Reviewer response for version 1

Advanced sequencing technologies and improvements in bioinformatics have provided a new way to study plant-associated microbial communities, including the use of host genomic sequencing. Our study focuses on the leaf microbiome of basin big sagebrush (Artemisia tridentata subsp. tridentata), a foundational shrub of western North America. We analyzed Illumina shotgun sequences from sagebrush leaves to investigate the metagenomes of leaf-associated microbes that were sequenced alongside their plant hosts. We aimed to profile the leaf microbiome across different sample sources (magenta box, greenhouse, and field/wild), reconstruct metagenome-assembled genomes (MAGs) where possible, and investigate functional gene annotations of the resulting MAGs, specifically with regard to the potential metabolism of sagebrush chemicals. To achieve this, Illumina shotgun sequence reads (containing both host and associated microbial reads) were mapped to the reference genomes of Artemisia tridentata, Artemisia annua, and the human reference genome to remove plant host and human-associated sequences. Host-cleaned reads were then analyzed using microbial metagenomics techniques. Taxonomic profiling revealed that Phyllobacterium and Sphingomonas were the most abundant microbial genera in greenhouse-grown plants, with very little variation among the samples. Wild, field-collected samples were much more variable and were dominated by Klebsiella and Aureobasidium species. From the co-assembly of greenhouse samples, we reconstructed two high-quality MAGs (a Phyllobacterium species and a Sphingomonas species) with >98% completion and <1% contamination. Functional annotation of these MAGs uncovered genes associated with the degradation and metabolism of camphor and other essential oils such as pinene, geraniol, and limonene, which are part of sagebrush leaf chemistry.IMPORTANCEBig sagebrush (Artemisia tridentata), the foundation species of the sagebrush steppe, has broad ecological importance because its evergreen leaves offer nutrients and shade that facilitate the establishment of diverse understory plants in arid environments. Sagebrush leaves contain various secondary metabolites, including terpenoids, flavonoids, and phenolic compounds. These chemicals contribute to the plant's defense mechanisms against herbivores and pathogens. Despite this, sagebrush hosts diverse bacterial and fungal communities. We found that the microbial metagenome-assembled genomes (MAGs) we recovered contained genes that have the potential to degrade some of the chemical compounds in sagebrush leaves that could inhibit the growth of other microbes. This is the first study to mine plant genome data using host-derived sequences to generate microbial MAGs. Our results showed that MAGs can be recovered from plant host-derived sequence data, providing a new way to explore the identity and functional capabilities of difficult-to-culture microbes.

A review of computational tools for generating metagenome-assembled genomes from metagenomic sequencing data

The widespread occurrence of sulfate-reducing microorganisms (SRMs) in temporarily oxic/hypoxic aquatic environments indicates an intriguing possibility that SRMs can prevail in constantly oxic/hypoxic terrestrial sulfate-rich environments. However, little attention has been given to this possibility, leading to an incomplete understanding of microorganisms driving the terrestrial part of the global sulfur (S) cycle. In this study, genome-centric metagenomics and metatranscriptomics were employed to explore the diversity, metabolic potential, and gene expression profile of SRMs in a revegetated acidic mine wasteland under constantly oxic/hypoxic conditions. We recovered 16 medium- to high-quality metagenome-assembled genomes (MAGs) containing reductive dsrAB. Among them, 12 and four MAGs belonged to Acidobacteria and Deltaproteobacteria, respectively, harboring three new SRM genera. Comparative genomic analysis based on seven high-quality MAGs (completeness >90% and contamination <10%; including six acidobacterial and one deltaproteobacterial) and genomes of three additional cultured model species showed that Acidobacteria-related SRMs had more genes encoding glycoside hydrolases, oxygen-tolerant hydrogenases, and cytochrome c oxidases than Deltaproteobacteria-related SRMs. The opposite pattern was observed for genes encoding superoxide reductases and thioredoxin peroxidases. Using VirSorter, viral genome sequences were found in five of the 16 MAGs and in all three cultured model species. These prophages encoded enzymes involved in glycoside hydrolysis and antioxidation in their hosts. Moreover, metatranscriptomic analysis revealed that 15 of the 16 SRMs reported here were active in situ. An acidobacterial MAG containing a prophage dominated the SRM transcripts, expressing a large number of genes involved in its response to oxidative stress and competition for organic matter.

The Gemmatimonadota phylum has been widely detected in diverse natural environments, yet their specific ecological roles in many habitats remain poorly investigated. Similarly, the Candidatus ARS69 phylum has been identified only in a few habitats, and literature on their metabolic functions is relatively scarce. In the present study, we investigated the ecological significance of phyla Ca. ARS69 and Gemmatimonadota in the Arctic glacier foreland (GF) ecosystems through genome-resolved metagenomics. We have reconstructed the first high-quality metagenome-assembled genome (MAG) belonging to Ca. ARS69 and 12 other MAGs belonging to phylum Gemmatimonadota from the three different Arctic GF samples. We further elucidated these two groups phylogenetic lineage and their metabolic function through phylogenomic and pangenomic analysis. The analysis showed that all the reconstructed MAGs potentially belonged to novel species. The MAGs belonged to Ca. ARS69 consist about 8296 gene clusters, of which only about 8% of single-copy core genes (n = 980) were shared among them. The study also revealed the potential ecological role of Ca. ARS69 is associated with carbon fixation, denitrification, sulfite oxidation, and reduction biochemical processes in the GF ecosystems. Similarly, the study demonstrates the widespread distribution of different classes of Gemmatimonadota across wide ranges of ecosystems and their metabolic functions, including in the polar region.Key points• Glacier foreland ecosystems act as a natural laboratory to study microbial community structure.• We have reconstructed 13 metagenome-assembled genomes from the soil samples.• All the reconstructed MAGs belonged to novel species with different metabolic processes.• Ca. ARS69 and Gemmatimonadota MAGs were found to participate in carbon fixation and denitrification processes.

Most of Earth’s bacteria have yet to be cultivated. The metabolic and functional potentials of these uncultivated microorganisms thus remain mysterious, and the metagenome-assembled genome (MAG) approach is the most robust method for uncovering these potentials. However, MAGs discovered by conventional metagenomic assembly and binning are usually highly fragmented genomes with heterogeneous sequence contamination. In this study, we combined Illumina and Nanopore data to develop a new workflow to reconstruct 233 MAGs—six novel bacterial orders, 20 families, 66 genera, and 154 species—from Lake Shunet, a secluded meromictic lake in Siberia. With our workflow, the average N50 of reconstructed MAGs greatly increased 10–40-fold compared to when the conventional Illumina assembly and binning method were used. More importantly, six complete MAGs were recovered from our datasets. The recovery of 154 novel species MAGs from a rarely explored lake greatly expands the current bacterial genome encyclopedia.

Metagenomic binning is an essential technique for genome-resolved characterization of uncultured microorganisms in various ecosystems but hampered by the low efficiency of binning tools in adequately recovering metagenome-assembled genomes (MAGs). Here, we introduce BASALT (Binning Across a Series of Assemblies Toolkit) for binning and refinement of short- and long-read sequencing data. BASALT employs multiple binners with multiple thresholds to produce initial bins, then utilizes neural networks to identify core sequences to remove redundant bins and refine non-redundant bins. Using the same assemblies generated from Critical Assessment of Metagenome Interpretation (CAMI) datasets, BASALT produces up to twice as many MAGs as VAMB, DASTool, or metaWRAP. Processing assemblies from a lake sediment dataset, BASALT produces ~30% more MAGs than metaWRAP, including 21 unique class-level prokaryotic lineages. Functional annotations reveal that BASALT can retrieve 47.6% more non-redundant opening-reading frames than metaWRAP. These results highlight the robust handling of metagenomic sequencing data of BASALT.

High-throughput sequencing is commonly used to study microorganisms in the environment without a need for culturing. Computational analysis of metagenomic has enabled the construction of large compendia of metagenome-assembled genomes (MAGs) from human-, animal-associated and environmental samples. Metagenomic binning is the step in the standard pipeline which groups the contigs into bins such that each bin is predicted to contain only contigs from the same genome.Binning methods can be divided into reference-dependent (based on reference genomes) and reference-independent methods (operating de novo). Compared to reference-dependent methods, reference-independent methods can discover new species or even new phyla, but completely ignore any background information. To combine the advantages of these two categories, we propose a novel binning method called SemiBin which uses a semi-supervised neural network to incorporate information from reference genomes, while still being capable of finding novel species. SemiBin outperforms existing state-of-the-art binning methods in simulated and real microbiome datasets across three different environments (human gut, dog gut, and marine environments). SemiBin returns more high-quality bins (48.0-99.2% more in single-sample mode; 11.0-30.7% more in multi-sample mode, compared to the second-best alternative). Compared to the alternatives, SemiBin captures a larger taxonomic diversity, including more distinct genera and species. In particular, we verified that SemiBin can retrieve more genomes from both known and novel species.SemiBin is available as open source software at https://github.com/BigDataBiology/SemiBin/.

Insights into the mobility and bacterial hosts of antibiotic resistance genes under dinotefuran selection pressure in aerobic granular sludge based on metagenomic binning and functional modules.

Little is known regarding the vaginal microbiota of sheep that undergo spontaneous abortions. The aim of this pilot study was to characterize, using 16S rRNA gene sequencing and shotgun metagenomics, the vaginal microbiota throughout the gestation of two ewes (Ewe1 and Ewe2) that spontaneously aborted. To achieve this, weekly vaginal swabs were collected from the ewes prior to breeding until pregnancy testing; thereafter, biweekly swabs were collected until the spontaneous abortion occurred. Based on the 16S rRNA sequencing data, Ewe1’s vaginal microbiota, overall, contained high abundances of Histophilus (12.9% relative abundance), Staphylococcus (10.8% relative abundance) and Unclassified Pasteurellaceae (8.7% relative abundance). Most notable was the high abundance of Campylobacter following the abortion in Ewe1’s vaginal microbiota. Ewe2’s vaginal microbiota was characterized by high abundances of Pasteurella (41.7% relative abundance) throughout gestation. Shotgun metagenomic sequencing produced two high-quality metagenome-assembled genomes (MAGs), identified as Campylobacter jejuni and Histophilus somni. The C. jejuni MAG had 99.95% average nucleotide identity to the most abundant sheep abortive C. jejuni clone in the USA. The H. somni MAG was most similar to a pathogenic H. somni strain and contained genes that contribute to serum resistance and sialic acid utilization. The results presented here demonstrate the need for continued research into the vaginal microbiota, specifically to identify potential predictors of spontaneous abortion.

Acinetobacter baumannii is an emerging nosocomial pathogen that poses a significant challenge to healthcare systems. Antibiotics remain the primary approach for treating A. baumannii infections. Clinical strains of A. baumannii may share ecological and genetic links with environmental isolates, particularly in aquatic environments, making the investigation of resistance profiles in environmental strains relevant to developing treatment strategies for A. baumannii-related infections. Metagenomic binning was employed to reconstruct two metagenome-assembled genomes (MAGs) of A. baumannii from surface water samples collected from the Yangtze River during the COVID-19 pandemic. Analyses revealed that these MAGs harbored antibiotic resistance genes associated with β-lactams, multidrug resistance, and tetracyclines. In addition, phylogenetic analysis showed that the assembled A. baumannii strains were closely related to the IC11 clade of clinical A. baumannii isolates, based on a comparison with 28 clinical strains. According to the resistance gene profiles of the MAGs, we found that 2 (13 in total) commonly used antibiotics in China and 20 (29) WHO-recommended “last-resort” antibiotics had potential efficacy in the treatment. The findings underscore the value of environmental surveillance for understanding resistance mechanisms in A. baumannii and informing strategies to mitigate its clinical and public health impact.

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.

Denitrification accounts for a substantial nitrogen loss from environmental systems, shifting microbial composition and impacting other biogeochemical cycles. In Antarctica, rising temperatures cause increased organic matter deposition in marine sediments, which can significantly alter microbially mediated denitrification. To examine the genetic potential of microorganisms driving N-cycling in these sediments, benthic sediment cores were collected at two sites in the Weddell Sea, Antarctica. DNA was extracted from multiple depths at each site, resulting in the reconstruction of 75 high-quality metagenome-assembled genomes (MAGs). Forty-seven of these MAGs contained reductases involved in denitrification. MAGs belonging to the genus Methyloceanibacter were the most abundant MAGs at both sites and all depths, except depth 3-6cmbsf at one site, where they were not identified. The abundance of these Methyloceanibacter MAGs suggests the potential for nitrate-driven methanol oxidation at both sites. MAGs belonging to Beggiatoaceae and Sedimenticolaceae were found to have the genetic potential to produce intermediates in denitrification and the complete pathway for dissimilatory nitrate reduction to ammonia. MAGs within Acidimicrobiia and Dadabacteria had the potential to complete the final denitrification step. Based on MAGs, Antarctic peninsula sediment communities have the potential for complete denitrification and dissimilatory nitrate reduction to ammonia via a consortium.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce foundational genomic work on the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzymes able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) suggest the potential for microbial transfer between marine sediment and Kyphosus digestive tracts. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.

Exercise reduces inflammation, fatigue, and aids overall health. Additionally, physical fitness has been associated with desirable changes in the community composition of the athlete gut microbiome, with health-associated taxa being shown to be increased in active individuals. Here, using a combination of in silico and in vitro methods, we investigate the antimicrobial activity of the athlete gut microbiome. In vitro approaches resulted in the generation of 284 gut isolates with inhibitory activity against Clostridioides difficile and/or Fusobacterium nucleatum, and the most potent isolates were further characterized, and potential bacteriocins were predicted using both MALDI-TOF MS and whole-genome sequencing. Additionally, metagenomic reads from the faecal samples were used to recover 770 Metagenome Assembled Genomes (MAGs), of which 148 were assigned to be high-quality MAGs and screened for the presence of putative bacteriocin gene clusters using BAGEL4 software, with 339 gene clusters of interest being identified. Class I was the most abundant bacteriocin class predicted, accounting for 91.3% of predictions, Class III had a predicted abundance of 7.5%, and Class II was represented by just 1% of all predictions.

The decay of animal corpses may release various nutrients into the surrounding environment and participate in the biogeochemical cycle. However, few studies have involved the sulphur cycle in animal corpse decay. Here, we conducted an indoor control experiment at five different temperature gradients (23, 26, 29, 32 and 35 °C) to explore the change of sulphur cycling microbes and genes under corpse decay by metagenomic sequencing, metagenomic binning, and 16S rRNA sequencing. Our results showed that total sulphur content decreased by 41.82% due to decomposition. The dominant phyla of the S-cycle microbes were Proteobacteria, followed by Bacteroidetes and Acidobacteria. Corpse decay decreased the pathways involved in assimilatory sulphate reduction, sulfur oxidizing (Sox) system, organic sulphur transformation, and sulphur oxidation pathway, but increased the links between inorganic and organic sulphur transformation. The alpha diversity of sulphur cycling genes decreased with increasing temperature and the deterministic process was dominant in most of temperature gradients. The diversity of microbial community was the main driving factors of sulphur cycling genes, followed by corpse decay and temperature. We obtained 257 high-quality metagenome assembled genomes (MAGs) containing sulphur cycling genes, and the results of metagenome-assembled genomes provide evidence for the covariation of sulphur cycling genes, such as sir, cysD, cysH, and ssuC, which shared the function of assimilatory sulphate reduction. Our research reveals the pathways of microbial sulphur cycling and contributes to the understanding of the elemental cycle associated with animal corpse decay.