The soil microbial communities in two Japanese paddy fields were compared: site PA, which emits methane at the domestic average level, and site PX, which emits more than five times that amount. The transcription levels of methyl-coenzyme M reductase (mcrA) significantly increased during peak methane emissions at site PX, but not at site PA. The anaerobic methanotroph “Candidatus Methanoperedens” exhibited low activity and abundance exclusively at site PX. The genus Methanoregula was the most active methanogen at both sites; however, the dominant methanotrophs differed, with Methylocystis dominating at site PA and Methylomonas at site PX.

Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms stem and root nodules on Sesbania rostrata. All tested A. caulinodans strains exhibited degradation activity against the quorum-sensing signaling compounds, N-acyl-l-homoserine lactones (AHL). The AHL-degrading gene homolog, attM, was identified in the genome sequences of A. caulinodans MAFF 210031^T and other A. caulinodans strains. Recombinant AttM functions as an AHL lactonase that hydrolyzes the lactone bond of AHL and retains its stable activity at environmental temperatures. The AHL-degrading activity of the attM-deletion mutant was completely diminished, which revealed that AHL degradation by MAFF 210031 was dependent on attM.

Recent warming and glacier retreat in the Svalbard Archipelago, part of the Arctic cryosphere, have become increasingly evident. The present study investigated the foreland of Austre Brøggerbreen near Ny-Ålesund to clarify how soil nitrification responds to changing conditions. Two sites, exposed for different periods following glacier retreat, were compared. A manipulation experiment using open-top chambers (OTCs) and homogenized initial soil conditions was conducted to assess the effects of site differences, the OTC treatment, soil depth, and interannual variations on soil and nitrification properties. Although the OTC treatment slightly increased soil temperature and moisture, its overall effect on soil properties, ammonia oxidation potential (AOP), and microbial properties was negligible. In contrast, homogenization markedly increased total nitrogen at both sites and temporarily boosted AOPs for two years before levels returned to baseline. Site 2, exposed for longer, contained more soil nitrogen and showed higher AOP than Site 1. For example, in 2015, AOPs at 10°C were 3.5 and 2.4‍ ‍ng N g^–1 dry soil h^–1 at Sites 2 and 1, respectively. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were both more abundant at Site 2, although AOB clearly dominated at both sites. While AOB-amoA operational taxonomic units (OTUs) were mostly shared between sites, community compositions differed: OTU2 was prevalent at Site 1, but minor at Site 2. OTU2 may act as a pioneer taxon that declines in later stages, or its pattern may reflect site-specific soil conditions. A phylogenetic anal­ysis showed that OTU2 and OTU3 belonged to Cluster ME found near Mount Everest.

Agricultural soils are an important source of nitrous oxide (N₂O), which has greenhouse and ozone-depleting effects. Bradyrhizobium ottawaense SG09 is a nitrogen-fixing rhizobium with high N₂O-reducing activity. Rhizobia form symbiotic nodules in leguminous plants. The initial physical attachment of bacteria to plant roots is a critical step in the establishment of symbiotic interactions. In the present study, we performed a microscopic anal­ysis using DsRed-expressing B. ottawaense SG09. We revealed that B. ottawaense SG09 attached to both the root surface and root hairs via single cellular poles. This polar attachment was observed not only to the symbiotic host soybean, but also to non-leguminous plants, such as Arabidopsis, rice, corn, and wheat. We identified and analyzed the unipolar polysaccharide (upp) gene cluster, which is proposed to be involved in the polar attachment of rhizobia, in the genome of B. ottawaense SG09. We established an Arabidopsis-based interaction assay and demonstrated that uppC and uppE play a critical role in attachment to both the root surface and root hairs.

The standardization of strain identification methods is essential for effectively managing the genetic resources of arbuscular mycorrhizal (AM) fungi. Due to their highly polymorphic rRNA sequence and multinucleate nature, conventional strain identification using a single sequence of the rRNA region is often inadequate for these fungi. Therefore, the present study exami­ned the use of genetic diversity information obtained through high-throughput sequencing to improve the strain identification of AM fungal isolates. Five previously reported primer pairs were used to amplify a portion of the rRNA region from DNA extracted from AM fungal spores, which was then sequenced using the Illumina MiSeq platform. The majority of amplicon sequence variants (ASVs) matched the same strain as the source isolate. A cluster anal­ysis indicated that strains of the same species generally grouped together, demonstrating the method’s effectiveness for species-level identification. Furthermore, a phylogenetic anal­ysis revealed some strain-specific ASVs that may be valuable for differentiating between different strains within the same species. Based on these results, it is feasible to develop a reliable identification protocol for AM fungal isolates using MiSeq sequencing.

Several enzymes have subunits that require the addition of cofactors or maturation of the active center, which is facilitated by other genes. Information on these functionally-related genes not only aids in the functional anal­ysis of target genes, but is also useful for heterologous expression. In the present study, we analyzed the homologs of a target gene and their relationships with adjacent genes within the genome by constructing clusters of neighboring genes, quantifying the number of clustered genes, and examining their conservation in a taxonomic clade of target gene homologs. [NiFe]-hydrogenase was selected as the target because of the availability of a concrete database for subsequent evaluations in our anal­ysis. The present results indicate that genes associated with target gene function were conserved according to the molecular phylogeny of the target gene. We subsequently introduced automated clustering of the phylogenetic tree clade of clustered genes and applied this method to large datasets not yet analyzed and our previous data. The results obtained suggest that this approach provides insights into a comprehensive set of genes involved in cellular functions, particularly when the genes being analyzed are complex and require maturation. The procedure developed herein also provided similar and reproducible results on previously analyzed succinate dehydrogenase, which was not arbitrary.

We herein exami­ned the effects of soil flooding treatments on the occupancy of adzuki bean-nodulated bradyrhizobia and how changes in its occupancy affects adzuki bean growth. Microcosms containing mixtures of four different strains were prepared and incubated under flooded and non-flooded conditions. These microcosms were then used to cultivate adzuki bean in culture pots. After assessing the growth parameters of the plants, nodules collected from the roots were analyzed to assess occupancy rates. Be31, which exhibited a similar restriction fragment length polymorphism (RFLP) pattern in the 16S–23S rRNA gene ITS region to Bradyrhizobium elkanii USDA 31, was dominant overall. However, its occupancy declined under flooded conditions, while the occupancy of Bd110, similar to Bradyrhizobium diazoefficiens USDA 110^T, increased. Furthermore, a non–metric multidimensional scaling anal­ysis showed that adzuki bean-nodulated bradyrhizobial communities were affected by changes in Bd110 and Be31 occupancies due to soil flooding. In terms of growth, shoot length and shoot dry weight generally increased in cultivars where Bd110 occupancy surpassed that of Be31 under flooding. A correlation anal­ysis revealed that Bd110 occupancy correlated with shoot dry weight. These results suggest that increased Bd110 occupancy through soil flooding enhanced adzuki bean growth. However, alternative methods need to be considered in order to more effectively regulate Be31 occupancy.

The anammox process using marine anammox bacteria is a promising nitrogen removal process for recirculating aquaculture system wastewater. Marine anammox bacteria are typically found in oxygen-deficient zones and coastal areas under low phosphate concentrations. The optimal phosphate concentration for marine anammox bacteria remains unknown because most laboratory studies on these bacteria have been conducted under high phosphate concentrations. Therefore, the present study investigated the long-term effects of varying phosphate concentrations on the marine anammox bacteria, Candidatus Scalindua sp., to identify the optimal range of phosphate. Anammox activity and average growth rates were evaluated under seven phosphate concentrations (0, 0.23, 0.46, 0.68, 1.14, 6.15 [control], and 15.48‍ ‍mg P L^–1) over a period of 70 days. After 50 days of reactor operation, reactor performance under phosphate concentrations ranging from 0.23 to 6.15‍ ‍mg P L^–1 stabilized at 70% of total nitrogen removal efficiency, indicating the successful establishment of the anammox process. Conversely, anammox reactor performance under conditions without phosphate addition (0‍ ‍mg P L^–1) and the highest phosphate concentration (15.48‍ ‍mg P L^–1) did not reach 70% of total nitrogen removal efficiency, indicating a suboptimal phosphate concentration for normal anammox activity. Average growth rates calculated from total biomass samples varied from 0.0006 to 0.0012 h^–1. These results indicate that Ca. Scalindua need to be kept at phosphate concentrations between 0.23 and 6.15‍ ‍mg P L^–1 for optimal functioning in wastewater treatment ecosystems.

Since nitrogenase is intrinsically sensitive to oxygen (O₂), diverse aerobic diazotrophs need strategies to cope with nitrogenase damage by O₂. In the present study, we investigated the mechanisms by which aerobic methane-oxidizing bacteria (methanotrophs) enable the concurrent activities of methane monooxygenase, which uses O₂, and nitrogenase in the cytoplasm of the same cell. By using ¹⁵N labeling, we confirmed the capacity of alphaproteobacterial methanotroph Methylosinus sp. 3S-1 for nitrogen fixation and diazotrophic growth across a wide range of O₂ concentrations <20%. When the initial O₂ concentration was increased from 2 to 20% in a diazotrophic culture, similar decreases were observed in fixed nitrogen and NifH protein levels. In contrast, the mRNA levels of nitrogen fixation genes (nif genes) markedly increased and remained elevated for the duration of slow growth at high O₂ concentrations. This pattern of nif expression in response to O₂ may be attributed to the properties of the nif-specific transcriptional regulator NifA. The present results suggest that the increase in nif transcription is one of the strategies by which this methanotroph maintains nitrogen fixation on the background of aerobic methane oxidation.

Legionella survive in the natural environment by remaining within protist host cells. Many protist species, including Paramecium spp., are potential hosts for Legionella. However, the factors and mechanisms involved in the establishment of this relationship are unknown. The advantages gained by Paramecium spp. when they maintain Legionella are also unclear, and the existence of these relationships has not been confirmed. In the present study, feeding with Legionella increased the number of Paramecium cells over time. However, the growth-promoting effect of Legionella was weaker than that of Klebsiella pneumoniae, which is considered the optimal bacterial feed for Paramecium. Phagocytosis was strongly inhibited in Paramecium cells fed Legionella, indicating that this relationship prevents the uptake of harmful organisms. The inhibition of phagocytosis was also observed when Paramecium cells were treated with the Legionella culture supernatant. Despite the inhibition of phagocytosis, the presence of live Legionella within host cells allowed Paramecium spp. to survive and even increase in number, as observed earlier. This result suggests that Legionella support the survival of Paramecium hosts from a nutritional aspect. Although it is difficult to definitively state whether the relationship between Legionella and Paramecium hosts is completely mutualistic, the present results provide one rationale for defining their relationship.

Leguminous plants establish root nodule symbiosis, which is initiated by the recognition of rhizobial nodulation factors by plant receptor kinases. However, other factors, such as Type III effector proteins, also affect host specificity. We herein investigated the role of nodulation outer protein M (NopM), a Type III effector of Bradyrhizobium elkanii USDA61, in symbiosis with Lotus japonicus MG-20 and Lotus burttii. NopM, annotated as an E3 ubiquitin ligase, triggers an early senescence-like response, inducing brown nodules that hinder effective symbiosis. NopM shares structural features with E3 ubiquitin ligases derived from both pathogenic and symbiotic bacteria, including a leucine-rich-repeat and E3 ubiquitin ligase domain. The deletion of these domains or substitution of the cysteine residue, predicted to be the active site of the ubiquitin ligase domain, suppressed the formation of brown nodules. These results suggest that NopM interacts with target proteins through its leucine-rich-repeat domain and mediates ubiquitination via its ligase domain, thereby contributing to the induction of brown nodules. A transcriptome ana­lysis further suggested that the early senescence-like response closely resembled the plant hypersensitive response, with the up-regulation of defense-related genes. Therefore, L. japonicus may recognize NopM in infected nodule cells, leading to an immune response that disrupts symbiosis. The present study provides insights into the mole­cular mechanisms by which rhizobial effectors modulate symbiotic interactions in infected nodule cells, highlighting the ability of L. japonicus to activate immune responses even in nodule cells where rhizobia have been accepted.

Flooded rice fields are a major source of atmospheric methane, a strong greenhouse gas second only to carbon dioxide. Rice roots are one of the most important hotspots for methane oxidation in rice fields. However, limited information is available on the physiological and genomic characteristics of methane-oxidizing bacteria (MOB) inhabiting rice roots. In the present study, we isolated MOB from rice roots and characterized the strains phenotypically and genomically. We obtained 100 MOB-enriched cultures from the roots of three rice cultivars (Oryza sativa L. subsp. japonica cv. Nipponbare, O. sativa L. subsp. indica cv. Muha, and Tupa 121-3), in which twelve MOB isolates, two Methylomonas sp., three Methylocystis sp., and seven Methylosinus sp., were successfully purified. They showed different morphological features (types of flagellation) and colony formation potentials within the same group in some cases. A genome sequencing ana­lysis revealed variations in the number of genes or the clusters of methane monooxygenase, methanol dehydrogenase, and nitrogenase. The number of plasmid DNAs also differed among the strains. Four strains belonging to the genus Methylomonas or Methylocystis represented putative novel species based on their phenotypic and genotypic characteristics. The present study largely expanded the eco-collection of MOB cultures inhabiting rice fields and rice roots.