Biological nitrogen fixation in the long-term nitrogen-fertilized and unfertilized paddy fields, with special reference to diazotrophic iron-reducing bacteria.

Biological nitrogen fixation (BNF) is important to sustain nitrogen fertility of paddy soil and rice yield, while could be affected by nitrogen fertilization. Iron-reducing bacteria, Anaeromyxobacter and Geobacter, are newly found diazotrophic bacteria predominant in paddy soil. Experimental field of this study is a long-term (35 years) nitrogen fertilized (6.0 g N/m2/year) and unfertilized paddy field, where ca. 70% of rice yield was obtained yearly in nitrogen unfertilized plot (443 ± 37 g/m2) compared to fertilized plot (642 ± 64 g/m2). Effects of long-term nitrogen fertilization/unfertilization on soil properties related to BNF were investigated with special reference to diazotrophic iron-reducing bacteria. Soil chemical/biochemical properties, soil nitrogen-fixing activity, and community composition of diazotrophic bacteria were similar between nitrogen fertilized and unfertilized plot soils. In both plot soils, Anaeromyxobacter and Geobacter were the most predominant diazotrophs. Their nifD transcripts were detected at similar level, while those of other general diazotrophs were under detection limit. It was concluded that long-term use/unuse of nitrogen fertilizer in this field did not affect the predominance and nitrogen-fixing activity of diazotrophic iron-reducing bacteria, composition of other general diazotrophs, and the resulting soil nitrogen-fixing activity. BNF, primarily driven by diazotrophic iron-reducing bacteria, might significantly contribute to sustain soil nitrogen fertility and rice yield in both plot soils. Appropriate soil management to maintain BNF, including diazotrophic iron-reducing bacteria, will be important for sustainable soil nitrogen fertility and rice production.

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.

Mitigation of N2O emission from granular organic fertilizer with alkali- and salt-resistant plant growth-promoting rhizobacteria.

AIM: Organic fertilizer application significantly stimulates nitrous oxide (N2O) emissions from agricultural soils. Plant growth-promoting rhizobacteria (PGPR) strains are the core of bio-fertilizer or bio-organic fertilizer, while their beneficial effects are inhibited by environmental conditions, such as alkali and salt stress observed in organic manure or soil. This study aims to screen alkali- and salt-resistant PGPR that could mitigate N2O emission after applying strain-inoculated organic fertilizer. METHODS AND RESULTS: Among the 29 candidate strains, 11 (7 Bacillus spp., 2 Achromobacter spp., 1 Paenibacillus sp., and 1 Pseudomonas sp.) significantly mitigated N2O emissions from the organic fertilizer after inoculation. Seven strains were alkali tolerant (pH 10) and five were salt tolerant (4% salinity) in pure culture. Seven strains were selected for further evaluation in two agricultural soils. Five of these seven strains could significantly decrease the cumulative N2O emissions from Anthrosol, while six could significantly decrease the cumulative N2O emissions from Cambisol after the inoculation into the granular organic fertilizer compared with the non-inoculated control. CONCLUSIONS: Inoculating alkali- and salt-resistant PGPR into organic fertilizer can reduce N2O emissions from soils under microcosm conditions. Further studies are needed to investigate whether these strains will work under field conditions, under higher salinity, or at different soil pH.

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.

Genome evolution related to γ-hexachlorocyclohexane metabolic function in the soil microbial population.

γ-Hexachlorocyclohexane (γ-HCH)-degrading strain, Sphingobium sp. TA15, was newly isolated from an experimental field soil from which the archetypal γ-HCH-degrading strain, S. japonicum UT26, was isolated previously. Comparison of the complete genome sequences of these 2 strains revealed that TA15 shares the same basic genome backbone with UT26, but also has the variable regions that are presumed to have changed either from UT26 or from a putative common ancestor. Organization and localization of lin genes of TA15 were different from those of UT26. It was inferred that transposition of IS6100 had played a crucial role in these genome rearrangements. The accumulation of toxic dead-end products in TA15 was lower than in UT26, suggesting that TA15 utilizes γ-HCH more effectively than UT26. These results suggested that genome evolution related to the γ-HCH metabolic function in the soil microbial population is ongoing.

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.

Potentially Mobile Denitrification Genes Identified in Azospirillum sp. Strain TSH58.

Denitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested potential occurrences of horizontal gene transfer (HGT) for the denitrification genes. However, evidence of HGT has not been clearly presented thus far. In this study, we identified the sequences and the localization of the nitrite reductase genes in the genomes of 41 denitrifying Azospirillum sp. strains and searched for mobile genetic elements that contain denitrification genes. All Azospirillum sp. strains examined in this study possessed multiple replicons (4 to 11 replicons), with their sizes ranging from 7 to 1,031 kbp. Among those, the nitrite reductase gene nirK was located on large replicons (549 to 941 kbp). Genome sequencing showed that Azospirillum strains that had similar nirK sequences also shared similar nir-nor gene arrangements, especially between the TSH58, Sp7T, and Sp245 strains. In addition to the high similarity between nir-nor gene clusters among the three Azospirillum strains, a composite transposon structure was identified in the genome of strain TSH58, which contains the nir-nor gene cluster and the novel IS6 family insertion sequences (ISAz581 and ISAz582). The nirK gene within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing the nir-nor gene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely related Azospirillum sp. denitrifiers.IMPORTANCE The evolutionary history of denitrification is complex. While the occurrence of horizontal gene transfer has been suggested for denitrification genes, most studies report circumstantial evidences, such as disagreement between denitrification gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses of Azospirillum sp. denitrifiers, we identified denitrification genes, including nirK and norCBQD, located on a mobile genetic element in the genome of Azospirillum sp. strain TSH58. The nirK was actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely benefitted by acquiring denitrification genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge regarding the ecology and evolution of denitrification.

Noviherbaspirillum denitrificans sp. nov., a denitrifying bacterium isolated from rice paddy soil and Noviherbaspirillum autotrophicum sp. nov., a denitrifying, facultatively autotrophic bacterium isolated from rice paddy soil and proposal to reclassify Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.

Thirty-nine denitrifying bacterial strains closely related to one another, represented by strains TSA40T and TSA66T, were isolated from rice paddy soils. Strains TSA40T and TSA66T were Gram-stain-negative, slightly curved rod-shaped, and motile by means of polar flagella. They were able to reduce nitrate, nitrite and nitrous oxide, but unable to fix atmospheric N2. While strain TSA66T was able to grow autotrophically by H2-dependent denitrification, strain TSA40T could not. Phylogenetic analysis suggested that they belong to the family Oxalobacteraceae, the order Burkholderiales in the class Betaproteobacteria. Major components in the fatty acids (C16 : 0, C17 : 0 cyclo, C18 : 1ω7c and summed feature 3) and quinone (Q-8) also supported the affiliation of strains TSA40T and TSA66T to the family Oxalobacteraceae. Based on 16S rRNA gene sequence comparisons, strains TSA40T and TSA66T showed the greatest degree of similarity to Herbaspirillum massiliense JC206T, Noviherbaspirillum malthae CC-AFH3T, Noviherbaspirillum humi U15T, Herbaspirillum seropedicae Z67T and Paucimonas lemoignei LMG 2207T, and lower similarities to the members of other genera. Average nucleotide identity values between the genomes of strain TSA40T, TSA66T and H. massiliense JC206T were 75-77 %, which was lower than the threshold value for species discrimination (95-96 %). Based on the 16S rRNA gene sequence analysis in combination with physiological, chemotaxonomic and genomic properties, strains TSA40T (=JCM 17722T=ATCC TSD-69T) and TSA66T (=JCM 17723T=DSM 25787T) are the type strains of two novel species within the genus Noviherbaspirillum, for which the names Noviherbaspirillum denitrificans sp. nov. and Noviherbaspirillum autotrophicum sp. nov. are proposed, respectively. We also propose the reclassification of Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.

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.

Brevifollis gellanilyticus gen. nov., sp. nov., a gellan-gum-degrading bacterium of the phylum Verrucomicrobia.

The taxonomic properties of strain DC2c-G4(T), a Gram-staining-negative, ovoid, gellan-gum-degrading bacterial isolate, were examined. Phylogenetic analysis based on 16S rRNA gene sequences identified this isolate as a member of the phylum Verrucomicrobia and closest to the genus Prosthecobacter. The 16S rRNA gene sequence similarities between this isolate and any of the type strains of species of the genus Prosthecobacter were less than 95 %. In addition, the absence of a single prostheca and the predominant menaquinone MK-7(H2) supported the differentiation of this isolate from the genus Prosthecobacter. Here, we propose Brevifollis gellanilyticus gen. nov., sp. nov. to accommodate the isolate. The type strain of the type species is DC2c-G4(T) (= NBRC 108608(T) = CIP 110457(T)).

Roseimicrobium gellanilyticum gen. nov., sp. nov., a new member of the class Verrucomicrobiae.

The taxonomic properties of strain DC2a-G7(T), a Gram-negative, ovoid to rod-shaped, gellan gum-lysing bacterium, were examined. The 16S rRNA gene sequence similarity showed that DC2a-G7(T) is a member of the phylum Verrucomicrobia and the closest type strain of a species with a validly published name is Verrucomicrobium spinosum DSM 4136(T), with a sequence similarity of 91.2%. In addition to this similarity value lower than 95%, the absence of prostheca, the orangey-red colony colour and the compositions of the major menaquinones and polar lipids also supported the differentiation of this bacterium from the genus Verrucomicrobium. Here, we propose the name Roseimicrobium gellanilyticum gen. nov., sp. nov. for the isolate. The type strain of Roseimicrobium gellanilyticum is DC2a-G7(T) (=NBRC 108606(T)=DSM 25532(T)).

Complete genome sequence of the denitrifying and N2O-reducing bacterium Azoarcus sp. strain KH32C.

We report the finished and annotated genome sequence of a denitrifying and N(2)O-reducing betaproteobacterium, Azoarcus sp. strain KH32C. The genome is composed of one chromosome and one megaplasmid and contains genes for plant-microbe interactions and the gene clusters for aromatic-compound degradations.

Mitigation of Paddy Field Soil Methane Emissions by Betaproteobacterium Azoarcus Inoculation of Rice Seeds.

Paddy fields are a major source of atmospheric methane, a greenhouse gas produced by methanogens and consumed by methanotrophs in flooded soil. The inoculation of rice seeds with the bacterium Azoarcus sp. KH32C alters the rice root-associated soil bacterial community composition. The present study investigated the effects of KH32C-inoculated rice cultivation on soil methanogens and methanotrophs involved in methane emissions from a rice paddy field. KH32C-inoculated and non-inoculated rice (cv. Nipponbare) were cultivated in a Japanese rice paddy with and without nitrogen fertilizer. Measurements of methane emissions and soil solution chemical properties revealed increases in methane flux over the waterlogged period with elevations in the concentrations of dissolved methane, dissolved organic carbon, and ferrous iron, which is an indicator of soil reduction levels. Reverse transcription quantitative PCR and amplicon sequencing were used to assess the transcription of the methyl-coenzyme M reductase gene (mcrA) from methanogens and the particulate methane monooxygenase gene (pmoA) from methanotrophs in paddy soil. The results obtained showed not only the transcript copy numbers, but also the compositions of mcrA and pmoA transcripts were related to methane flux. KH32C-inoculated rice cultivation recruited soil methanogens and methanotrophs that suppressed high methane synthesis, increased methane consumption, and decreased methane emissions by 23.5 and 17.2% under non-fertilized and nitrogen-fertilized conditions, respectively, while maintaining rice grain yield. The present study demonstrated the mitigation of paddy field methane emissions arising from the use of KH32C in rice cultivation due to its influence on the compositions of soil methanogen and methanotroph populations.

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.

Simultaneous Nitrogen Removal and Plant Growth Promotion Using Salt-|tolerant Denitrifying Bacteria in Agricultural Wastewater.

Excess nitrate (NO3-) and nitrite (NO2-) in surface waters adversely affect human and environmental health. Bacteria with the ability to remove nitrogen (N) have been isolated to reduce water pollution caused by the excessive use of N fertilizer. To obtain plant growth-promoting rhizobacteria (PGPR) with salt tolerance and NO3--N removal abilities, bacterial strains were isolated from plant rhizosphere soils, their plant growth-promoting effects were evaluated using tomato in plate assays, and their NO3--N removal abilities were tested under different salinity, initial pH, carbon source, and agriculture wastewater conditions. The results obtained showed that among the seven strains examined, five significantly increased the dry weight of tomato plants. Two strains, Pseudomonas stutzeri NRCB010 and Bacillus velezensis NRCB026, showed good plant growth-promoting effects, salinity resistance, and NO3--N removal abilities. The maximum NO3--N removal rates from denitrifying medium were recorded by NRCB010 (90.6%) and NRCB026 (92.0%) at pH 7.0. Higher NO3--N removal rates were achieved using glucose or glycerin as the sole carbon source. The total N (TN) removal rates of NRCB010 and NRCB026 were 90.6 and 66.7% in farmland effluents, respectively, and 79.9 and 81.6% in aquaculture water, respectively. These results demonstrate the potential of NRCB010 and NRCB026 in the development of novel biofertilizers and their use in reducing N pollution in water.

Complete genome sequence of the denitrifying and N(2)O-reducing bacterium Pseudogulbenkiania sp. strain NH8B.

Pseudogulbenkiania sp. strain NH8B is a Neisseriales bacterium isolated from an agricultural field. This strain has strong denitrification and N(2)O reduction activities. Here, we report the finished and annotated genome sequence of this organism.

Isolation of oligotrophic denitrifiers carrying previously uncharacterized functional gene sequences.

Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N(2)O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.

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.