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.

Obligate Gut Symbiotic Association with Caballeronia in the Mulberry Seed Bug Paradieuches dissimilis (Lygaeoidea: Rhyparochromidae).

Many insects possess symbiotic bacteria in their bodies, and microbial symbionts play pivotal metabolic roles for their hosts. Members of the heteropteran superfamilies Coreoidea and Lygaeoidea stinkbugs harbor symbionts of the genus Caballeronia in their intestinal tracts. Compared with symbiotic associations in Coreoidea, those in Lygaeoidea insects are still less understood. Here, we investigated a symbiotic relationship involving the mulberry seed bug Paradieuches dissimilis (Lygaeoidea: Rhyparochromidae) using histological observations, cultivation of the symbiont, 16S rRNA gene amplicon sequencing, and infection testing of cultured symbionts. Histological observations and cultivation revealed that P. dissimilis harbors Caballeronia symbionts in the crypts of its posterior midgut. 16S rRNA gene amplicon sequencing of field-collected P. dissimilis confirmed that the genus Caballeronia is dominant in the midgut of natural populations of P. dissimilis. In addition, PCR diagnostics showed that the eggs were free of symbiotic bacteria, and hatchlings horizontally acquired the symbionts from ambient soil. Infection and rearing experiments revealed that symbiont-free aposymbiotic individuals had abnormal body color, small body size, and, strikingly, a low survival rate, wherein no individuals reached adulthood, indicating an obligate cooperative mutualism between the mulberry seed bug and Caballeronia symbionts.

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.

Investigation of Rice Yields and Critical N Losses from Paddy Soil under Different N Fertilization Rates with Iron Application.

The application of iron powder stimulated the growth of iron-reducing bacteria as a respiratory substrate and enhanced their nitrogen (N)-fixing activity in flooded paddy soils. High N fertilization (urea) in the flooded paddy soils has caused adverse environmental impacts such as ammonia (NH3) volatilization, nitrous oxide (N2O) emissions, and nitrate (NO3-) leaching. This study aims to investigate the effects of N fertilization rates in combination with an iron amendment on rice yields and N losses from flooded paddy fields. We performed a 2-year field plot experiment with traditional rice-wheat rotation in China's Yangtze River Delta. The investigation consisted of seven treatments, including 100%, 80%, 60%, and 0% of the conventional N (urea and commercial organic manure) fertilization rate, and 80%, 60%, and 0% of the conventional N with the iron powder (≥99% purity) amendment. The rice yields decreased with a reduction in the conventional N fertilization rate, whereas they were comparable after the iron application under the 80% and 60% conventional N rate. The critical N losses, including NH3 volatilization, N2O emissions, and NO3- and NH4+ leaching, generally decreased with a reduction in the conventional N fertilization rate. These N losses were significantly greater after the iron amendment compared with the non-amended treatments under the 80% and 60% conventional N fertilization rate in the first rice-growing season. However, it was comparable between the iron-amended and the non-amended treatments in the second season. Furthermore, NO3- leaching was the most significant N loss throughout the two rice seasons, followed by NH3 volatilization. The iron amendment significantly increased soil Fe2+ content compared with the non-amended treatments irrespective of N fertilization, suggesting the reduction of amended iron by iron-reducing bacteria and their simultaneous N fixation. A combination of the iron application with 60-80% of the conventional N fertilization rate could maintain rice yields similar to the conventional N fertilization rate while reducing the critical N losses in the flooded paddy field tested in this study. Our study leads to the establishment of novel and practical rice cultivation, which is a step towards the development of green agriculture.

Insecticide resistance by a host-symbiont reciprocal detoxification.

Insecticide resistance is one of the most serious problems in contemporary agriculture and public health. Although recent studies revealed that insect gut symbionts contribute to resistance, the symbiont-mediated detoxification process remains unclear. Here we report the in vivo detoxification process of an organophosphorus insecticide, fenitrothion, in the bean bug Riptortus pedestris. Using transcriptomics and reverse genetics, we reveal that gut symbiotic bacteria degrade this insecticide through a horizontally acquired insecticide-degrading enzyme into the non-insecticidal but bactericidal compound 3-methyl-4-nitrophenol, which is subsequently excreted by the host insect. This integrated "host-symbiont reciprocal detoxification relay" enables the simultaneous maintenance of symbiosis and efficient insecticide degradation. We also find that the symbiont-mediated detoxification process is analogous to the insect genome-encoded fenitrothion detoxification system present in other insects. Our findings highlight the capacity of symbiosis, combined with horizontal gene transfer in the environment, as a powerful strategy for an insect to instantly eliminate a toxic chemical compound, which could play a critical role in the human-pest arms race.

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.

Genome-Based Taxonomic Rearrangement of the Order Geobacterales Including the Description of Geomonas azotofigens sp. nov. and Geomonas diazotrophica sp. nov.

Geobacterales is a recently proposed order comprising members who originally belonged to the well-known family Geobacteraceae, which is a key group in terrestrial ecosystems involved in biogeochemical cycles and has been widely investigated in bioelectrochemistry and bioenergy fields. Previous studies have illustrated the taxonomic structure of most members in this group based on genomic phylogeny; however, several members are still in a pendent or chaotic taxonomic status owing to the lack of genome sequences. To address this issue, we performed this taxonomic reassignment using currently available genome sequences, along with the description of two novel paddy soil-isolated strains, designated Red51T and Red69T, which are phylogenetically located within this order. Phylogenomic analysis based on 120 ubiquitous single-copy proteins robustly separated the species Geobacter luticola from other known genera and placed the genus Oryzomonas (fam. Geobacteraceae) into the family 'Pseudopelobacteraceae'; thus, a novel genus Geomobilimonas is proposed, and the family 'Pseudopelobacteraceae' was emended. Moreover, genomic comparisons with similarity indexes, including average amino acid identity (AAI), percentage of conserved protein (POCP), and average nucleotide identity (ANI), showed proper thresholds as genera boundaries in this order with values of 70%, 65%, and 74% for AAI, POCP, and ANI, respectively. Based on this, the three species Geobacter argillaceus, Geobacter pelophilus, and Geobacter chapellei should be three novel genera, for which the names Geomobilibacter, Geoanaerobacter, and Pelotalea are proposed, respectively. In addition, the two novel isolated strains phylogenetically belonged to the genus Geomonas, family Geobacteraceae, and shared genomic similarity values higher than those of genera boundaries, but lower than those of species boundaries with each other and their neighbors. Taken together with phenotypic and chemotaxonomic characteristics similar to other Geomonas species, these two strains, Red51T and Red69T, represent two novel species in the genus Geomonas, for which the names Geomonas azotofigens sp. nov. and Geomonas diazotrophica sp. nov. are proposed, respectively.

Geomesophilobacter sediminis gen. nov., sp. nov., Geomonas propionica sp. nov. and Geomonas anaerohicana sp. nov., three novel members in the family Geobacterecace isolated from river sediment and paddy soil.

Bacteria in the family Geobacteraceae have been proven to fill important niches in a diversity of anaerobic environments and global biogeochemical processes. Here, three bacterial strains in this family, designated Red875T, Red259T, and Red421T were isolated from river sediment and paddy soils in Japan. All of them are Gram-staining-negative, strictly anaerobic, motile, flagellum-harboring cells that form red colonies on agar plates and are capable of utilizing Fe(III)-NTA, Fe(III) citrate, ferrihydrite, MnO2, fumarate, and nitrate as electron acceptors with acetate, propionate, pyruvate, and glucose as electron donors. Phylogenetic analysis based on the 16S rRNA gene and 92 concatenated core proteins sequences revealed that strains Red259T and Red421T clustered with the type strains of Geomonas species, whereas strain Red875T formed an independent lineage within the family Geobacteraceae. Genome comparison based on  average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values clearly distinguished these three strains from other Geobacteraceae members, with lower values than the thresholds for species delineation. Moreover, strain Red875T also shared low average amino acid identity (AAI) and percentage of conserved proteins (POCP) values with the type species of the family Geobacteraceae. Based on these physiological, chemotaxonomic, and phylogenetic distinctions, we propose that strain Red875T (=NBRC 114290T = MCCC 1K04407T) represents a novel genus in the family Geobacteraceae, namely, Geomesophilobacter sediminis gen. nov., sp. nov., and strains Red259T (=NBRC 114288T = MCCC 1K05016T) and Red421T (=NBRC 114289T = MCCC 1K06216T) represent two novel independent species in the genus Geomonas, namely, Geomonas propionica sp. nov. and Geomonas anaerohicana sp. nov., respectively.

Insecticide resistance governed by gut symbiosis in a rice pest, Cletus punctiger, under laboratory conditions.

Resistance to toxins in insects is generally thought of as their own genetic trait, but recent studies have revealed that gut microorganisms could mediate resistance by detoxifying phytotoxins and man-made insecticides. By laboratory experiments, we here discovered a striking example of gut symbiont-mediated insecticide resistance in a serious rice pest, Cletus punctiger. The rice bug horizontally acquired fenitrothion-degrading Burkholderia through oral infection and housed it in midgut crypts. Fenitrothion-degradation test revealed that the gut-colonizing Burkholderia retains a high degrading activity of the organophosphate compound in the insect gut. This gut symbiosis remarkably increased resistance against fenitrothion treatment in the host rice bug. Considering that many stinkbug pests are associated with soil-derived Burkholderia, our finding strongly supports that a number of stinkbug species could gain resistance against insecticide simply by acquiring insecticide-degrading gut bacteria.

Dual oxidase enables insect gut symbiosis by mediating respiratory network formation.

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.

Worker-dependent gut symbiosis in an ant.

The hallmark of eusocial insects, honeybees, ants, and termites, is division of labor between reproductive and non-reproductive worker castes. In addition, environmental adaption and ecological dominance are also underpinned by symbiotic associations with beneficial microorganisms. Microbial symbionts are generally considered to be maintained in an insect colony in two alternative ways: shared among all colony members or inherited only by a specific caste. Especially in ants, the reproductive caste plays a crucial role in transmission of the symbionts shared among colony members over generations. Here, we report an exceptional case, the worker-dependent microbiota in an ant, Diacamma cf. indicum from Japan. By collecting almost all the individuals from 22 colonies in the field, we revealed that microbiota of workers is characterized by a single dominant bacterium localized at the hindgut. The bacterium belonging to an unclassified member within the phylum Firmicutes, which is scarce or mostly absent in the reproductive castes. Furthermore, we show that the gut symbiont is acquired at the adult stage. Collectively, our findings strongly suggest that the specific symbiont is maintained by only workers, demonstrating a novel pattern of ant-associated bacterial symbiosis, and thus further our understanding of host-microbe interactions in the light of sociobiology.

Geomonas silvestris sp. nov., Geomonas paludis sp. nov. and Geomonas limicola sp. nov., isolated from terrestrial environments, and emended description of the genus Geomonas.

Three bacterial strains, designated Red330T, Red736T and Red745T, were isolated from forest and paddy soils in Japan. Strains Red330T, Red736T and Red745T are flagella-harbouring and strictly anaerobic bacteria forming red colonies. A 16S rRNA gene sequence-based phylogenetic tree showed that all three strains were located in a cluster, including the type strains of Geomonas species, which were recently separated from the genus Geobacter within the family Geobacteraceae. Similarities of the 16S rRNA gene sequences among the three strains and Geomonas oryzae S43T, the type species of the genus Geomonas, were 96.3-98.5 %. The genome-related indexes, average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity, among the three strains and G. oryzae S43T were 74.7-86.8 %, 21.2-33.3 % and 70.4-89.8 %, respectively, which were lower than the species delineation thresholds. Regarding the phylogenetic relationships based on genome sequences, the three strains clustered with the type strains of Geomonas species, which were independent from the type strains of Geobacter species. The distinguishableness of the three isolated strains was supported by physiological and chemotaxonomic properties, with the profile of availability of electron donors and cellular fatty acids composition being particularly different among them. Based on genetic, phylogenetic and phenotypic properties, the three isolates represent three novel independent species in the genus Geomonas, for which the names Geomonas silvestris sp. nov., Geomonas paludis sp. nov. and Geomonas limicola sp. nov. are proposed. The type strains are Red330T (=NBRC 114028T=MCCC 1K03949T), Red736T (=NBRC 114029T=MCCC 1K03950T) and Red745T (=NBRC 114030T=MCCC 1K03951T), respectively.

Diazotrophic Anaeromyxobacter Isolates from Soils.

Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of Anaeromyxobacter (in the class Deltaproteobacteria), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of Anaeromyxobacter in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of Anaeromyxobacter to fix nitrogen. Therefore, we verified the diazotrophy of Anaeromyxobacter based on both genomic and culture-dependent analyses using Anaeromyxobacter sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of nif gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic Geobacter and Pelobacter in the class Deltaproteobacteria contain the minimum set of genes for nitrogenase (nifBHDKEN). These results imply that Anaeromyxobacter species have the ability to fix nitrogen. In fact, Anaeromyxobacter PSR-1 and Red267 exhibited N2-dependent growth and acetylene reduction activity (ARA) in vitro Transcriptional activity of the nif gene was also detected when both strains were cultured with N2 gas as a sole nitrogen source, indicating that Anaeromyxobacter can fix and assimilate N2 gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that Anaeromyxobacter can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of Anaeromyxobacter, which is a common soil bacterium.IMPORTANCEAnaeromyxobacter is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of Anaeromyxobacter from various soil environments. Although the importance of Anaeromyxobacter as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that Anaeromyxobacter fixes nitrogen. This study demonstrates that Anaeromyxobacter harboring nitrogenase genes exhibits diazotrophic ability; moreover, N2-dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for Anaeromyxobacter to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of Anaeromyxobacter, which is a common bacterium in soils.

Patiriisocius marinistellae gen. nov., sp. nov., isolated from the starfish Patiria pectinifera, and reclassification of Ulvibacter marinus as a member of the genus Patiriisocius comb. nov.

A marine strain, designated KK4T, was isolated from the surface of a starfish, Patiria pectinifera, which was collected from seawater off the coast of Hokkaido, Japan. Strain KK4T is a Gram-stain-negative, non-spore-forming, rod-shaped, aerobic bacterium that forms yellow-pigmented colonies. A phylogenetic relationship analysis, based on 16S rRNA gene sequences, revealed that strain KK4T was closely related to Ulvibacter marinus IMCC12008T, Ulvibacter antarcticus IMCC3101T and Ulvibacter litoralis KMM 3912T, with similarities of 96.9, 95.8 and 95.6 %, respectively, but low sequence similarities (<94 %) among other genera in the family Flavobacteriaceae. Genomic similarities between strain KK4T and the three Ulvibacter type strains based on average nucleotide identity and digital DNA-DNA hybridization values were lower than the species delineation thresholds. Moreover, phylogenetic tree based on genome sequences showed that strain KK4T was clustered with U. marinus IMCC12008T and formed a branch independent from the cluster including type species of the genera Ulvibacter, Marixanthomonas, Marinirhabdus, Aureitalea and Aequorivita. Amino acid identity values between strain KK4T/U. marinus IMCC12008T and the neighbour type species/strains were 61.9-68.2% and 61.5-67.4 %, which were lower than the genus delineation threshold, implying the novel genus status of strain KK4T. Strain KK4T growth occurred at pH 6.0-9.0, 4-30 °C and in NaCl concentrations of 0.5-5.0 %, and optimally at pH 7.0, 25 °C and 3.0 %, respectively. Unlike Ulvibacter strains, strain KK4T could assimilate glucose, mannose, galactose and acetate. The major quinone and fatty acids were menaquinone-6 and iso-C15 : 0 (27.5 %), iso-C15 : 1 G (22.5 %) and iso-C17 : 0 3-OH (12.8 %), respectively. Based on genetic, phylogenetic and phenotypic properties, strain KK4T represents a novel species of the genus Patiriisocius, for which the name Patiriisocius marinistellae gen. nov., sp. nov. is proposed. The type strain is KK4T (=JCM 33344T=KCTC 72225T). In addition, based on the current data, Ulvibacter marinus should be reclassified as Patiriisocius marinus comb. nov.

Description of Three Novel Members in the Family Geobacteraceae, Oryzomonas japonicum gen. nov., sp. nov., Oryzomonas sagensis sp. nov., and Oryzomonas ruber sp. nov.

Bacteria of the family Geobacteraceae are particularly common and deeply involved in many biogeochemical processes in terrestrial and freshwater environments. As part of a study to understand biogeochemical cycling in freshwater sediments, three iron-reducing isolates, designated as Red96T, Red100T, and Red88T, were isolated from the soils of two paddy fields and pond sediment located in Japan. The cells were Gram-negative, strictly anaerobic, rod-shaped, motile, and red-pigmented on agar plates. Growth of these three strains was coupled to the reduction of Fe(III)-NTA, Fe(III) citrate, and ferrihydrite with malate, methanol, pyruvate, and various organic acids and sugars serving as alternate electron donors. Phylogenetic analysis based on the housekeeping genes (16S rRNA gene, gyrB, rpoB, nifD, fusA, and recA) and 92 concatenated core genes indicated that all the isolates constituted a coherent cluster within the family Geobacteraceae. Genomic analyses, including average nucleotide identity and DNA-DNA hybridization, clearly differentiated the strains Red96T, Red100T, and Red88T from other species in the family Geobacteraceae, with values below the thresholds for species delineation. Along with the genomic comparison, the chemotaxonomic features further helped distinguish the three isolates from each other. In addition, the lower values of average amino acid identity and percentage of conserved protein, as well as biochemical differences with their relatives, indicated that the three strains represented a novel genus in the family Geobacteraceae. Hence, we concluded that strains Red96T, Red100T, and Red88T represented three novel species of a novel genus in the family Geobacteraceae, for which the names Oryzomonas japonicum gen. nov., sp. nov., Oryzomonas sagensis sp. nov., and Oryzomonas ruber sp. nov. are proposed, with type strains Red96T (= NBRC 114286T = MCCC 1K04376T), Red100T (= NBRC 114287T = MCCC 1K04377T), and Red88T (= MCCC 1K03694T = JCM 33033T), respectively.

Host-symbiont specificity determined by microbe-microbe competition in an insect gut.

Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.

Geomonas oryzae gen. nov., sp. nov., Geomonas edaphica sp. nov., Geomonas ferrireducens sp. nov., Geomonas terrae sp. nov., Four Ferric-Reducing Bacteria Isolated From Paddy Soil, and Reclassification of Three Species of the Genus Geobacter as Members of the Genus Geomonas gen. nov.

In paddy soil, bacteria from the family Geobacteraceae have been shown to strongly contribute to the biogeochemical cycle. However, no Geobacteraceae species with validly published names have been isolated from paddy soil. In this study, we isolated and characterized four novel ferric reducing bacteria in the family Geobacteraceae from the paddy soils of three different fields in Japan. The four strains, S43T, Red53T, S62T, and Red111T, were Gram-stain negative, strictly anaerobic, chemoheterotrophic, and motile with peritrichous flagella. Phylogenetic studies based on 16S rRNA gene sequences, five concatenated housekeeping genes (fusA, rpoB, recA, nifD, and gyrB) and 92 concatenated core genes revealed that the four strains belong to the family Geobacteraceae and are most closely related to Geobacter bemidjiensis BemT (97.4-98.2%, 16S rRNA gene sequence similarities) and Geobacter bremensis Dfr1T (97.1-98.0%). Genomic analysis with average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) calculations clearly distinguished the four isolated strains from other species of the family Geobacteraceae and indicated that strains S43T, Red53T, S62T, and Red111T represent independent species, with values below the thresholds for species delineation. Chemotaxonomic characteristics, including major fatty acid and whole cell protein profiles, showed differences among the isolates and their closest relatives, which were consistent with the results of DNA fingerprints and physiological characterization. Additionally, each of the four isolates shared a low 16S rRNA gene sequence similarity (92.4%) and average amino acid identity (AAI) with the type strain of the type species Geobacter metallireducens. Overall, strains S43T, Red53T, S62T, and Red111T represent four novel species, which we propose to classify in a novel genus of the family Geobacteraceae, and the names Geomonas oryzae gen. nov., sp. nov. (type strain S43T), Geomonas edaphica sp. nov. (type strain Red53T), Geomonas ferrireducens sp. nov. (type strain S62T), and Geomonas terrae sp. nov. (type strain Red111T) are proposed. Based on phylogenetic and genomic analyses, we also propose the reclassification of Geobacter bremensis as Geomonas bremensis comb. nov., Geobacter pelophilus as Geomonas pelophila comb. nov., and Geobacter bemidjiensis as Geomonas bemidjiensis comb. nov.

Burkholderia Gut Symbionts Associated with European and Japanese Populations of the Dock Bug Coreus marginatus (Coreoidea: Coreidae).

Insects of the heteropteran superfamilies Coreoidea and Lygaeoidea are consistently associated with symbionts of a specific group of the genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group. The symbiosis is maintained by the environmental transmission of symbionts. We investigated European and Japanese populations of the dock bug Coreus marginatus (Coreoidea: Coreidae). High nymphal mortality in reared aposymbiotic insects suggested an obligate host-symbiont association in this species. Molecular phylogenetic analyses based on 16S rRNA gene sequences revealed that all 173 individuals investigated were colonized by Burkholderia, which were further assigned to different subgroups of the SBE in a region-dependent pattern.