Metagenomics-resolved genomics provides novel insights into chitin turnover, metabolic specialization, and niche partitioning in the octocoral microbiome

The role of bacterial symbionts that populate octocorals (Cnidaria, Octocorallia) is still poorly understood. To shed light on their metabolic capacities, we examined 66 high-quality metagenome-assembled genomes (MAGs) spanning 30 prokaryotic species, retrieved from microbial metagenomes of three octocoral species and seawater. Results Symbionts of healthy octocorals were affiliated with the taxa Endozoicomonadaceae, Candidatus Thioglobaceae, Metamycoplasmataceae, unclassified Pseudomonadales, Rhodobacteraceae, unclassified Alphaproteobacteria and Ca. Rhabdochlamydiaceae. Phylogenomics inference revealed that the Endozoicomonadaceae symbionts uncovered here represent two species of a novel genus unique to temperate octocorals, here denoted Ca. Gorgonimonas eunicellae and Ca. Gorgonimonas leptogorgiae. Their genomes revealed metabolic capacities to thrive under suboxic conditions and high gene copy numbers of serine-threonine protein kinases, type 3-secretion system, type-4 pili, and ankyrin-repeat proteins, suggesting excellent capabilities to colonize, aggregate, and persist inside their host. Contrarily, MAGs obtained from seawater frequently lacked symbiosis-related genes. All Endozoicomonadaceae symbionts harbored endo-chitinase and chitin-binging protein-encoding genes, indicating that they can hydrolyze the most abundant polysaccharide in the oceans. Other symbionts, including Metamycoplasmataceae and Ca. Thioglobaceae, may assimilate the smaller chitin oligosaccharides resulting from chitin breakdown and engage in chitin deacetylation, respectively, suggesting possibilities for substrate cross-feeding and a role for the coral microbiome in overall chitin turnover. We also observed sharp differences in secondary metabolite production potential between symbiotic lineages. Specific Proteobacteria taxa may specialize in chemical defense and guard other symbionts, including Endozoicomonadaceae, which lack such capacity. Conclusion This is the first study to recover MAGs from dominant symbionts of octocorals, including those of so-far unculturable Endozoicomonadaceae, Ca. Thioglobaceae and Metamycoplasmataceae symbionts. We identify a thus-far unanticipated, global role for Endozoicomonadaceae symbionts of corals in the processing of chitin, the most abundant natural polysaccharide in the oceans and major component of the natural zoo- and phytoplankton feed of octocorals. We conclude that niche partitioning, metabolic specialization, and adaptation to low oxygen conditions among prokaryotic symbionts likely contribute to the plasticity and adaptability of the octocoral holobiont in changing marine environments. These findings bear implications not only for our understanding of symbiotic relationships in the marine realm but also for the functioning of benthic ecosystems at large.info:eu-repo/semantics/publishedVersio

Metagenome-assembled genomes indicate that antimicrobial resistance genes are highly prevalent among urban bacteria and multidrug and glycopeptide resistances are ubiquitous in most taxa

IntroductionEvery year, millions of deaths are associated with the increased spread of antimicrobial resistance genes (ARGs) in bacteria. With the increasing urbanization of the global population, the spread of ARGs in urban bacteria has become a more severe threat to human health.MethodsIn this study, we used metagenome-assembled genomes (MAGs) recovered from 1,153 urban metagenomes in multiple urban locations to investigate the fate and occurrence of ARGs in urban bacteria. Additionally, we analyzed the occurrence of these ARGs on plasmids and estimated the virulence of the bacterial species.ResultsOur results showed that multidrug and glycopeptide ARGs are ubiquitous among urban bacteria. Additionally, we analyzed the deterministic effects of phylogeny on the spread of these ARGs and found ARG classes that have a non-random distribution within the phylogeny of our recovered MAGs. However, few ARGs were found on plasmids and most of the recovered MAGs contained few virulence factors.DiscussionOur results suggest that the observed non-random spreads of ARGs are not due to the transfer of plasmids and that most of the bacteria observed in the study are unlikely to be virulent. Additional research is needed to evaluate whether the ubiquitous and widespread ARG classes will become entirely prevalent among urban bacteria and how they spread among phylogenetically distinct species

Desenvolvimento de abordagem automatizada para construção de banco de dados de preferências conformacionais de carboidratos

Carboidratos são as moléculas mais abundantes na natureza. Constituídos, basicamente, por hidrogênio, oxigênio e carbono, numa razão aproximada de 2:1:1, estão intrinsecamente relacionados a uma miríade de funções biológicas. Interagindo como moeda energética metabólica e alicerce estrutural, também protagonizam em edições pós-traducionais de proteínas, modificando suas propriedades físico-químicas, padrões de enovelamento e, consequentemente, função protéica. Embora de suma importância, a compreensão das estruturas moleculares de sacarídeos é obscura devido a limitação dos métodos experimentais em detectar e resolver o espectro conformacional destas moléculas, dada a sua flexibilidade e riqueza de átomos de hidrogênio, produzindo imagens difusas e sem resolução. A estrutura molecular de polissacarídeos conta ainda com polimerização ramificada, diferentes tipos de ligação e isômeros ativos, corroborando em um objeto de pesquisa de alta complexidade. Considerando o exposto, o presente trabalho visa aplicar em larga escala uma metodologia previamente desenvolvida na UFRGS para explorar os padrões conformacionais de carboidratos, levando em conta diferentes unidades monoméricas (monossacarídeos) e suas formas anoméricas e diferentes tipos de ligação glicosídica, considerando sua flexibilidade. Espera-se, através destes métodos de simulação computacional baseados em Mecânica Molecular, alicerçar o desenvolvimento de um banco de dados de conformações preferenciais para carboidratos.Carbohydrates are the most abundant molecules in nature. Consisting basically of hydrogen, oxygen and carbon in a 2:1:1 ratio, they are intrinsically associated with a myriad of biological functions. Interplaying as energetic currency and structural scaffold, they also play the main role in post-translation protein editing, altering polypeptide’s physical and chemical properties, folding patterns and, consequently, protein function. The understanding of their molecular structure is of paramount importance, yet its full comprehension is far from being achieved, for the experimental methods fail to access saccharides’ conformational aspects and oscillatory features, due to the high presence of hydrogen in its molecular structure, yielding indistinct images of low resolution. Carbohydrates become even more complex if one considers their branching capabilities, optical isomers and different types of bonding, behaving as a remarkably complex subject of study. Considering the above mentioned, this work aims to apply in large scale the previously developed methodology at UFRGS, in order to unveil and unravel the conformational patterns of carbohydrates, taking into account its numerous monomeric unities (monossacharides) and anomeric forms and different glycosidic bondings, considering their dynamic properties. Hopefully, through methods of molecular mechanics-based computer simulation, this study proposes a consistent automated methodology, aiming to provide the foundations for the development of a data bank of conformational preferences of carbohydrates

Desenvolvimento de abordagem automatizada para construção de banco de dados de preferências conformacionais de carboidratos

Carboidratos são as moléculas mais abundantes na natureza. Constituídos, basicamente, por hidrogênio, oxigênio e carbono, numa razão aproximada de 2:1:1, estão intrinsecamente relacionados a uma miríade de funções biológicas. Interagindo como moeda energética metabólica e alicerce estrutural, também protagonizam em edições pós-traducionais de proteínas, modificando suas propriedades físico-químicas, padrões de enovelamento e, consequentemente, função protéica. Embora de suma importância, a compreensão das estruturas moleculares de sacarídeos é obscura devido a limitação dos métodos experimentais em detectar e resolver o espectro conformacional destas moléculas, dada a sua flexibilidade e riqueza de átomos de hidrogênio, produzindo imagens difusas e sem resolução. A estrutura molecular de polissacarídeos conta ainda com polimerização ramificada, diferentes tipos de ligação e isômeros ativos, corroborando em um objeto de pesquisa de alta complexidade. Considerando o exposto, o presente trabalho visa aplicar em larga escala uma metodologia previamente desenvolvida na UFRGS para explorar os padrões conformacionais de carboidratos, levando em conta diferentes unidades monoméricas (monossacarídeos) e suas formas anoméricas e diferentes tipos de ligação glicosídica, considerando sua flexibilidade. Espera-se, através destes métodos de simulação computacional baseados em Mecânica Molecular, alicerçar o desenvolvimento de um banco de dados de conformações preferenciais para carboidratos.Carbohydrates are the most abundant molecules in nature. Consisting basically of hydrogen, oxygen and carbon in a 2:1:1 ratio, they are intrinsically associated with a myriad of biological functions. Interplaying as energetic currency and structural scaffold, they also play the main role in post-translation protein editing, altering polypeptide’s physical and chemical properties, folding patterns and, consequently, protein function. The understanding of their molecular structure is of paramount importance, yet its full comprehension is far from being achieved, for the experimental methods fail to access saccharides’ conformational aspects and oscillatory features, due to the high presence of hydrogen in its molecular structure, yielding indistinct images of low resolution. Carbohydrates become even more complex if one considers their branching capabilities, optical isomers and different types of bonding, behaving as a remarkably complex subject of study. Considering the above mentioned, this work aims to apply in large scale the previously developed methodology at UFRGS, in order to unveil and unravel the conformational patterns of carbohydrates, taking into account its numerous monomeric unities (monossacharides) and anomeric forms and different glycosidic bondings, considering their dynamic properties. Hopefully, through methods of molecular mechanics-based computer simulation, this study proposes a consistent automated methodology, aiming to provide the foundations for the development of a data bank of conformational preferences of carbohydrates

Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters

Viral ecology of terrestrial habitats is yet-to be extensively explored, in particular the terrestrial subsurface. One problem in obtaining viral sequences from groundwater aquifer samples is the relatively low amount of virus particles. As a result, the amount of extracted DNA may not be sufficient for direct sequencing of such samples. Here we compared three DNA amplification methods to enrich viral DNA from three pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory to evaluate potential bias created by the different amplification methods as determined by viral metagenomics. Linker amplification shotgun libraries resulted in lowest redundancy among the sequencing reads and showed the highest diversity, while multiple displacement amplification produced the highest number of contigs with the longest average contig size, suggesting a combination of these two methods is suitable for the successful enrichment of viral DNA from pristine groundwater samples. In total, we identified 27,173, 5,886 and 32,613 viral contigs from the three samples from which 11.92 to 18.65% could be assigned to taxonomy using blast. Among these, members of the Caudovirales order were the most abundant group (52.20 to 69.12%) dominated by Myoviridae and Siphoviridae. Those, and the high number of unknown viral sequences, substantially expand the known virosphere

Combining Flow Cytometry and Metagenomics Improves Recovery of Metagenome-Assembled Genomes in a Cell Culture from Activated Sludge

The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting

Metagenomics-resolved genomics provides novel insights into chitin turnover, metabolic specialization, and niche partitioning in the octocoral microbiome.

BackgroundThe role of bacterial symbionts that populate octocorals (Cnidaria, Octocorallia) is still poorly understood. To shed light on their metabolic capacities, we examined 66 high-quality metagenome-assembled genomes (MAGs) spanning 30 prokaryotic species, retrieved from microbial metagenomes of three octocoral species and seawater.ResultsSymbionts of healthy octocorals were affiliated with the taxa Endozoicomonadaceae, Candidatus Thioglobaceae, Metamycoplasmataceae, unclassified Pseudomonadales, Rhodobacteraceae, unclassified Alphaproteobacteria and Ca. Rhabdochlamydiaceae. Phylogenomics inference revealed that the Endozoicomonadaceae symbionts uncovered here represent two species of a novel genus unique to temperate octocorals, here denoted Ca. Gorgonimonas eunicellae and Ca. Gorgonimonas leptogorgiae. Their genomes revealed metabolic capacities to thrive under suboxic conditions and high gene copy numbers of serine-threonine protein kinases, type 3-secretion system, type-4 pili, and ankyrin-repeat proteins, suggesting excellent capabilities to colonize, aggregate, and persist inside their host. Contrarily, MAGs obtained from seawater frequently lacked symbiosis-related genes. All Endozoicomonadaceae symbionts harbored endo-chitinase and chitin-binging protein-encoding genes, indicating that they can hydrolyze the most abundant polysaccharide in the oceans. Other symbionts, including Metamycoplasmataceae and Ca. Thioglobaceae, may assimilate the smaller chitin oligosaccharides resulting from chitin breakdown and engage in chitin deacetylation, respectively, suggesting possibilities for substrate cross-feeding and a role for the coral microbiome in overall chitin turnover. We also observed sharp differences in secondary metabolite production potential between symbiotic lineages. Specific Proteobacteria taxa may specialize in chemical defense and guard other symbionts, including Endozoicomonadaceae, which lack such capacity.ConclusionThis is the first study to recover MAGs from dominant symbionts of octocorals, including those of so-far unculturable Endozoicomonadaceae, Ca. Thioglobaceae and Metamycoplasmataceae symbionts. We identify a thus-far unanticipated, global role for Endozoicomonadaceae symbionts of corals in the processing of chitin, the most abundant natural polysaccharide in the oceans and major component of the natural zoo- and phytoplankton feed of octocorals. We conclude that niche partitioning, metabolic specialization, and adaptation to low oxygen conditions among prokaryotic symbionts likely contribute to the plasticity and adaptability of the octocoral holobiont in changing marine environments. These findings bear implications not only for our understanding of symbiotic relationships in the marine realm but also for the functioning of benthic ecosystems at large. Video Abstract