous microbial mat communities and provides a framework for future microbial studies in the Comau fjord.
We describe a deep-branching lineage of marine Actinobacteria with very low GC content (33%) and the smallest free living cells described yet (cell volume ca. 0.013 μm3), even smaller than the cosmopolitan marine photoheterotroph, ‘Candidatus Pelagibacter ubique'. These microbes are highly related to 16S rRNA sequences retrieved by PCR from the Pacific and Atlantic oceans 20 years ago. Metagenomic fosmids allowed a virtual genome reconstruction that also indicated very small genomes below 1 Mb. A new kind of rhodopsin was detected indicating a photoheterotrophic lifestyle. They are estimated to be ~4% of the total numbers of cells found at the site studied (the Mediterranean deep chlorophyll maximum) and similar numbers were estimated in all tropical and temperate photic zone metagenomes available. Their geographic distribution mirrors that of picocyanobacteria and there appears to be an association between these microbial groups. A new sub-class, ‘Candidatus Actinomarinidae' is proposed to designate these microbes.
Cellular metagenomes are primarily used for investigating microbial community structure and function. However, cloned fosmids from such metagenomes capture phage genome fragments that can be used as a source of phage genomes. We show that fosmid cloning from cellular metagenomes and sequencing at a high coverage is a credible alternative to constructing metaviriomes and allows capturing and assembling novel, complete phage genomes. It is likely that phages recovered from cellular metagenomes are those replicating within cells during sample collection and represent “active” phages, naturally amplifying their genomic DNA and increasing chances for cloning. We describe five sets of siphoviral contigs (MEDS1, MEDS2, MEDS3, MEDS4, and MEDS5), obtained by sequencing fosmids from the cellular metagenome of the deep chlorophyll maximum in the Mediterranean. Three of these represent complete siphoviral genomes and two represent partial ones. This is the first set of phage genomes assembled directly from cellular metagenomic fosmid libraries. They exhibit low sequence similarities to one another and to known siphoviruses but are remarkably similar in overall genome architecture. We present evidence suggesting they infect picocyanobacteria, likely Synechococcus. Four of these sets also define a novel branch in the phylogenetic tree of phage large subunit terminases. Moreover, some of these siphoviral groups are globally distributed and abundant in the oceans, comparable to some known myoviruses and podoviruses. This suggests that, as more siphoviral genomes become available, we will be better able to assess the abundance and influence of this diverse and polyphyletic group in the marine habitat.
The genome of Alteromonas macleodii strain ATCC 27126T has been resequenced and closed into a single contig. We describe here the genome of this important and globally distributed marine bacterium.
We have compared genomes of Alteromonas macleodii “deep ecotype” isolates from two deep Mediterranean sites and two surface samples from the Aegean and the English Channel. A total of nine different genomes were analyzed. They belong to five clonal frames (CFs) that differ among them by approximately 30,000 single-nucleotide polymorphisms (SNPs) over their core genomes. Two of the CFs contain three strains each with nearly identical genomes (∼100 SNPs over the core genome). One of the CFs had representatives that were isolated from samples taken more than 1,000 km away, 2,500 m deeper, and 5 years apart. These data mark the longest proven persistence of a CF in nature (outside of clinical settings). We have found evidence for frequent recombination events between or within CFs and even with the distantly related A. macleodii surface ecotype. The different CFs had different flexible genomic islands. They can be classified into two groups; one type is additive, that is, containing different numbers of gene cassettes, and is very variable in short time periods (they often varied even within a single CF). The other type was more stable and produced the complete replacement of a genomic fragment by another with different genes. Although this type was more conserved within each CF, we found examples of recombination among distantly related CFs including English Channel and Mediterranean isolates.
Alteromonas macleodii; SNPs; microevolution; recombination; horizontal gene transfer
Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne‘ohe Bay, Hawai‘i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.
We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.
Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.
We have analyzed a natural population of the marine bacterium, Alteromonas macleodii, from a single sample of seawater to evaluate the genomic diversity present. We performed full genome sequencing of four isolates and 161 metagenomic fosmid clones, all of which were assigned to A. macleodii by sequence similarity. Out of the four strain genomes, A. macleodii deep ecotype (AltDE1) represented a different genome, whereas AltDE2 and AltDE3 were identical to the previously described AltDE. Although the core genome (∼80%) had an average nucleotide identity of 98.51%, both AltDE and AltDE1 contained flexible genomic islands (fGIs), that is, genomic islands present in both genomes in the same genomic context but having different gene content. Some of the fGIs encode cell surface receptors known to be phage recognition targets, such as the O-chain of the lipopolysaccharide, whereas others have genes involved in physiological traits (e.g., nutrient transport, degradation, and metal resistance) denoting microniche specialization. The presence in metagenomic fosmids of genomic fragments differing from the sequenced strain genomes, together with the presence of new fGIs, indicates that there are at least two more A. macleodii clones present. The availability of three or more sequences overlapping the same genomic region also allowed us to estimate the frequency and distribution of recombination events among these different clones, indicating that these clustered near the genomic islands. The results indicate that this natural A. macleodii population has multiple clones with a potential for different phage susceptibility and exploitation of resources, within a seemingly unstructured habitat.
Alteromonas macleodii; metagenome; population genomics; genomic island; constant-diversity; phage
Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24°C throughout the year, and a remarkable uniform temperature (∼22°C) and salinity (∼41 psu) from the mixed layer (∼200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea’s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.
16S rRNA gene (rrs) is considered of low taxonomic interest in the genus Aeromonas. Here, 195 Aeromonas strains belonging to populations structured by multilocus phylogeny were studied using an original approach that considered Ribosomal Multi-Operon Diversity. This approach associated pulsed-field gel electrophoresis (PFGE) to assess rrn operon number and distribution across the chromosome and PCR-temporal temperature gel electrophoresis (TTGE) to assess rrs V3 region heterogeneity. Aeromonads harbored 8 to 11 rrn operons, 10 operons being observed in more than 92% of the strains. Intraspecific variability was low or nul except for A. salmonicida and A. aquariorum suggesting that large chromosomic rearrangements might occur in these two species while being extremely rarely encountered in the evolution of other taxa. rrn operon number at 8 as well as PFGE patterns were shown valuable for taxonomic purpose allowing resolution of species complexes. PCR-TTGE revealed a high rate of strains (41.5%) displaying intragenomic rrs heterogeneity. Strains isolated from human samples more frequently displayed intragenomic heterogeneity than strains recovered from non-human and environmental specimens. Intraspecific variability ranged from 0 to 76.5% of the strains. The observation of species-specific TTGE bands, the recovery of identical V3 regions in different species and the variability of intragenomic heterogeneity (1–13 divergent nucleotides) supported the occurrence of mutations and horizontal transfer in aeromonad rrs evolution. Altogether, the presence of a high number of rrn operon, the high proportion of strains harboring divergent rrs V3 region and the previously demonstrated high level of genetic diversity argued in favor of highly adaptative capabilities of aeromonads. Outstanding features observed for A. caviae supported the ongoing process of adaptation to a specialized niche represented by the gut, previously hypothesized. 16S rRNA gene is an informative marker in the genus Aeromonas for both evolutionary and polyphasic taxonomic studies provided that multi-operon fingerprinting approaches are used.
Alteromonas macleodii is a marine gammaproteobacterium with widespread distribution in temperate or tropical waters. We describe three genomes of isolates from surface waters around Europe (Atlantic, Mediterranean and Black Sea) and compare them with a previously described deep Mediterranean isolate (AltDE) that belongs to a widely divergent clade. The surface isolates are quite similar, the most divergent being the Black Sea (BS11) isolate. The genomes contain several genomic islands with different gene content. The recruitment of very similar genomic fragments from metagenomes in different locations indicates that the surface clade is globally abundant with little effect of geography, even the AltDE and the BS11 genomes recruiting from surface samples in open ocean locations. The finding of CRISPR protospacers of AltDE in a lysogenic phage in the Atlantic (English Channel) isolate illustrates a flow of genetic material among these clades and a remarkably wide distribution of this phage.
Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.
Coastal lagoons, both hypersaline and freshwater, are common, but still understudied ecosystems. We describe, for the first time, using high throughput sequencing, the extant microbiota of two large and representative Mediterranean coastal lagoons, the hypersaline Mar Menor, and the freshwater Albufera de Valencia, both located on the south eastern coast of Spain. We show there are considerable differences in the microbiota of both lagoons, in comparison to other marine and freshwater habitats. Importantly, a novel uncultured sulfur oxidizing Alphaproteobacteria was found to dominate bacterioplankton in the hypersaline Mar Menor. Also, in the latter prokaryotic cyanobacteria were almost exclusively comprised by Synechococcus and no Prochlorococcus was found. Remarkably, the microbial community in the freshwaters of the hypertrophic Albufera was completely in contrast to known freshwater systems, in that there was a near absence of well known and cosmopolitan groups of ultramicrobacteria namely Low GC Actinobacteria and the LD12 lineage of Alphaproteobacteria.
Direct sequencing of environmental DNA (metagenomics) has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.
Among small photosynthetic eukaryotes that play a key role in oceanic food webs, picoplanktonic Mamiellophyceae such as Bathycoccus, Micromonas, and Ostreococcus are particularly important in coastal regions. By using a combination of cell sorting by flow cytometry, whole genome amplification (WGA), and 454 pyrosequencing, we obtained metagenomic data for two natural picophytoplankton populations from the coastal upwelling waters off central Chile. About 60% of the reads of each sample could be mapped to the genome of Bathycoccus strain from the Mediterranean Sea (RCC1105), representing a total of 9 Mbp (sample T142) and 13 Mbp (sample T149) of non-redundant Bathycoccus genome sequences. WGA did not amplify all regions uniformly, resulting in unequal coverage along a given chromosome and between chromosomes. The identity at the DNA level between the metagenomes and the cultured genome was very high (96.3% identical bases for the three larger chromosomes over a 360 kbp alignment). At least two to three different genotypes seemed to be present in each natural sample based on read mapping to Bathycoccus RCC1105 genome.
Sequencing of microbial community RNA (metatranscriptome) is a useful approach for assessing gene expression in microorganisms from the natural environment. This method has revealed transcriptional patterns in situ, but can also be used to detect transcriptional cascades in microcosms following experimental perturbation. Unambiguously identifying differential transcription between control and experimental treatments requires constraining effects that are simply due to sampling and bottle enclosure. These effects remain largely uncharacterized for “challenging” microbial samples, such as those from anoxic regions that require special handling to maintain in situ conditions. Here, we demonstrate substantial changes in microbial transcription induced by sample collection and incubation in experimental bioreactors. Microbial communities were sampled from the water column of a marine oxygen minimum zone by a pump system that introduced minimal oxygen contamination and subsequently incubated in bioreactors under near in situ oxygen and temperature conditions. Relative to the source water, experimental samples became dominated by transcripts suggestive of cell stress, including chaperone, protease, and RNA degradation genes from diverse taxa, with strong representation from SAR11-like alphaproteobacteria. In tandem, transcripts matching facultative anaerobic gammaproteobacteria of the Alteromonadales (e.g., Colwellia) increased 4–13 fold up to 43% of coding transcripts, and encoded a diverse gene set suggestive of protein synthesis and cell growth. We interpret these patterns as taxon-specific responses to combined environmental changes in the bioreactors, including shifts in substrate or oxygen availability, and minor temperature and pressure changes during sampling with the pump system. Whether such changes confound analysis of transcriptional patterns may vary based on the design of the experiment, the taxonomic composition of the source community, and on the metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations.
Viruses are ubiquitous in the oceans and critical components of marine microbial communities, regulating nutrient transfer to higher trophic levels or to the dissolved organic pool through lysis of host cells. Hydrothermal vent systems are oases of biological activity in the deep oceans, for which knowledge of biodiversity and its impact on global ocean biogeochemical cycling is still in its infancy. In order to gain biological insight into viral communities present in hydrothermal vent systems, we developed a method based on deep-sequencing of pulsed field gel electrophoretic bands representing key viral fractions present in seawater within and surrounding a hydrothermal plume derived from Loki's Castle vent field at the Arctic Mid-Ocean Ridge. The reduction in virus community complexity afforded by this novel approach enabled the near-complete reconstruction of a lambda-like phage genome from the virus fraction of the plume. Phylogenetic examination of distinct gene regions in this lambdoid phage genome unveiled diversity at loci encoding superinfection exclusion- and integrase-like proteins. This suggests the importance of fine-tuning lyosgenic conversion as a viral survival strategy, and provides insights into the nature of host-virus and virus-virus interactions, within hydrothermal plumes. By reducing the complexity of the viral community through targeted sequencing of prominent dsDNA viral fractions, this method has selectively mimicked virus dominance approaching that hitherto achieved only through culturing, thus enabling bioinformatic analysis to locate a lambdoid viral “needle" within the greater viral community “haystack". Such targeted analyses have great potential for accelerating the extraction of biological knowledge from diverse and poorly understood environmental viral communities.
Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases.
Methodology and Principal Findings
Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales).
Conclusions and Significance
We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.
The disaccharide trehalose is considered as a universal stress molecule, protecting cells and biomolecules from injuries imposed by high osmolarity, heat, oxidation, desiccation and freezing. Chromohalobacter salexigens is a halophilic and extremely halotolerant γ-proteobacterium of the family Halomonadaceae. In this work, we have investigated the role of trehalose as a protectant against salinity, temperature and desiccation in C. salexigens. A mutant deficient in the trehalose-6-phosphate synthase gene (otsA::Ω) was not affected in its salt or heat tolerance, but double mutants ectoine- and trehalose-deficient, or hydroxyectoine-reduced and trehalose-deficient, displayed an osmo- and thermosensitive phenotype, respectively. This suggests a role of trehalose as a secondary solute involved in osmo- (at least at low salinity) and thermoprotection of C. salexigens. Interestingly, trehalose synthesis was osmoregulated at the transcriptional level, and thermoregulated at the post-transcriptional level, suggesting that C. salexigens cells need to be pre-conditioned by osmotic stress, in order to be able to quickly synthesize trehalose in response to heat stress. C. salexigens was more sensitive to desiccation than E. coli and desiccation tolerance was slightly improved when cells were grown at high temperature. Under these conditions, single mutants affected in the synthesis of trehalose or hydroxyectoine were more sensitive to desiccation than the wild-type strain. However, given the low survival rates of the wild type, the involvement of trehalose and hydroxyectoine in C. salexigens response to desiccation could not be firmly established.
Microbial metagenomes are DNA samples of the most abundant, and therefore most successful organisms at the sampling time and location for a given cell size range. The study of microbial communities via their DNA content has revolutionized our understanding of microbial ecology and evolution. Iron availability is a critical resource that limits microbial communities' growth in many oceanic areas. Here, we built a database of 2319 sequences, corresponding to 140 gene families of iron metabolism with a large phylogenetic spread, to explore the microbial strategies of iron acquisition in the ocean's bacterial community. We estimate iron metabolism strategies from metagenome gene content and investigate whether their prevalence varies with dissolved iron concentrations obtained from a biogeochemical model. We show significant quantitative and qualitative variations in iron metabolism pathways, with a higher proportion of iron metabolism genes in low iron environments. We found a striking difference between coastal and open ocean sites regarding Fe2+ versus Fe3+ uptake gene prevalence. We also show that non-specific siderophore uptake increases in low iron open ocean environments, suggesting bacteria may acquire iron from natural siderophore-like organic complexes. Despite the lack of knowledge of iron uptake mechanisms in most marine microorganisms, our approach provides insights into how the iron metabolic pathways of microbial communities may vary with seawater iron concentrations.
Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ∼90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R2>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ∼1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ∼3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.
Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a potentially significant contribution of non-fungal eukaryotes in the actual hydrolysis of soil organic matter.