The gill-associated microbiome is the main source of wood plant polysaccharide hydrolases and secondary metabolite gene clusters in the mangrove shipworm Neoteredo reynei.

Teredinidae are a family of highly adapted wood-feeding and wood-boring bivalves, commonly known as shipworms, whose evolution is linked to the acquisition of cellulolytic gammaproteobacterial symbionts harbored in bacteriocytes within the gills. In the present work we applied metagenomics to characterize microbiomes of the gills and digestive tract of Neoteredo reynei, a mangrove-adapted shipworm species found over a large range of the Brazilian coast. Comparative metagenomics grouped the gill symbiont community of different N. reynei specimens, indicating closely related bacterial types are shared. Similarly, the intestine and digestive gland communities were related, yet were more diverse than and showed no overlap with the gill community. Annotation of assembled metagenomic contigs revealed that the gill symbiotic community of N. reynei encodes a plethora of plant cell wall polysaccharides degrading glycoside hydrolase encoding genes, and Biosynthetic Gene Clusters (BGCs). In contrast, the digestive tract microbiomes seem to play little role in wood digestion and secondary metabolites biosynthesis. Metagenome binning recovered the nearly complete genome sequences of two symbiotic Teredinibacter strains from the gills, a representative of Teredinibacter turnerae "clade I" strain, and a yet to be cultivated Teredinibacter sp. type. These Teredinibacter genomes, as well as un-binned gill-derived gammaproteobacteria contigs, also include an endo-ß-1,4-xylanase/acetylxylan esterase multi-catalytic carbohydrate-active enzyme, and a trans-acyltransferase polyketide synthase (trans-AT PKS) gene cluster with the gene cassette for generating ß-branching on complex polyketides. Finally, we use multivariate analyses to show that the secondary metabolome from the genomes of Teredinibacter representatives, including genomes binned from N. reynei gills' metagenomes presented herein, stands out within the Cellvibrionaceae family by size, and enrichments for polyketide, nonribosomal peptide and hybrid BGCs. Results presented here add to the growing characterization of shipworm symbiotic microbiomes and indicate that the N. reynei gill gammaproteobacterial community is a prolific source of biotechnologically relevant enzymes for wood-digestion and bioactive compounds production.

Optimizing and evaluating the reconstruction of Metagenome-assembled microbial genomes.

BACKGROUND: Microbiome/host interactions describe characteristics that affect the host's health. Shotgun metagenomics includes sequencing a random subset of the microbiome to analyze its taxonomic and metabolic potential. Reconstruction of DNA fragments into genomes from metagenomes (called metagenome-assembled genomes) assigns unknown fragments to taxa/function and facilitates discovery of novel organisms. Genome reconstruction incorporates sequence assembly and sorting of assembled sequences into bins, characteristic of a genome. However, the microbial community composition, including taxonomic and phylogenetic diversity may influence genome reconstruction. We determine the optimal reconstruction method for four microbiome projects that had variable sequencing platforms (IonTorrent and Illumina), diversity (high or low), and environment (coral reefs and kelp forests), using a set of parameters to select for optimal assembly and binning tools. METHODS: We tested the effects of the assembly and binning processes on population genome reconstruction using 105 marine metagenomes from 4 projects. Reconstructed genomes were obtained from each project using 3 assemblers (IDBA, MetaVelvet, and SPAdes) and 2 binning tools (GroopM and MetaBat). We assessed the efficiency of assemblers using statistics that including contig continuity and contig chimerism and the effectiveness of binning tools using genome completeness and taxonomic identification. RESULTS: We concluded that SPAdes, assembled more contigs (143,718 ± 124 contigs) of longer length (N50 = 1632 ± 108 bp), and incorporated the most sequences (sequences-assembled = 19.65%). The microbial richness and evenness were maintained across the assembly, suggesting low contig chimeras. SPAdes assembly was responsive to the biological and technological variations within the project, compared with other assemblers. Among binning tools, we conclude that MetaBat produced bins with less variation in GC content (average standard deviation: 1.49), low species richness (4.91 ± 0.66), and higher genome completeness (40.92 ± 1.75) across all projects. MetaBat extracted 115 bins from the 4 projects of which 66 bins were identified as reconstructed metagenome-assembled genomes with sequences belonging to a specific genus. We identified 13 novel genomes, some of which were 100% complete, but show low similarity to genomes within databases. CONCLUSIONS: In conclusion, we present a set of biologically relevant parameters for evaluation to select for optimal assembly and binning tools. For the tools we tested, SPAdes assembler and MetaBat binning tools reconstructed quality metagenome-assembled genomes for the four projects. We also conclude that metagenomes from microbial communities that have high coverage of phylogenetically distinct, and low taxonomic diversity results in highest quality metagenome-assembled genomes.

Bacterial Community Associated with the Reef Coral Mussismilia braziliensis's Momentum Boundary Layer over a Diel Cycle.

Corals display circadian physiological cycles, changing from autotrophy during the day to heterotrophy during the night. Such physiological transition offers distinct environments to the microbial community associated with corals: an oxygen-rich environment during daylight hours and an oxygen-depleted environment during the night. Most studies of coral reef microbes have been performed on samples taken during the day, representing a bias in the understanding of the composition and function of these communities. We hypothesized that coral circadian physiology alters the composition and function of microbial communities in reef boundary layers. Here, we analyzed microbial communities associated with the momentum boundary layer (MBL) of the Brazilian endemic reef coral Mussismilia braziliensis during a diurnal cycle, and compared them to the water column. We determined microbial abundance and nutrient concentration in samples taken within a few centimeters of the coral's surface every 6 h for 48 h, and sequenced microbial metagenomes from a subset of the samples. We found that dominant taxa and functions in the coral MBL community were stable over the time scale of our sampling, with no significant shifts between night and day samples. Interestingly, the two water column metagenomes sampled 1 m above the corals were also very similar to the MBL metagenomes. When all samples were analyzed together, nutrient concentration significantly explained 40% of the taxonomic dissimilarity among dominant genera in the community. Functional profiles were highly homogenous and not significantly predicted by any environmental variables measured. Our data indicated that water flow may overrule the effects of coral physiology in the MBL bacterial community, at the scale of centimeters, and suggested that sampling resolution at the scale of millimeters may be necessary to address diurnal variation in community composition.

Complete Genome Sequencing of a Multidrug-Resistant and Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Genotype.

Salmonella enterica subsp. enterica serovar Typhimurium strain YU39 was isolated in 2005 in the state of Yucatán, Mexico, from a human systemic infection. The YU39 strain is representative of the multidrug-resistant emergent sequence type 213 (ST213) genotype. The YU39 complete genome is composed of a chromosome and seven plasmids.

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum.

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.

Microbial community diversity and physical-chemical features of the Southwestern Atlantic Ocean.

Microbial oceanography studies have demonstrated the central role of microbes in functioning and nutrient cycling of the global ocean. Most of these former studies including at Southwestern Atlantic Ocean (SAO) focused on surface seawater and benthic organisms (e.g., coral reefs and sponges). This is the first metagenomic study of the SAO. The SAO harbors a great microbial diversity and marine life (e.g., coral reefs and rhodolith beds). The aim of this study was to characterize the microbial community diversity of the SAO along the depth continuum and different water masses by means of metagenomic, physical-chemical and biological analyses. The microbial community abundance and diversity appear to be strongly influenced by the temperature, dissolved organic carbon, and depth, and three groups were defined [1. surface waters; 2. sub-superficial chlorophyll maximum (SCM) (48-82 m) and 3. deep waters (236-1,200 m)] according to the microbial composition. The microbial communities of deep water masses [South Atlantic Central water, Antarctic Intermediate water and Upper Circumpolar Deep water] are highly similar. Of the 421,418 predicted genes for SAO metagenomes, 36.7 % had no homologous hits against 17,451,486 sequences from the North Atlantic, South Atlantic, North Pacific, South Pacific and Indian Oceans. From these unique genes from the SAO, only 6.64 % had hits against the NCBI non-redundant protein database. SAO microbial communities share genes with the global ocean in at least 70 cellular functions; however, more than a third of predicted SAO genes represent a unique gene pool in global ocean. This study was the first attempt to characterize the taxonomic and functional community diversity of different water masses at SAO and compare it with the microbial community diversity of the global ocean, and SAO had a significant portion of endemic gene diversity. Microbial communities of deep water masses (236-1,200 m) are highly similar, suggesting that these water masses have very similar microbiological attributes, despite the common knowledge that water masses determine prokaryotic community and are barriers to microbial dispersal. The present study also shows that SCM is a clearly differentiated layer within Tropical waters with higher abundance of phototrophic microbes and microbial diversity.

Comparative genomics of 274 Vibrio cholerae genomes reveals mobile functions structuring three niche dimensions.

BACKGROUND: Vibrio cholerae is a globally dispersed pathogen that has evolved with humans for centuries, but also includes non-pathogenic environmental strains. Here, we identify the genomic variability underlying this remarkable persistence across the three major niche dimensions space, time, and habitat. RESULTS: Taking an innovative approach of genome-wide association applicable to microbial genomes (GWAS-M), we classify 274 complete V. cholerae genomes by niche, including 39 newly sequenced for this study with the Ion Torrent DNA-sequencing platform. Niche metadata were collected for each strain and analyzed together with comprehensive annotations of genetic and genomic attributes, including point mutations (single-nucleotide polymorphisms, SNPs), protein families, functions and prophages. CONCLUSIONS: Our analysis revealed that genomic variations, in particular mobile functions including phages, prophages, transposable elements, and plasmids underlie the metadata structuring in each of the three niche dimensions. This underscores the role of phages and mobile elements as the most rapidly evolving elements in bacterial genomes, creating local endemicity (space), leading to temporal divergence (time), and allowing the invasion of new habitats. Together, we take a data-driven approach for comparative functional genomics that exploits high-volume genome sequencing and annotation, in conjunction with novel statistical and machine learning analyses to identify connections between genotype and phenotype on a genome-wide scale.

A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes.

Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the majority of published human faecal metagenomes, which we refer to as crAssphage. Its ~97 kbp genome is six times more abundant in publicly available metagenomes than all other known phages together; it comprises up to 90% and 22% of all reads in virus-like particle (VLP)-derived metagenomes and total community metagenomes, respectively; and it totals 1.68% of all human faecal metagenomic sequencing reads in the public databases. The majority of crAssphage-encoded proteins match no known sequences in the database, which is why it was not detected before. Using a new co-occurrence profiling approach, we predict a Bacteroides host for this phage, consistent with Bacteroides-related protein homologues and a unique carbohydrate-binding domain encoded in the phage genome.

Genomic taxonomy of the genus prochlorococcus.

The genus Prochlorococcus is globally abundant and dominates the total phytoplankton biomass and production in the oligotrophic ocean. The single species, Prochlorococcus marinus, comprises six named ecotypes. Our aim was to analyze the taxonomic structure of the genus Prochlorococcus. We analyzed the complete genomes of 13 cultured P. marinus type and reference strains by means of several genomic taxonomy tools (i.e., multilocus sequence analysis, amino acid identity, Karlin genomic signature, and genome to genome distance). In addition, we estimated the diversity of Prochlorococcus species in over 100 marine metagenomes from all the major oceanic provinces. According to our careful taxonomic analysis, the 13 strains corresponded, in fact, to ten different Prochlorococcus species. This analysis establishes a new taxonomic framework for the genus Prochlorococcus. Further, the analysis of the metagenomic data suggests that, in total, there may only be 35 Prochlorococcus species in the world's oceans. We propose that the dearth of species observed in this study is driven by high selective pressures that limit diversification in the global ocean.

Metagenomic analysis of healthy and white plague-affected Mussismilia braziliensis corals.

Coral health is under threat throughout the world due to regional and global stressors. White plague disease (WP) is one of the most important threats affecting the major reef builder of the Abrolhos Bank in Brazil, the endemic coral Mussismilia braziliensis. We performed a metagenomic analysis of healthy and WP-affected M. braziliensis in order to determine the types of microbes associated with this coral species. We also optimized a protocol for DNA extraction from coral tissues. Our taxonomic analysis revealed Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Actinomycetes as the main groups in all healthy and WP-affected corals. Vibrionales, members of the Cytophaga-Flavobacterium-Bacteroides complex, Rickettsiales, and Neisseriales were more abundant in the WP-affected corals. Diseased corals also had more eukaryotic metagenomic sequences identified as Alveolata and Apicomplexa. Our results suggest that WP disease in M. braziliensis is caused by a polymicrobial consortium.

Polyketide synthase gene diversity within the microbiome of the sponge Arenosclera brasiliensis, endemic to the Southern Atlantic Ocean.

Microbes associated with marine sponges are considered important producers of bioactive, structurally unique polyketides. The synthesis of such secondary metabolites involves type I polyketide synthases (PKSs), which are enzymes that reach a maximum complexity degree in bacteria. The Haplosclerida sponge Arenosclera brasiliensis hosts a complex microbiota and is the source of arenosclerins, alkaloids with cytotoxic and antibacterial activity. In the present investigation, we performed high-throughput sequencing of the ketosynthase (KS) amplicon to investigate the diversity of PKS genes present in the metagenome of A. brasiliensis. Almost 4,000 ketosynthase reads were recovered, with about 90% annotated automatically as bacterial. A total of 235 bacterial KS contigs was rigorously assembled from this sequence pool and submitted to phylogenetic analysis. A great diversity of six type I PKS groups has been consistently detected in our phylogenetic reconstructions, including a novel and A. brasiliensis-exclusive group. Our study is the first to reveal the diversity of type I PKS genes in A. brasiliensis as well as the potential of its microbiome to serve as a source of new polyketides.

Oxygen minimum zones harbour novel viral communities with low diversity.

Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral-to-microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non-redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non-redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.

Taxonomic and functional microbial signatures of the endemic marine sponge Arenosclera brasiliensis.

The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (∼150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome.

Genome sequences of the ethanol-tolerant Lactobacillus vini strains LMG 23202T and JP7.8.9.

We report on the genome sequences of Lactobacillus vini type strain LMG 23202(T) (DSM 20605) (isolated from fermenting grape musts in Spain) and the industrial strain L. vini JP7.8.9 (isolated from a bioethanol plant in northeast Brazil). All contigs were assembled using gsAssembler, and genes were predicted and annotated using Rapid Annotation using Subsystem Technology (RAST). The identified genome sequence of LMG 23202(T) had 2.201.333 bp, 37.6% G+C, and 1,833 genes, whereas the identified genome sequence of JP7.8.9 had 2.301.037 bp, 37.8% G+C, and 1,739 genes. The gene repertoire of the species L. vini offers promising opportunities for biotechnological applications.