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The microbial communities associated with marine sediments are critical for ecosystem function yet remain poorly characterized. While culture-independent (CI) techniques capture the broadest perspective on community composition, culture-dependent (CD) methods can select for low abundance taxa that are missed using CI approaches. This study aimed to assess microbial diversity in tropical marine sediments at five shallow-water sites in Belize using both CD and CI techniques. The CD methods captured approximately 3% of the >800 genera detected across all sites using the CI approach. Additionally, 39 genera were only detected in culture, revealing rare taxa that were missed with the CI approach. Significantly different communities were detected across sites, with rare taxa playing an important role in distinguishing among communities. This study provides important baseline data describing shallow-water sediment microbial communities, evidence that standard cultivation techniques may be more effective than previously recognized, and the first steps towards identifying new taxa that are amenable to agar plate cultivation.
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Sedimentos Geológicos , MicrobiotaRESUMO
Thousands of natural products have been identified from cultured microorganisms, yet evidence of their production in the environment has proven elusive. Technological advances in mass spectrometry, combined with public databases, now make it possible to address this disparity by detecting compounds directly from environmental samples. Here, we used adsorbent resins, tandem mass spectrometry, and next-generation sequencing to assess the metabolome of marine sediments and its relationship to bacterial community structure. We identified natural products previously reported from cultured bacteria, providing evidence they are produced in situ, and compounds of anthropogenic origin, suggesting this approach can be used as an indicator of environmental impact. The bacterial metabolite staurosporine was quantified and shown to reach physiologically relevant concentrations, indicating that it may influence sediment community structure. Staurosporine concentrations were correlated with the relative abundance of the staurosporine-producing bacterial genus Salinispora and production confirmed in strains cultured from the same location, providing a link between compound and candidate producer. Metagenomic analyses revealed numerous biosynthetic gene clusters related to indolocarbazole biosynthesis, providing evidence for noncanonical sources of staurosporine and a path forward to assess the relationships between natural products and the organisms that produce them. Untargeted environmental metabolomics circumvents the need for laboratory cultivation and represents a promising approach to understanding the functional roles of natural products in shaping microbial community structure in marine sediments.IMPORTANCE Natural products are readily isolated from cultured bacteria and exploited for useful purposes, including drug discovery. However, these compounds are rarely detected in the environments from which the bacteria are obtained, thus limiting our understanding of their ecological significance. Here, we used environmental metabolomics to directly assess chemical diversity in marine sediments. We identified numerous metabolites and, in one case, isolated strains of bacteria capable of producing one of the compounds detected. Coupling environmental metabolomics with community and metagenomic analyses provides opportunities to link compounds and producers and begin to assess the complex interactions mediated by specialized metabolites in marine sediments.
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Bactérias/metabolismo , Produtos Biológicos/isolamento & purificação , Oceanos e Mares , Bactérias/genética , Produtos Biológicos/química , Descoberta de Drogas , Sedimentos Geológicos/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Metaboloma , Metabolômica , Metagenoma , Microbiota/fisiologia , Micromonosporaceae/metabolismo , Família Multigênica , Água do Mar/microbiologiaRESUMO
Bacterial genome sequences consistently contain many more biosynthetic gene clusters encoding specialized metabolites than predicted by the compounds discovered from the respective strains. One hypothesis invoked to explain the cryptic nature of these gene clusters is that standard laboratory conditions do not provide the environmental cues needed to trigger gene expression. A potential source of such cues is other members of the bacterial community, which are logical targets for competitive interactions. In this study, we examined the effects of such interactions on specialized metabolism in the marine actinomycete Salinispora tropica. The results show that antibiotic activities and the concentration of some small molecules increase in the presence of co-occurring bacterial strains relative to monocultures. Some increases in antibiotic activity could be linked to nutrient depletion by the competitor as opposed to the production of a chemical cue. Other increases were correlated with the production of specific compounds by S. tropica. In particular, one interaction with a Vibrio sp. consistently induced antibiotic activity and was associated with parent ions that were unique to this interaction, although the associated compound could not be identified. This study provides insight into the metabolomic complexities of bacterial interactions and baseline information for future genome mining efforts.
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Interações Microbianas/fisiologia , Micromonosporaceae/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Técnicas de Cocultura , Metabolômica , Micromonosporaceae/crescimento & desenvolvimento , Espectrometria de Massas em TandemRESUMO
Artificial habitats for animals have high commercial and societal value. Microbial communities (microbiomes) in such habitats may play ecological roles similar to those in nature. However, this hypothesis remains largely untested. Georgia Aquarium's Ocean Voyager (OV) exhibit is a closed-system aquatic habitat that mimics the oligotrophic open ocean and houses thousands of large marine animals, including fish, sea turtles, and whale sharks. We present a 14-month time series characterizing the OV water column microbiome. The composition and stability of the microbiome differed from those of natural marine environments with similar chemical features. The composition shifted dramatically over the span of 2 weeks and was characterized by bloom events featuring members of two heterotrophic bacterial lineages with cosmopolitan distributions in the oceans. The relative abundances of these lineages were inversely correlated, suggesting an overlap in ecological niches. Transcript mapping to metagenome-assembled genomes (MAGs) of these taxa identified unique characteristics, including the presence and activity of genes for the synthesis and degradation of cyanophycin, an amino acid polymer linked to environmental stress and found frequently in cyanobacteria but rarely in heterotrophic bacteria. The dominant MAGs also contained and transcribed plasmid-associated sequences, suggesting a role for conjugation in adaptation to the OV environment. These findings indicate a highly dynamic microbiome despite the stability of the physical and chemical parameters of the water column. Characterizing how such fluctuations affect microbial function may inform our understanding of animal health in closed aquaculture systems.IMPORTANCE Public aquariums play important societal roles, for example, by promoting science education and helping conserve biodiversity. The health of aquarium animals depends on interactions with the surrounding microbiome. However, the extent to which aquariums recreate a stable and natural microbial ecosystem is uncertain. This study describes the taxonomic composition of the water column microbiome over 14 months in a large indoor aquatic habitat, the Ocean Voyager exhibit at the Georgia Aquarium. Despite stable water column conditions, the exhibit experienced blooms in which the abundance of a single bacterial strain increased to over 65% of the community. Genome analysis indicated that the OV's dominant strains share unique adaptations, notably genes for storage polymers associated with environmental stress. These results, interpreted alongside data from natural ocean systems and another artificial seawater aquarium, suggest a highly dynamic aquarium microbiome and raise questions of how microbiome stability may affect the ecological health of the habitat.
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Bactérias/isolamento & purificação , Ecossistema , Microbiota , Água do Mar/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Peixes/crescimento & desenvolvimento , Georgia , Metagenoma , FilogeniaRESUMO
Marine sediments harbor complex microbial communities that remain poorly studied relative to other biomes such as seawater. Moreover, bacteria in these communities produce antibiotics and other bioactive secondary metabolites, yet little is known about how these compounds affect microbial community structure. In this study, we used next-generation amplicon sequencing to assess native microbial community composition in shallow tropical marine sediments. The results revealed complex communities comprised of largely uncultured taxa, with considerable spatial heterogeneity and known antibiotic producers comprising only a small fraction of the total diversity. Organic extracts from cultured strains of the sediment-dwelling actinomycete genus Salinispora were then used in mesocosm studies to address how secondary metabolites shape sediment community composition. We identified predatory bacteria and other taxa that were consistently reduced in the extract-treated mesocosms, suggesting that they may be the targets of allelopathic interactions. We tested related taxa for extract sensitivity and found general agreement with the culture-independent results. Conversely, several taxa were enriched in the extract-treated mesocosms, suggesting that some bacteria benefited from the interactions. The results provide evidence that bacterial secondary metabolites can have complex and significant effects on sediment microbial communities. IMPORTANCE: Ocean sediments represent one of Earth's largest and most poorly studied biomes. These habitats are characterized by complex microbial communities where competition for space and nutrients can be intense. This study addressed the hypothesis that secondary metabolites produced by the sediment-inhabiting actinomycete Salinispora arenicola affect community composition and thus mediate interactions among competing microbes. Next-generation amplicon sequencing of mesocosm experiments revealed complex communities that shifted following exposure to S. arenicola extracts. The results reveal that certain predatory bacteria were consistently less abundant following exposure to extracts, suggesting that microbial metabolites mediate competitive interactions. Other taxa increased in relative abundance, suggesting a benefit from the extracts themselves or the resulting changes in the community. This study takes a first step toward assessing the impacts of bacterial metabolites on sediment microbial communities. The results provide insight into how low-abundance organisms may help structure microbial communities in ocean sediments.
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Actinobacteria/metabolismo , Sedimentos Geológicos/microbiologia , Água do Mar/microbiologia , Metabolismo Secundário/fisiologia , Actinobacteria/classificação , Actinobacteria/genética , Biodiversidade , Sequenciamento de Nucleotídeos em Larga Escala , Microbiota/fisiologia , Oceanos e Mares , Microbiologia da ÁguaRESUMO
Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites.
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Actinobacteria/genética , Actinobacteria/isolamento & purificação , Produtos Biológicos/metabolismo , Genoma Bacteriano , Água do Mar/microbiologia , Actinobacteria/classificação , Actinobacteria/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Filogenia , Análise de Sequência de DNARESUMO
Next-generation sequencing has increased the coverage of microbial diversity surveys by orders of magnitude, but differentiating artifacts from rare environmental sequences remains a challenge. Clustering 16S rRNA sequences into operational taxonomic units (OTUs) organizes sequence data into groups of 97 % identity, helping to reduce data volumes and avoid analyzing sequencing artifacts by grouping them with real sequences. Here, we analyze sequence abundance distributions across environmental samples and show that 16S rRNA sequences of >99 % identity can represent functionally distinct microorganisms, rendering OTU clustering problematic when the goal is an accurate analysis of organism distribution. Strict postsequencing quality control (QC) filters eliminated the most prevalent artifacts without clustering. Further experiments proved that DNA polymerase errors in polymerase chain reaction (PCR) generate a significant number of substitution errors, most of which pass QC filters. Based on our findings, we recommend minimizing the number of PCR cycles in DNA library preparation and applying strict postsequencing QC filters to reduce the most prevalent artifacts while maintaining a high level of accuracy in diversity estimates. We further recommend correlating rare and abundant sequences across environmental samples, rather than clustering into OTUs, to identify remaining sequence artifacts without losing the resolution afforded by high-throughput sequencing.
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Actinomycetales/genética , Biodiversidade , Reação em Cadeia da Polimerase/normas , Actinomycetales/classificação , Actinomycetales/isolamento & purificação , Primers do DNA/genética , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genéticaRESUMO
Marine bacteria are known to produce a wide variety of structurally diverse and biologically active secondary metabolites. Considerably less is known about the ecological functions of these compounds, in part due to methodological challenges associated with this field of research. Here, we review the antagonistic activities mediated by marine bacteria with a focus on activities linked to structurally defined secondary metabolites. Bacterial antagonism has been documented against other marine bacteria as well as eukaryotes, and includes antibiosis, the inhibition of quorum sensing, larval settlement deterrence, and defense against predation. These compounds likely play important ecological roles that ultimately affect ecosystem structure and function, however, much remains to be learned before these roles can be fully appreciated. Recent technological advances coupled with a better understanding of the diverse processes mediated by secondary metabolites provide new opportunities to expand our understanding of the chemical ecology of bacterial antagonism in the marine environment.
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Antibiose , Organismos Aquáticos , Ecossistema , Metabolismo Secundário , AnimaisRESUMO
The expanding interest in marine microbiome and eDNA sequence data has led to a demand for sample collection and preservation standard practices to enable comparative assessments of results across studies and facilitate meta-analyses. We support this effort by providing guidelines based on a review of published methods and field sampling experiences. The major components considered here are environmental and resource considerations, sample processing strategies, sample storage options, and eDNA extraction protocols. It is impossible to provide universal recommendations considering the wide range of eDNA applications; rather, we provide information to design fit-for-purpose protocols. To manage scope, the focus here is on sampling collection and preservation of prokaryotic and microeukaryotic eDNA. Even with a focused view, the practical utility of any approach depends on multiple factors, including habitat type, available resources, and experimental goals. We broadly recommend enacting rigorous decontamination protocols, pilot studies to guide the filtration volume needed to characterize the target(s) of interest and minimize PCR inhibitor collection, and prioritizing sample freezing over (only) the addition of preservation buffer. An annotated list of studies that test these parameters is included for more detailed investigation on specific steps. To illustrate an approach that demonstrates fit-for-purpose methodologies, we provide a protocol for eDNA sampling aboard an oceanographic vessel. These guidelines can aid the decision-making process for scientists interested in sampling and sequencing marine microbiomes and/or eDNA.
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BACKGROUND: Amplicon sequencing (metabarcoding) is a common method to survey diversity of environmental communities whereby a single genetic locus is amplified and sequenced from the DNA of whole or partial organisms, organismal traces (e.g., skin, mucus, feces), or microbes in an environmental sample. Several software packages exist for analyzing amplicon data, among which QIIME 2 has emerged as a popular option because of its broad functionality, plugin architecture, provenance tracking, and interactive visualizations. However, each new analysis requires the user to keep track of input and output file names, parameters, and commands; this lack of automation and standardization is inefficient and creates barriers to meta-analysis and sharing of results. FINDINGS: We developed Tourmaline, a Python-based workflow that implements QIIME 2 and is built using the Snakemake workflow management system. Starting from a configuration file that defines parameters and input files-a reference database, a sample metadata file, and a manifest or archive of FASTQ sequences-it uses QIIME 2 to run either the DADA2 or Deblur denoising algorithm; assigns taxonomy to the resulting representative sequences; performs analyses of taxonomic, alpha, and beta diversity; and generates an HTML report summarizing and linking to the output files. Features include support for multiple cores, automatic determination of trimming parameters using quality scores, representative sequence filtering (taxonomy, length, abundance, prevalence, or ID), support for multiple taxonomic classification and sequence alignment methods, outlier detection, and automated initialization of a new analysis using previous settings. The workflow runs natively on Linux and macOS or via a Docker container. We ran Tourmaline on a 16S ribosomal RNA amplicon data set from Lake Erie surface water, showing its utility for parameter optimization and the ability to easily view interactive visualizations through the HTML report, QIIME 2 viewer, and R- and Python-based Jupyter notebooks. CONCLUSION: Automated workflows like Tourmaline enable rapid analysis of environmental amplicon data, decreasing the time from data generation to actionable results. Tourmaline is available for download at github.com/aomlomics/tourmaline.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Ribossômico 16S/genética , Silicatos , Fluxo de TrabalhoRESUMO
BACKGROUND: Antibiotic resistance is a growing problem that can be ameliorated by the discovery of novel drug candidates. Bacterial associates are often the source of pharmaceutically active natural products isolated from marine invertebrates, and thus, important targets for drug discovery. While the microbiomes of many marine organisms have been extensively studied, microbial communities from chemically-rich nudibranchs, marine invertebrates that often possess chemical defences, are relatively unknown. METHODS: We applied both culture-dependent and independent approaches to better understand the biochemical potential of microbial communities associated with nudibranchs. Gram-positive microorganisms isolated from nudibranchs collected in the Red Sea were screened for antibacterial and antitumor activity. To assess their biochemical potential, the isolates were screened for the presence of natural product biosynthetic gene clusters, including polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, using PCR. The microbiomes of the nudibranchs were investigated by high-throughput sequencing of 16S rRNA amplicons. RESULTS: In screens against five model microorganisms, 51% of extracts displayed antimicrobial activity against more than one organism, and 19% exhibited antitumor activity against Ehrlich's ascites carcinoma. Sixty-four percent of isolates contained PKS and NRPS genes, suggesting their genomes contain gene clusters for natural product biosynthesis. Thirty-five percent were positive for more than one class of biosynthetic gene. These strains were identified as belonging to the Firmicutes and Actinobacteria phyla via 16S rRNA gene sequencing. In addition, 16S rRNA community amplicon sequencing revealed all bacterial isolates were present in the uncultured host-associated microbiome, although they were a very small percentage of the total community. Taken together, these results indicate that bacteria associated with marine nudibranchs are potentially a rich source of bioactive compounds and natural product biosynthetic genes.
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Harmful Algal Blooms (HABs) exert considerable ecological and economic damage and are becoming increasingly frequent worldwide. However, the biological factors underlying HABs remain uncertain. Relationships between algae and bacteria may contribute to bloom formation, strength, and duration. We investigated the microbial communities and metabolomes associated with a HAB of the toxic dinoflagellate Karenia brevis off the west coast of Florida in June 2018. Microbial communities and intracellular metabolite pools differed based on both bacterial lifestyle and bloom level, suggesting a complex role for blooms in reshaping microbial processes. Network analysis identified K. brevis as an ecological hub in the planktonic ecosystem, with significant connections to diverse microbial taxa. These included four flavobacteria and one sequence variant unidentified past the domain level, suggesting uncharacterized diversity in phytoplankton-associated microbial communities. Additionally, intracellular metabolomic analyses associated high K. brevis levels with higher levels of aromatic compounds and lipids. These findings reveal water column microbial and chemical characteristics with potentially important implications for understanding HAB onset and duration.
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Although competition, niche partitioning, and spatial isolation have been used to describe the ecology and evolution of macro-organisms, it is less clear to what extent these principles account for the extraordinary levels of bacterial diversity observed in nature. Ecological interactions among bacteria are particularly challenging to address due to methodological limitations and uncertainties over how to recognize fundamental units of diversity and link them to the functional traits and evolutionary processes that led to their divergence. Here we show that two closely related marine actinomycete species can be differentiated based on competitive strategies. Using a direct challenge assay to investigate inhibitory interactions with members of the bacterial community, we observed a temporal difference in the onset of inhibition. The majority of inhibitory activity exhibited by Salinispora arenicola occurred early in its growth cycle and was linked to antibiotic production. In contrast, most inhibition by Salinispora tropica occurred later in the growth cycle and was more commonly linked to nutrient depletion or other sources. Comparative genomics support these differences, with S. arenicola containing nearly twice the number of secondary metabolite biosynthetic gene clusters as S. tropica, indicating a greater potential for secondary metabolite production. In contrast, S. tropica is enriched in gene clusters associated with the acquisition of growth-limiting nutrients such as iron. Coupled with differences in growth rates, the results reveal that S. arenicola uses interference competition at the expense of growth, whereas S. tropica preferentially employs a strategy of exploitation competition. The results support the ecological divergence of two co-occurring and closely related species of marine bacteria by providing evidence they have evolved fundamentally different strategies to compete in marine sediments.