Chironomids (Diptera; Chironomidae), non-biting midges, are a highly diverse family of holometabolous insects, many of which are known for their tolerance to extreme environmental conditions, such as desiccation, pollution, and high acidity. The contribution of microbial symbionts to these adaptations was recently suggested. Therefore, we herein exami­ned the microbiome associated with the larvae of the undescribed acid-tolerant chironomid species, Polypedilum sp., which inhabits the Yukawa River (Gunma, Japan), an environment that is characterized by an extremely low pH (≤2) and high concentrations of heavy metal ions (including arsenic). Amplicon sequencing of the 16S rRNA gene revealed a distinct larval microbiome with a lower alpha diversity value and more enriched and specific bacterial taxa than the surrounding river water and detritus. Full-length 16S rRNA gene sequencing using nanopore long-read technology identified several previously undescribed operational taxonomic units (OTUs), among which OTU_Bacillaceae_Yukawa was consistently present in larvae reared in the laboratory for more than 4 months, suggesting persistent, possibly vertically transmitted, symbiosis. An inferred pathway ana­lysis suggested the contribution of the larval microbiome to host nutritional physiology. The possibly acid-sensitive OTU_Bacillaceae_Yukawa localized to midgut segments, indicating internal pH-buffered niches for microbial survival. These results provide novel insights into the ecology of acid-tolerant chironomids and lay the groundwork for further examinations of holobiont-based stress tolerance.

Marine anammox bacteria have been an exciting research area in recent years due to their high effectiveness in treating ammonia-containing saline wastewater. However, their direct implementation in the wastewater industry faces challenges due to slow growth, difficulty obtaining pure cultures, and their tendency to exist as part of an anammox consortium, interacting symbiotically with other bacteria. In the present study, 91 draft genome metagenome-assembled genomes (MAGs) from a long-term-operated reactor were recovered to clarify detailed symbiotic interactions within an anammox consortium. One marine anammox bacterial MAG, identified as Candidatus Scalindua, was successfully recovered and was abundant within the sampled microbial community. A comprehensive metabolic pathway ana­lysis revealed that Ca. Scalindua exhibited the complete anammox pathway and the Wood–Ljungdahl pathway for carbon fixation. The folate biosynthesis pathway in Ca. Scalindua was incomplete, lacking dihydrofolate reductase, a key enzyme for tetrahydrofolate (THF) production. The folate biopterin transporter, essential for transporting folate-related metabolites among coexisting bacteria, was identified exclusively in Ca. Scalindua. In addition, the impact of exogenously supplied THF on microbial activity and carbon uptake rates was investigated in batch experiments using ¹⁴C-labeled bicarbonate. The results obtained revealed that 2‍ ‍mg L^–1 of exogenous THF resulted in a 43% increase in the carbon uptake rate, while anammox activity remained unaffected. The present results suggest that THF is a key intermediate for carbon fixation in Ca. Scalindua and may be essential for their growth.

Nitrite is a key intermediate in global nitrogen cycles. It has been widely recognized that the accumulation of nitrite is often not appreciable in environments, and nitrite concentrations in canonical media for the cultivation of nitrite-oxidizing bacteria (NOB) in laboratories may not be low enough to recover oligotrophic NOB. We herein report the isolation, physiology, and genomics of oligotrophic NOB from a Japanese forest soil. NOB in soil samples were enumerated using the most probable number method with a medium containing urea for enriching oligotrophic NOB. Urea was completely converted into nitrate, and nitrite was not detected in any nitrifier-positive tubes cultivated after 9‍ ‍weeks of incubation. After subculturing NOB several times in a medium supplemented with 1‍ ‍mM nitrite and performing the extinction-dilution procedure, a novel strain oxidizing nitrite to nitrate was obtained and designated as strain CN101, which was affiliated with the genus Nitrobacter at the 16S rRNA gene level. The half-saturation constant of strain CN101 was lower than other known Nitrobacter strains, suggesting that Nitrobacter strains do not always exhibit low affinity for nitrite. The complete genome of strain CN101 included a larger number of nitrite/nitrate transporters than other Nitrobacter strains, which may serve as tools for flexibly adapting to varying nitrite concentrations in soils. Therefore, the physiological and genomic characteristics of strain CN101 will expand knowledge of the ecologically important but understudied genus Nitrobacter.

Over the past 10 centuries, sake brewing methods have been developed in stages, including doburoku, mizumoto, kimoto, yamahaimoto, and sokujyomoto. Mizumoto-sake is considered the oldest prototype. The brewing process involves lactic acid fermentation and multiple parallel saccharification and alcoholic fermentation by indigenous microbes, which has been operated based on a sense of craftsmanship. The processes involved lead to the creation of extreme conditions characterized by low pH levels and high alcohol concentrations. The characteristic feature of mizumoto-sake is that it begins with fermentation by indigenous lactic acid bacteria to produce acidic water for yeasts to ferment alcohol by inhibiting the growth of undesirable microbes. In the present study, we investigated changes in the microbial community and the transition of metabolites that affect taste and flavor during processes from the initiation of mizumoto-sake brewing to the final product. In the lactic acid fermentation phase, bacteria, including those in the genera Lactococcus, Leuconostoc, and Lactobacillus, produced lactic acid and contributed to the production of acidic water (pH of approximately 4) called soyashimizu. A heating process, known as “Anka”, which increased the brewing temperature, then switched the relative abundance of 18S rRNA from 75.0% Pichia to 72.3% Saccharomycetaceae. Alcohol fermentation was accelerated by the Saccharomyces family (relative abundance: 89.8%), reaching alcohol concentrations >15%.

Bacteria with endosymbiotic lifestyles often show marked genome reduction. While the shrinkage of genomes in intracellular symbionts of animals, including parasitic bacteria, has been extensively exami­ned, less is known about symbiotic bacteria associated with single-celled eukaryotes. We herein report the genomes of two novel gammaproteobacterial lineages, RS3 and XS4, identified as putative parasitic endosymbionts of the dinoflagellate Citharistes regius. Phylogenetic ana­lyses suggest that RS3 and XS4 belong to the family Fastidiosibacteraceae within the order Beggiatoales, forming independent lineages therein. The genomes of RS3 and XS4 are 529 and 436‍ ‍kbp in size, respectively, revealing marked reductions from related bacterial genomes. XS4, which has a very reduced genome with a low GC content, uses a different genetic code, in which UGA assigned tryptophan. The small genomes of RS3 and XS4 encode a limited number of proteins, retaining only approximately 20% of the predicted ancestral proteome. Metabolic reconstruction suggests that RS3 and XS4 are parasitic symbionts that are heavily dependent on their host for essential metabolites. Furthermore, we found that the ancestor of both genomes likely acquired an ADP:ATP antiporter gene via horizontal gene transfer, an event that may have enabled their evolution as energy parasites by facilitating the acquisition of ATP from their host. These results on novel bacteria with highly reduced genomes expand our understanding of the phylogenetic and genomic diversities of endosymbiotic bacteria in protists.

Improving the reliability of a metagenomic sequencing ana­lysis requires the use of control samples, known as mock communities. Therefore, mock communities must be prepared with high accuracy and reproducibility, which is particularly challenging for cellular mock communities. In the present study, we prepared a cellular mock community consisting of bacterial strains representative of the human and surrounding environmental microbiomes to demonstrate the suitability of a HPLC-based method that measures the genome number of cells. This method proved to be more accurate and reproducible for preparing cellular mock communities than traditional cell counting-based enumeration methods.

Digital PCR is a technique that quantifies target genes based on the absence or presence of the targets in PCR amplicons. The present study exami­ned group-specific probes for the quantification of mcrA genes in six methanogenic archaeal groups and “Candidatus Methanoperedens” by digital PCR with the universal primers ML-f and ML-r. A digital PCR ana­lysis of paddy field soil detected all the targets, with the dominant and minor groups being Methanomicrobiales and Methanobrevibacter spp., respectively (10⁷ and 10⁴ copies [g dry soil]^–1). This method has the potential to reveal the dynamics of specific methanogenic archaeal groups in the environment.

Nitrous oxide (N₂O) is a key atmospheric greenhouse gas that contributes to global warming, with anthropogenic N₂O emissions from agriculture being a particular concern. Among agricultural sources, unknown soil organisms in the legume rhizosphere emit N₂O from degraded root nodules. To discriminate between fungal and bacterial N₂O emissions, we adopted an isotopomer ana­lysis, which provides site preference values (the difference in ¹⁵N abundance of the central and terminal N atoms in the N₂O molecule). The addition of nitrite instead of nitrate to soybean nodulated roots significantly increased SP_N2O from –3.5‰ to 4.2‰ in a pot system. Moreover, a mutation of the nirK gene (encoding dissimilatory nitrite reductase) in symbiotic bradyrhizobia significantly increased SP_N2O from 4.2‰ to 13.9‰ with nitrite. These results suggest that nitrite-utilizing N₂O emissions via fungal denitrification occurred in the model pot system of the soybean rhizosphere. Microscopic observations showed fungal hyphae and crescent spores around N₂O-emitting nodules. Therefore, we isolated single spores from soybean nodules under a microscope. A phylogenetic ana­lysis revealed that all 12 fungal isolates were Fusarium species, which exist in soybean field soil. When these isolates were cultivated in glycerol-peptone medium supplemented with nitrate or nitrite (1‍ ‍mM), 11 of the 12 isolates strongly converted nitrite to N₂O; however, no N₂O emissions were noted in the presence of nitrate. A ¹⁵N-nitrite tracer experiment revealed that one N₂O molecule was derived exclusively from two molecules of nitrite (NO₂^–) in the fungal culture. These results suggest that nitrite-utilizing Fusarium fungi mediate N₂O emissions in the soybean rhizosphere.

The whitefly, Bemisia tabaci, is a notorious insect pest that transmits plant pathogenic viruses to a wide range of economically important crops. An invasive genetic group of B. tabaci, Mediterranean Q2 (MED Q2), has recently spread to Europe, USA, and Asia. In the present study, we investigated the prevalence of MED Q2 in Japanese agricultural sites and found that its distribution has expanded since it was initially detected in 2013. A polymerase chain reaction ana­lysis revealed that all MED Q2 individuals were infected with Rickettsia. Rickettsia titers increased during nymphal development, presumably in response to the nutritional needs of the host. A fluorescence in situ hybridization ana­lysis revealed that Rickettsia was densely located near Portiera-containing bacteriocytes at all growth stages. Therefore, Rickettsia may play an important role, such as supplying nutrients to the host, in cooperation with Portiera. Transfer experiments indicated that MED Q2 was as effective a vector for Tomato yellow leaf curl virus as MED Q1 and, thus, is a high-risk agricultural pest. These results provide important insights into the biology and ecology of invasive MED Q2 to effectively control its spread and minimize its impact on crops.

Phytophthora root and stem rot (PRSR) caused by Phytophthora sojae is a major concern for global soybean production. To identify a bacterial biocontrol agent against PRSR, 73 rhizobacterial strains were isolated from wild and cultivated legumes and screened for their protective activities against PRSR in pot experiments. Strain GVv1 was selected for its consistent protective effect through repeated pot experiments. The protective effect of this strain was similar to that of the fungicide mancozeb-metalaxyl. A dual-culture assay showed that GVv1 produced antifungal metabolites effective against P. sojae. To evaluate the potential adaptability of GVv1 to the soybean rhizosphere environment, its growth was exami­ned in soybean root exudates and nutrient medium, both supplemented with daidzein, an antimicrobial isoflavone secreted by soybean roots. GVv1 proliferated using soybean root exudates and had sufficient tolerance to daidzein to colonize the soybean rhizosphere. The plant growth-promoting effect of GVv1 on soybean plants was also investigated. GVv1 significantly increased shoot and root dry weights, indicating its plant growth-promoting activity. In vitro assays showed that GVv1 produced indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylate deaminase and solubilized insoluble phosphates. A taxonogenomic ana­lysis of the draft genome identified GVv1 as Enterobacter pseudoroggenkampii with high similarity (98.32% average nucleotide identity) to E. pseudoroggenkampii strain 155092^T. To the best of our knowledge, this is the first study to report the biocontrol and plant growth-promoting activities of E. pseudoroggenkampii.

Microbial community structures in mesophilic and low-temperature anammox and partial nitrification–anammox reactors were exami­ned by a 16S rRNA–gene amplicon sequencing ana­lysis. The anammox bacterium, Jettenia sp., was dominant, and nitrifying bacteria, including Nitrosomonas sp. (aerobic ammonia–oxidizing bacterium) and Nitrospira sp., (nitrite–oxidizing bacterium) coexisted in the bioreactors. Core coexisting bacteria, such as Sulfurisoma sp. and Zeimonas sp., showed oxygen-scavenging and NO₃^– reduction potentials. Sulfurisoma-related bacteria are distributed across wastewater treatment plants worldwide, particularly in denitrification systems. These results underscore the ecological and functional importance of microbial consortia in enhancing nitrogen removal efficiency.