CoMet: A workflow using contig coverage and composition for binning a metagenomic sample with high precision

Background: In metagenomics, the separation of nucleotide sequences belonging to an individual or closely matched populations is termed binning. Binning helps the evaluation of underlying microbial population structure as well as the recovery of individual genomes from a sample of uncultivable microbial organisms. Both supervised and unsupervised learning methods have been employed in binning; however, characterizing a metagenomic sample containing multiple strains remains a significant challenge. In this study, we designed and implemented a new workflow, Coverage and composition based binning of Metagenomes (CoMet), for binning contigs in a single metagenomic sample. CoMet utilizes coverage values and the compositional features of metagenomic contigs. The binning strategy in CoMet includes the initial grouping of contigs in guanine-cytosine (GC) content-coverage space and refinement of bins in tetranucleotide frequencies space in a purely unsupervised manner. With CoMet, the clustering algorithm DBSCAN is employed for binning contigs. The performances of CoMet were compared against four existing approaches for binning a single metagenomic sample, including MaxBin, Metawatt, MyCC (default) and MyCC (coverage) using multiple datasets including a sample comprised of multiple strains. Results: Binning methods based on both compositional features and coverages of contigs had higher performances than the method which is based only on compositional features of contigs. CoMet yielded higher or comparable precision in comparison to the existing binning methods on benchmark datasets of varying complexities. MyCC (coverage) had the highest ranking score in F1-score. However, the performances of CoMet were higher than MyCC (coverage) on the dataset containing multiple strains. Furthermore, CoMet recovered contigs of more species and was 18 - 39% higher in precision than the compared existing methods in discriminating species from the sample of multiple strains. CoMet resulted in higher precision than MyCC (default) and MyCC (coverage) on a real metagenome. Conclusions: The approach proposed with CoMet for binning contigs, improves the precision of binning while characterizing more species in a single metagenomic sample and in a sample containing multiple strains. The F1-scores obtained from different binning strategies vary with different datasets; however, CoMet yields the highest F1-score with a sample comprised of multiple strains

High-resolution spatial and genomic characterization of coral-associated microbial aggregates in the coral Stylophora pistillata

Bacteria commonly form aggregates in a range of coral species [termed coral-associated microbial aggregates (CAMAs)], although these structures remain poorly characterized despite extensive efforts studying the coral microbiome. Here, we comprehensively characterize CAMAs associated with Stylophora pistillata and quantify their cell abundance. Our analysis reveals that multiple Endozoicomonas phylotypes coexist inside a single CAMA. Nanoscale secondary ion mass spectrometry imaging revealed that the Endozoicomonas cells were enriched with phosphorus, with the elemental compositions of CAMAs different from coral tissues and endosymbiotic Symbiodiniaceae, highlighting a role in sequestering and cycling phosphate between coral holobiont partners. Consensus metagenome--assembled genomes of the two dominant Endozoicomonas phylotypes confirmed their metabolic potential for polyphosphate accumulation along with genomic signatures including type VI secretion systems allowing host association. Our findings provide unprecedented insights into Endozoicomonas-dominated CAMAs and the first direct physiological and genomic linked evidence of their biological role in the coral holobiont

Long-Term Survey Is Necessary to Reveal Various Shifts of Microbial Composition in Corals

The coral holobiont is the assemblage of coral host and its microbial symbionts, which functions as a unit and is responsive to host species and environmental factors. Although monitoring surveys have been done to determine bacteria associated with coral, none have persisted for >1 year. Therefore, potential variations in minor or dominant community members that occur over extended intervals have not been characterized. In this study, 16S rRNA gene amplicon pyrosequencing was used to investigate the relationship between bacterial communities in healthy Stylophora pistillata in tropical and subtropical Taiwan over 2 years, apparently one of the longest surveys of coral-associated microbes. Dominant bacterial genera in S. pistillata had disparate changes in different geographical setups, whereas the constitution of minor bacteria fluctuated in abundance over time. We concluded that dominant bacteria (Acinetobacter, Propionibacterium, and Pseudomonas) were stable in composition, regardless of seasonal and geographical variations, whereas Endozoicomonas had a geographical preference. In addition, by combining current data with previous studies, we concluded that a minor bacteria symbiont, Ralstonia, was a keystone species in coral. Finally, we concluded that long-term surveys for coral microbial communities were necessary to detect compositional shifts, especially for minor bacterial members in corals

Additional file 1 of CoMet: a workflow using contig coverage and composition for binning a metagenomic sample with high precision

Supplementary Material file contains the details of recall values obtained in this study and the GC content distributions and the GC content - log(Coverage) distributions of the contigs in the simulated datasets considered in this study. (PDF 203 kb

Genomic exploration of coral-associated bacteria: identifying probiotic candidates to increase coral bleaching resilience in Galaxea fascicularis

Abstract Background Reef-building corals are acutely threatened by ocean warming, calling for active interventions to reduce coral bleaching and mortality. Corals associate with a wide diversity of bacteria which can influence coral health, but knowledge of specific functions that may be beneficial for corals under thermal stress is scant. Under the oxidative stress theory of coral bleaching, bacteria that scavenge reactive oxygen (ROS) or nitrogen species (RNS) are expected to enhance coral thermal resilience. Further, bacterial carbon export might substitute the carbon supply from algal photosymbionts, enhance thermal resilience and facilitate bleaching recovery. To identify probiotic bacterial candidates, we sequenced the genomes of 82 pure-cultured bacteria that were isolated from the emerging coral model Galaxea fascicularis. Results Genomic analyses showed bacterial isolates were affiliated with 37 genera. Isolates such as Ruegeria, Muricauda and Roseovarius were found to encode genes for the synthesis of the antioxidants mannitol, glutathione, dimethylsulfide, dimethylsulfoniopropionate, zeaxanthin and/or β-carotene. Genes involved in RNS-scavenging were found in many G. fascicularis-associated bacteria, which represents a novel finding for several genera (including Pseudophaeobacter). Transporters that are suggested to export carbon (semiSWEET) were detected in seven isolates, including Pseudovibrio and Roseibium. Further, a range of bacterial strains, including strains of Roseibium and Roseovarius, revealed genomic features that may enhance colonisation and association of bacteria with the coral host, such as secretion systems and eukaryote-like repeat proteins. Conclusions Our work provides an in-depth genomic analysis of the functional potential of G. fascicularis-associated bacteria and identifies novel combinations of traits that may enhance the coral’s ability to withstand coral bleaching. Identifying and characterising bacteria that are beneficial for corals is critical for the development of effective probiotics that boost coral climate resilience. Video Abstrac

Metagenomic, phylogenetic, and functional characterization of predominant endolithic green sulfur bacteria in the coral Isopora palifera

Abstract Background Endolithic microbes in coral skeletons are known to be a nutrient source for the coral host. In addition to aerobic endolithic algae and Cyanobacteria, which are usually described in the various corals and form a green layer beneath coral tissues, the anaerobic photoautotrophic green sulfur bacteria (GSB) Prosthecochloris is dominant in the skeleton of Isopora palifera. However, due to inherent challenges in studying anaerobic microbes in coral skeleton, the reason for its niche preference and function are largely unknown. Results This study characterized a diverse and dynamic community of endolithic microbes shaped by the availability of light and oxygen. In addition, anaerobic bacteria isolated from the coral skeleton were cultured for the first time to experimentally clarify the role of these GSB. This characterization includes GSB’s abundance, genetic and genomic profiles, organelle structure, and specific metabolic functions and activity. Our results explain the advantages endolithic GSB receive from living in coral skeletons, the potential metabolic role of a clade of coral-associated Prosthecochloris (CAP) in the skeleton, and the nitrogen fixation ability of CAP. Conclusion We suggest that the endolithic microbial community in coral skeletons is diverse and dynamic and that light and oxygen are two crucial factors for shaping it. This study is the first to demonstrate the ability of nitrogen uptake by specific coral-associated endolithic bacteria and shed light on the role of endolithic bacteria in coral skeletons

Raw datasets from Cellular mechanisms underlying extraordinary sulphide tolerance in a crustacean holobiont from hydrothermal vents

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulphide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulphur-rich vent systems. Despite the toxicity of hydrogen sulphide, X. testudinatus occupies an ecological niche in a sulphide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulphide detoxification. Here sulphide is oxidized to thiosulphate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulphate and hypotaurine is mediated by the sodium-independent sulphate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulphide-oxidizing endo-symbiotic bacteria, creating an effective sulphide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet

Supplemental Figures from Cellular mechanisms underlying extraordinary sulphide tolerance in a crustacean holobiont from hydrothermal vents

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulphide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulphur-rich vent systems. Despite the toxicity of hydrogen sulphide, X. testudinatus occupies an ecological niche in a sulphide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulphide detoxification. Here sulphide is oxidized to thiosulphate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulphate and hypotaurine is mediated by the sodium-independent sulphate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulphide-oxidizing endo-symbiotic bacteria, creating an effective sulphide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet

Supplemental Information for Materials and Methods from Cellular mechanisms underlying extraordinary sulphide tolerance in a crustacean holobiont from hydrothermal vents

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulphide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulphur-rich vent systems. Despite the toxicity of hydrogen sulphide, X. testudinatus occupies an ecological niche in a sulphide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulphide detoxification. Here sulphide is oxidized to thiosulphate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulphate and hypotaurine is mediated by the sodium-independent sulphate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulphide-oxidizing endo-symbiotic bacteria, creating an effective sulphide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet