Culture Enrichment Combined With Long-Read Sequencing Facilitates Genomic Understanding of Hadal Sediment Microbes

Abstract

Culture enrichment was developed to discover the uncharted microbial species in the environmental microbiota. Yet this strategy has not been widely used to study microbes of deep-sea environments. Here, we report the cultivation and metagenomic analysis of oceanic sediment microbiota collected from 6,477 m deep in the Mariana Trench. The sediment samples were cultured anaerobically in the laboratory for 4 months, before being subjected to full-length 16S rRNA gene sequencing using the PacBio technique and metagenome sequencing using both the Illumina and Oxford Nanopore techniques. The 16S rRNA gene analyses revealed 437 operational taxonomic units specific to the cultured microbes, despite the lower diversity of the cultured microbiota in comparison with the original. Metagenome analyses revealed the prevalence of functions related to respiration, energy production, and stress response in the cultured microbes, suggesting these functions may contribute to microbial proliferation under laboratory conditions. Binning of the assembled metagenome contigs of the cultured microbiota generated four nearly complete genomes affiliated to yet unclassified species under the genera Alcanivorax, Idiomarina, Sulfitobacter, and Erythrobacter. Excepting Alcanivorax, the other three genera were almost undetectable in the original samples and largely enriched in the cultured samples. The four genomes possessed a variety of genes for carbohydrate utilization and nitrite reduction, pointing to an ability to respire diverse carbon sources using nitrite as the final electron acceptor. Taken together, the findings suggest that a combination of culture enrichment and long-read sequencing is an ideal way to mine novel microbial species in the hadal environment, particularly species that are rare in their native environmental niches, and thus expand our understanding of the hadal microbial diversity and function.

Capturing the microbial dark matter in desert soils using culturomics-based metagenomics and high-resolution analysis

“…In addition to improve taxonomic classification, access to more comprehensive recovery of MAGs (as well as ASVs) via culture-enriched metagenomic sequencing fundamentally alters the resolution of functional analysis, which has been demonstrated in this study (Fig. 9 ), as well as previous microbiome studies 37 , 38 , 45 , 46 . An extended collection of HQ MAGs by culture-enriched metagenomic sequencing and genome-resolved metagenomic analysis is essential for the in-depth functional profiling of desert microorganisms.…”

“…In this study, we conducted a systematic culturomic approach and proposed the use of culturomics-based metagenomics (CBM) to both magnify signals of specific taxa selectively enriched by different culture conditions and considerably simplify microbial diversity of metagenomic samples (Fig. 1 ), as recently achieved when applied to the gut 38 , 44 , lung 37 , wastewater 45 , and sediment 46 samples. We demonstrate that the CBM strategy which integrates large-scale culturomics, full-length 16S rRNA gene amplicon, and shotgun metagenomic sequencing can greatly improve the taxonomic and functional resolution, shedding light on the undiscovered novel microbial resources in desert soils.…”

A bacterial symbiont in the gill of the marine scallop Argopecten irradians irradians metabolizes dimethylsulfoniopropionate

“…To explore the composition and diversity of the microbiomes in scallop gills and adjacent seawater, amplification of long reads of the 16S rRNA genes from the microbial communities in the gill tissue and surrounding seawater was performed using barcoded primers of 27F/1492R (Table S1 ) 41 , 42 . The PCR reaction mixtures contained 25 μl Prime Star Max premix (R045; Takara), 2.5 μl F/R primers, and 1 μl DNA template, made up to a final volume of 50 µl with distilled H 2 O.…”

Top abundant deep ocean heterotrophic bacteria can be retrieved by cultivation

“…Moreover, when targeting the marine heterotrophic culturable bacteria from marine ecosystems, most of the studies have focused on the upper ocean (0–200 m depth) or on specific oceanographic regions [ 16 , 19 – 21 ], while studies covering different depths are less frequent [ 22 – 24 ]. Efforts to culture bacteria from the deep ocean (>200 m) have focused mostly on isolates from hydrothermal vents [ 25 – 27 ], whale carcasses [ 28 ], trenches [ 29 ], and deep-sea sediments [ 30 – 33 ]. However, very few studies have attempted to isolate bacteria from the mesopelagic [ 34 – 36 ], or the bathypelagic waters [ 23 , 37 – 39 ], and those available were mainly done at a local or regional scale but not a large scale.…”