Global soil metagenomics reveals distribution and predominance of Deltaproteobacteria in nitrogen-fixing microbiome.

BACKGROUND: Biological nitrogen fixation is a fundamental process sustaining all life on earth. While distribution and diversity of N2-fixing soil microbes have been investigated by numerous PCR amplicon sequencing of nitrogenase genes, their comprehensive understanding has been hindered by lack of de facto standard protocols for amplicon surveys and possible PCR biases. Here, by fully leveraging the planetary collections of soil shotgun metagenomes along with recently expanded culture collections, we evaluated the global distribution and diversity of terrestrial diazotrophic microbiome. RESULTS: After the extensive analysis of 1,451 soil metagenomic samples, we revealed that the Anaeromyxobacteraceae and Geobacteraceae within Deltaproteobacteria are ubiquitous groups of diazotrophic microbiome in the soils with different geographic origins and land usage types, with particular predominance in anaerobic soils (paddy soils and sediments). CONCLUSION: Our results indicate that Deltaproteobacteria is a core bacterial taxon in the potential soil nitrogen fixation population, especially in anaerobic environments, which encourages a careful consideration on deltaproteobacterial diazotrophs in understanding terrestrial nitrogen cycling. Video Abstract.

Lysinibacillus piscis sp. nov. isolated from the gut of mottled spinefoot Siganus fuscescens.

A novel Lysinibacillus strain, designated KH24T, was isolated from the gut of Siganus fuscescens, a herbivorous fish, which was captured off the coast of Okinawa, Japan. Strain KH24T is a rod-shaped, Gram-stain-positive, spore-forming, and motile bacterium that forms off-white colonies. The 16S rRNA gene sequence of strain KH24T showed the highest similarity (97.4%) with Lysinibacillus pakistanensis JCM 18776T and L. irui IRB4-01T. Genomic similarities between strain KH24T and Lysinibacillus type strains, based on average nucleotide identity, digital DNA-DNA hybridization (genome-to-genome distance calculation), and average amino acid identity were 70.4-77.7%, 17.1-24.4%, and 69.2-81.2%, respectively, which were lower than species delineation thresholds. Strain KH24T growth occurred at pH values of 5.5-8.5, temperatures of 20-40 °C, and NaCl concentrations of 0-4.0%, and optimally at pH 7.0, 30 °C, and 0%, respectively. Unlike related Lysinibacillus type strains, strain KH24T could assimilate D-glucose, D-fructose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, ferric citrate, salicin, D-cellobiose, D-maltose, D-sucrose, and gentiobiose. Major fatty acids included iso-C15:0 (45.8%), anteiso-C15:0 (15.1%), iso-C17:0 (12.6%), and anteiso-C17:0 (10.9%). Menaquinone-7 was the predominant quinone, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and lysophosphatidylethanolamine. Based on its genetic and phenotypic properties, strain KH24T represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus piscis sp. nov. is proposed. The type strain is KH24T (= JCM 36611 T = KCTC 43676 T).

Mesoterricola silvestris gen. nov., sp. nov., Mesoterricola sediminis sp. nov., Geothrix oryzae sp. nov., Geothrix edaphica sp. nov., Geothrix rubra sp. nov., and Geothrix limicola sp. nov., six novel members of Acidobacteriota isolated from soils.

Forty-eight Acidobacteriota strains were isolated from soils and sediments in Japan. Among them, six representative strains, designated W79T, W786T, Red222T, Red802T, Red803T, and Red804T, were subjected to the taxonomic classification. These six strains are Gram-stain-negative, non-spore-forming, rod-shaped, and facultative anaerobic bacterium that can reduce ferric iron. Phylogenetic and phylogenomic trees based on 16S rRNA genes and multiple single-copy gene sequences showed that strains Red222T, Red802T, Red803T, and Red804T formed a cluster with the type strains of Geothrix species, but strains W79T and W786T created an independent cluster from any other type strains. The former four strains shared 97.95-99.08% similarities of 16S rRNA gene sequence with the type strains of the genus Geothrix, whereas the latter two strains 94.86-95.49% similarities. The average amino acid identity of strains W79T and W786T were <63 % to any other type strains, which were below the genus delineation thresholds. Moreover, colonies of these two strains were white, while those of the other four isolated strains were reddish-yellow as well as the type strain Geothrix fermentans H-5T. Although the known type strains of Geothrix species have been reported to be non-motile, five strains (W79T, W786T, Red222T, Red803T, and Red804T) except for strain Red802T displayed motility. Furthermore, multiple genomic, phylogenetic, and phenotypic features supported the discrimination between these isolated strains. Based on the study evidence, we propose these six isolates as novel members within the Acidobacteriota/Holophagae/Holophagales/Holophagaceae, comprising two novel species of a novel genus, Mesoterricola silvestris gen. nov., sp. nov., and Mesoterricola sediminis sp. nov., and four novel species of the genus Geothrix: Geothrix oryzae sp. nov., Geothrix edaphica sp. nov., Geothrix rubra sp. nov., and Geothrix limicola sp. nov.

Anaeromyxobacter oryzae sp. nov., Anaeromyxobacter diazotrophicus sp. nov. and Anaeromyxobacter paludicola sp. nov., isolated from paddy soils.

Three bacterial strains (Red232T, Red267T and Red630T) were isolated from paddy soils sampled in Japan. Cells of these strains were Gram-stain-negative, facultative anaerobic, long rod-shaped with monotrichous flagella or pilus-like structures for motility, and formed red colonies on agar plates. Phylogenetic trees based on 16S rRNA gene and multiple single-copy gene sequences showed that the three strains formed a cluster with the type strains of Anaeromyxobacter species, independent from any other strain genera. Similarity values of the 16S rRNA gene sequences and genomes among the three isolated strains and the type strain of Anaeromyxobacter, Anaeromyxobacter dehalogenans 2CP-1T, were 95.4-97.4% for 16S rRNA gene sequence, 75.3-79.5% for average nucleotide identity, 19.6-21.7% for digital DNA-DNA hybridization and 64.1-72.6% for average amino acid identity, all of which are below the species delineation thresholds. Nitrogenase genes were observed in the genomes of the three novel strains, but not in A. dehalogenans 2CP-1T. Moreover, multiple genomic, physiological and chemotaxonomic features supported the discrimination between these three strains. Based on the evidence in this study, the three isolates represent three novel independent species for which the following names are proposed: Anaeromyxobacter oryzae sp. nov., Anaeromyxobacter diazotrophicus sp. nov. and Anaeromyxobacter paludicola sp. nov. The type strains are Red232T (=NBRC 114074T=MCCC 1K03954T), Red267T (=NBRC 114075T=MCCC 1K04211T), and Red630T (=NBRC 114076T=MCCC 1K03957T), respectively.

Unexpected absence of ribosomal protein genes from metagenome-assembled genomes.

Metagenome-assembled genomes (MAGs) have revealed the hidden diversity and functions of uncultivated microbes, but their reconstruction from metagenomes remains a computationally difficult task. Repetitive or exogenous sequences, such as ribosomal RNA and horizontally transferred genes, are frequently absent from MAGs because of misassembly and binning errors. Here, we report that ribosomal protein genes are also often absent from MAGs, although they are neither repetitive nor exogenous. Comprehensive analyses of more than 190,000 MAGs revealed that these genes could be missing in more than 20-40% of near-complete (i.e., with completeness of 90% or higher) MAGs. While some uncultivated environmental microbes intrinsically lack some ribosomal protein genes, we found that this unexpected absence is largely due to special evolutionary patterns of codon usage bias in ribosomal protein genes and algorithmic characteristics of metagenomic binning, which is dependent on tetranucleotide frequencies of contigs. This problem reflects the microbial life-history strategy. Fast-growing microbes tend to have this difficulty, likely because of strong evolutionary pressures on ribosomal protein genes toward the efficient assembly of ribosomes. Our observations caution those who study genomics and phylogeny of uncultivated microbes, the diversity and evolution of microbial genes in the central dogma, and bioinformatics in metagenomics.

Undervalued Pseudo-nifH Sequences in Public Databases Distort Metagenomic Insights into Biological Nitrogen Fixers.

Nitrogen fixation, a distinct process incorporating the inactive atmospheric nitrogen into the active biological processes, has been a major topic in biological and geochemical studies. Currently, insights into diversity and distribution of nitrogen-fixing microbes are dependent upon homology-based analyses of nitrogenase genes, especially the nifH gene, which are broadly conserved in nitrogen-fixing microbes. Here, we report the pitfall of using nifH as a marker of microbial nitrogen fixation. We exhaustively analyzed genomes in RefSeq (231,908 genomes) and KEGG (6,509 genomes) and cooccurrence and gene order patterns of nitrogenase genes (including nifH) therein. Up to 20% of nifH-harboring genomes lacked nifD and nifK, which encode essential subunits of nitrogenase, within 10 coding sequences upstream or downstream of nifH or on the same genome. According to a phenotypic database of prokaryotes, no species and strains harboring only nifH possess nitrogen-fixing activities, which shows that these nifH genes are "pseudo"-nifH genes. Pseudo-nifH sequences mainly belong to anaerobic microbes, including members of the class Clostridia and methanogens. We also detected many pseudo-nifH reads from metagenomic sequences of anaerobic environments such as animal guts, wastewater, paddy soils, and sediments. In some samples, pseudo-nifH overwhelmed the number of "true" nifH reads by 50% or 10 times. Because of the high sequence similarity between pseudo- and true-nifH, pronounced amounts of nifH-like reads were not confidently classified. Overall, our results encourage reconsideration of the conventional use of nifH for detecting nitrogen-fixing microbes, while suggesting that nifD or nifK would be a more reliable marker. IMPORTANCE Nitrogen-fixing microbes affect biogeochemical cycling, agricultural productivity, and microbial ecosystems, and their distributions have been investigated intensively using genomic and metagenomic sequencing. Currently, insights into nitrogen fixers in the environment have been acquired by homology searches against nitrogenase genes, particularly the nifH gene, in public databases. Here, we report that public databases include a significant amount of incorrectly annotated nifH sequences (pseudo-nifH). We exhaustively investigated the genomic structures of nifH-harboring genomes and found hundreds of pseudo-nifH sequences in RefSeq and KEGG. Over half of these pseudo-nifH sequences belonged to members of the class Clostridia, which is supposed to be a prominent nitrogen-fixing clade. We also found that the abundance of nitrogen fixers in metagenomes could be overestimated by 1.5 to >10 times due to pseudo-nifH recorded in public databases. Our results encourage reconsideration of the prevalent use of nifH as a marker of nitrogen-fixing microbes.

Environmental Atlas of Prokaryotes Enables Powerful and Intuitive Habitat-Based Analysis of Community Structures.

The recent prevalence of high-throughput sequencing has been producing numerous prokaryotic community structure datasets. Although the trait-based approach is useful to interpret those datasets from ecological perspectives, available trait information is biased toward culturable prokaryotes, especially those of clinical and public health relevance, and thus may not represent the breadth of microbiota found across many of Earth's environments. To facilitate habitat-based analysis free of such bias, here we report a ready-to-use prokaryotic habitat database, ProkAtlas. ProkAtlas comprehensively links 16S rRNA gene sequences to prokaryotic habitats, using public shotgun metagenome datasets. We also developed a computational pipeline for habitat-based analysis of given prokaryotic community structures. After confirmation of the method effectiveness using 16S rRNA gene sequence datasets from individual genomes and the Earth Microbiome Project, we showed its validness and effectiveness in drawing ecological insights by applying it to six empirical prokaryotic community datasets from soil, aquatic, and human gut samples.

Invention of Artificial Rice Field Soil: A Tool to Study the Effect of Soil Components on the Activity and Community of Microorganisms Involved in Anaerobic Organic Matter Decomposition.

Soils are characterized by diverse biotic and abiotic constituents, and this complexity hinders studies on the effects of individual soil components on microorganisms in soil. Although artificial soils have been used to overcome this issue, anoxic soils have not yet been examined. We herein aimed to create artificial soil that reproduces anaerobic methane production by soil from a rice field. Organic materials and mineral particles separated from rice field soil were mixed to prepare an artificial soil matrix; the matrix was added with a small volume of a soil suspension as a microbial inoculum. When the microbial inoculum was added immediately after matrix preparation, anaerobic decomposition was markedly less than that by original soil. When the inoculum was added 9-15 days after soil matrix preparation, anaerobic CO2 and methane production was markedly activated, similar to that by original soil after 40 days of incubation, which suggested that the maturation of the soil matrix was crucial for the reproduction of anaerobic microbial activities. The diversity of the microbial community that developed in artificial soil was markedly less than that in original soil, whereas their predicted functional profiles were similar. Humic substances altered the composition and network patterns of the microbial community. These results suggested that the functional redundancy of soil microorganisms was sustained by different microbial sub-communities. The present study demonstrated that artificial soil is a useful tool for investigating the effects of soil components on microorganisms in anoxic soil.

Eukaryotic Microbial Communities in Japanese Arable Andisols Investigated by Amplicon Sequencing of 18S rRNA Genes.

Compared with the well-studied soil prokaryotic communities, little is known about soil eukaryotic communities. Here, we investigated the eukaryotic community structures in 43 arable soils using amplicon sequencing of 18S rRNA genes. Major taxonomic groups, such as Fungi, Holozoa, and Stramenopiles, were detected in all samples.

Prokaryotic Community Structure of Long-Term Fertilization Field Andisols in Central Japan.

Long-term fertilization experiments are a useful way to elucidate the impacts of fertilization on soil ecosystems. Here, we report the prokaryotic community structure in experimental field soil after 80 years of successive fertilization. Our 16S rRNA gene sequencing detected 20,996 amplicon sequence variants, including major phyla such as Proteobacteria, Acidobacteria, and Actinobacteria.

Phosphorus-mineralizing Communities Reflect Nutrient-Rich Characteristics in Japanese Arable Andisols.

Elucidating the soil phosphorus cycle driven by soil microbes is a vital question in soil microbial ecology. The Japanese arable Andisols, occupying half of the Japanese cropland, are known for their high phosphorus sorption capacity. However, limited information is currently available on microbially driven phosphorus mineralization in arable Andisols. We herein report that the phosphorus-mineralizing community in the Japanese arable Andisols showed characteristic distribution and composition patterns, from those in other types of soils. We performed a chemical analysis and microbial community analysis of 43 arable Andisols along the Japanese archipelago. Soil phosphomonoesterase activities measured at pH 11 were approximately 70% of those at pH 6.5, which indicates that alkaline phosphatase contributes to phosphorus cycling, although most soil samples were acidic. Functional gene predictions based on 16S rRNA gene sequencing indicated that the alkaline phosphatase gene phoD was more abundant than other alkaline phosphatase genes and, thus, plays major roles. Hence, amplicon sequencing targeting phoD was performed and the results obtained showed that alphaproteobacterial phoD was dominant. This is in contrast to previously reported phoD compositions in other soils and may be attributed to the nutrient conditions in arable Andisols, which favor copiotrophic Alphaproteobacteria. Furthermore, the composition of phoD correlated with soil pH and bioavailable phosphorus concentrations rather than carbon or nitrogen concentrations. These results were partly different from previous findings, varying in the soil types and geographic ranges of sampling sites. Collectively, the present results indicate that the phosphorus-mineralizing community in the Japanese arable Andisols is regulated differently from those in other soil types.