Despite its ecological importance, the gut microbiota of whale sharks (Rhincodon typus) remains poorly understood. Therefore, the present study exami­ned how environmental differences affect the fecal microbiota by comparing long-term captive and newly captured individuals. Fecal samples were collected over time from four long-term captive and two newly captured whale sharks, with seawater also being sampled from their respective tanks. Using 16S rRNA sequencing, 12,497 amplicon sequence variants (ASVs) were identified, including 6,976 classified as major ASVs. There were no significant differences in alpha diversity indexes between long-term captive and newly captured sharks; however, the latter showed slightly larger variance in four indexes. The ASV count per individual was slightly lower in long-term captive sharks than in their newly captured counterparts. In long-term captive individuals, Photobacterium was highly abundant. Conversely, Ureaplasma was dominant in newly captured individuals, but was barely detected in long-term captive sharks. Although alpha diversity did not differ significantly between the groups, a beta diversity ana­lysis showed clear distinctions. The high abundance of Ureaplasma in newly captured sharks suggests its involvement in nitrogen metabolism, possibly through urea recycling. Although further research is needed to clarify the taxonomic position and ecological functions of these Ureaplasma populations, the present study provides key insights for the conservation of wild whale sharks and improving health management for captive individuals.

There is a growing demand for the reuse of sediment basin soil in cabbage fields; however, reusing soil poses a potential challenge of spreading Verticillium wilt to the fields via pathogen-infested sediments. We evaluated the density of the Verticillium wilt pathogen in sediment basin soil using a quantitative nested real-time polymerase chain reaction assay and its incidence using pot cultivation tests. We detected low pathogenic DNA levels in the sediment, coupled with a low Verticillium wilt incidence and severity in the pot experiment, indicating a low risk of spreading Verticillium wilt with the reuse of sediment basin soil.

Frankia, a nitrogen-fixing actinobacterium, forms a unique multicellular structure known as a vesicle that is dedicated to nitrogen fixation. The vesicle is surrounded by a thick hopanoid lipid envelope that acts as a barrier against oxygen penetration, preventing nitrogenase inactivation. Five mutants produced a similar number of vesicles to the wild type; however, they failed to fix N₂. The thickness of vesicle envelopes was reduced in all five mutants, and the oxygen concentration increased inside the vesicles of four mutants. Therefore, these mutants were unable to fix N₂ due to the inactivation of nitrogenase caused by oxygen penetration into the vesicles.

Naturally occurring caves are sites of significant cultural value, while also displaying the unique biodiversity of associated microbiomes that may provide an untapped source of potentially beneficial organisms. However, the touristic exploitation of show caves may ultimately result in the biodeterioration of speleothems, primarily through the introduction and establishment of alien microbiota or the uncontrolled growth of indigenous species, exacerbated by the use of artificial lighting. These habitat characteristics are present in the Dictean cave, also known as “Diktaion Andron”, a highly visited cave in eastern Crete, Greece, which was regarded in ancient Greek mythology as one of the putative sites of the birth of Zeus. Therefore, an efficient approach to controlling these ecological niches without irreversibly disturbing microbial diversity is needed, and essential oils are currently being investigated as a mild cleaning method. The present study exami­ned the microbial diversity of the Dictean cave using 16S and 18S rRNA gene amplicon sequencing and methods for quantitative metabolic activity estimations and also investigated the application of a formulation containing specific essential oils as a mild cleaning method. Amplicon sequencing ana­lyses revealed distinct profiles among the different sample sites, with species of the genera Pseudomonas, Sporosarcina, Butiauxella, Glutamicibacter, Paenibacillus, Mortierella, and Jenufa being the most abundant, while uncharacterized microorganisms were also detected. The single simultaneous application of a formulation of 0.2% (v/v) oregano and 0.4% (v/v) cinnamon essential oils was effective at significantly reducing microbial metabolic activity by up to 89.2% within 24 h, without adversely affecting the coloration of speleothems.

Nitrogen-fixing sulfate-reducing bacteria have not yet been exami­ned in thermal environments. In this study, strain TK110, belonging to the genus Thermodesulfovibrio, was successfully isolated from a geothermal spring using an NH₃-free inorganic medium. Strain TK110 harbored genes associated with the Calvin–Benson–Bassham cycle and nitrogen fixation-related genes, nifHDKENXIIB. Nitrogenase activity was assessed using an acetylene reduction assay and detected in strain TK110 under autotrophic conditions, as well as in Thermodesulfovibrio yellowstonii DSM 11347^T under heterotrophic conditions at 65°C. To the best of our knowledge, this is the first study to demonstrate nitrogen fixation by thermophilic sulfate-reducing bacteria.

Nitrifying communities in activated sludge play a crucial role in biological nitrogen removal processes in municipal wastewater treatment plants. While extensive research has been conducted in temperate regions, limited information is available on nitrifiers in tropical regions. The present study investigated all currently known nitrifying communities in two full-scale municipal wastewater treatment plants in Malaysia operated under low-dissolved oxygen (DO) (0.2–0.7‍ ‍mg‍ ‍DO‍ ‍L^–1) or high-DO (2.0–5.5‍ ‍mg‍ ‍DO‍ ‍L^–1) conditions at 30°C. The core nitrifiers in the municipal wastewater treatment plants were Nitrosomonas (ammonia-oxidizing bacteria, AOB), Nitrospira (nitrite-oxidizing or complete ammonia-oxidizing, comammox, bacteria), and ammonia-oxidizing archaea (AOA) as identified by a 16S rRNA gene amplicon sequencing ana­lysis and corroborated by 16S rRNA-targeted fluorescence in situ hybridization. A quantitative polymerase chain reaction (qPCR) targeting ammonia monooxygenase subunit A (amoA) genes revealed stable populations of comammox Nitrospira and AOB in both wastewater treatment plants. AOA were detected in only one of the plants and their population sizes fluctuated, with higher temporary abundance under high-DO conditions. These results provide important insights into the composition and dynamics of nitrifying communities in tropical municipal wastewater treatment plants.

Sweet potato foot rot disease caused by Diaporthe destruens (formerly Plenodomus destruens) severely affects the yield and quality of sweet potatoes. To gain basic knowledge on regulating the pathogen using indigenous soil bacteria, the following organic materials were applied to potted soils collected from a sweet potato field contaminated with D. destruens: Kuroihitomi (compost made from shochu waste and chicken manure), Soil–fine (material made by adsorbing shochu waste on rice bran), and rice bran. Soil samples were periodically collected during an incubation for bacterial colony counts and a community ana­lysis using a meta 16S amplicon ana­lysis. The number of bacterial colonies was significantly higher with the Soil–fine and rice bran treatments and slightly higher with the Kuroihitomi treatment than with a chemical fertilizer as the control, and then gradually decreased over time. An amplicon ana­lysis showed that the Soil–fine and rice bran treatments increased the relative abundance of Streptomycetaceae and Micrococcaceae belonging to Actinobacteria and Burkholderiaceae belonging to Beta–proteobacteria. The Kuroihitomi treatment also increased the relative abundance of Streptomycetaceae. The dominant amplicon sequencing variant (ASV) sequences among these families were affiliated with the genera Kitasatospora, Arthrobacter, and Paraburkholderia. Bacteria with sequences identical to these ASVs were isolated from the incubated soils using selective media for dual culture assays. Bacterial isolates in a cluster of Kitasatospora exhibited antagonistic activity against D. destruens. The present results suggest that combining organic materials with antagonistic bacteria may be an effective approach to regulating the growth of D. destruens.

Soybean (Glycine max) is one of the most important crops worldwide. Root nodule symbiosis between soybean and rhizobia has been extensively exami­ned due to its significance for agricultural productivity and environmental sustainability. Recent advances have enhanced our understanding of the soybean genotypes known as the Rj/rj genotypes, which play a critical role in regulating root nodule symbiosis. Furthermore, the function of rhizobium-secreted proteins, termed effectors, in eliciting specific responses in soybean Rj/rj genotypes has been elucidated. This review summarizes the involvement of soybean Rj/rj genotypes and their corresponding root nodule bacterial effectors in the regulation of nodule formation. We also discussed the potential for manipulating root nodule symbiosis by applying Rj/rj genotypes in soybean breeding programs, which may enhance nitrogen fixation efficiency and subsequently reduce the need for chemical fertilizers and greenhouse gas emissions from agricultural land.

We recently proposed a novel microbial isolation technique, the “duckweed-microbe co-cultivation method”, for isolating a wide variety of microbes, including rarely cultivated microbes. This method involves the inoculation of aseptic duckweed with environmental microbes followed by co-cultivation for a set period. Plants and their surrounding medium are then used as microbial sources for isolation in the conventional agar plate method. In the present study, we improved the method by using microfilter membranes (pore sizes of 0.8–1.2‍ ‍μm) to pretreat microbial inocula, which increased the isolation efficiency of rarely cultivated microbes representing the phylum Verrucomicrobiota.

Chemotaxis is essential for infection by plant pathogenic bacteria. The causal agent of tobacco wildfire disease, Pseudomonas syringae pv. tabaci 6605 (Pta6605), is known to cause severe leaf disease and is highly motile. The requirement of chemotaxis for infection has been demonstrated through the inoculation of mutant strains lacking chemotaxis sensory component proteins. Pta6605 possesses 54 genes that encode chemoreceptors (known as methyl-accepting chemotaxis proteins, MCPs). Chemoreceptors are classified into several groups based on the type and localization of ligand-binding domains (LBD). Cache LBD-type chemoreceptors have been reported to recognize formate in several bacterial species. In the present study, we identified Cache_3 Cache_2 LBD-type Mcp26 encoded by Pta6605_RS00335 as a chemoreceptor for formate using a quantitative capillary assay, and named it McpF. Although the deletion mutant of mcpF (ΔmcpF) retained attraction to 1% yeast extract, its chemotactic response to formate was markedly reduced. Swimming and swarming motilities were also impaired in the mutant. To investigate the effects of McpF on bacterial virulence, we conducted inoculations on tobacco plants using several methods. The ΔmcpF mutant exhibited weaker virulence in flood and spray assays than wild-type and complemented strains, highlighting not only the involvement of McpF in formate recognition, but also its critical role in leaf entry during the early stages of infection.

Circumneutral iron-rich hot springs may represent analogues of Neoarchean to Paleoproterozoic oceans of early Earth, potentially providing windows into ancient microbial ecology. Here we sampled five Japanese hot springs to gain insights into functional processes and taxonomic diversity in these analog environments. Amplicon and metagenomic sequencing confirm a hypothesis where taxonomy is distinct between sites and linked to the geochemical setting. Metabolic functions shared among the springs include carbon fixation via the reductive pentose phosphate cycle, nitrogen fixation, and dissimilatory iron oxidation/reduction. Among the sites, Kowakubi was unique in that it was dominated by Hydrogenophilaceae, a group known for performing hydrogen oxidation, motivating a hypothesis that H₂ as an electron donor may shape community composition even in the presence of abundant ferrous iron. Evidence for nitrogen cycling across the springs included N₂ fixation, dissimilatory nitrate reduction to ammonia (DNRA), and denitrification. The low-salinity springs Furutobe and OHK lacked evidence for ammonium oxidation by ammonia monooxygenase, but evidence for complete nitrification existed at Kowakubi, Jinata, and Tsubakiyama. In most sites, the microaerophilic iron-oxidizing bacteria from the Zetaproteobacteria or Gammaproteobacteria classes had higher relative abundances than Cyanobacteria. Microaerophilic iron oxidizers may outcompete abiotic Fe oxidation, while being fueled by oxy-phototrophic Cyanobacteria. Our data provide a foundation for considering which factors may have controlled productivity and elemental cycling as Earth’s oceans became oxygenated at the onset of the Great Oxidation Event.

Many plant pathogenic bacteria regulate the expression of virulence factors via N-acylhomoserine lactone (AHL), a quorum-sensing signaling compound. When numerous spore-forming bacteria were isolated from a natural environment, Priestia megaterium was the dominant species, and some P. megaterium strains exhibited AHL-degrading activity. The results of a HPLC ana­lysis of AHL degradation products demonstrated that P. megaterium degraded AHL by AHL lactonase, which hydrolyzes the lactone ring of AHL. The novel AHL lactonase gene, aiiB, was found in the whole genome sequence of AHL-degrading P. megaterium. The relationship between the presence of aiiB and AHL-degrading activity in P. megaterium strains revealed that P. megaterium may be classified into three AHL degradation groups: Group 1 (with AHL-degrading activity and aiiB), Group 2 (with neither AHL-degrading activity nor aiiB), and Group 3 (without AHL-degrading activity, but with aiiB). A comparative genome ana­lysis suggested that aiiB was obtained or missed by a non-transpositional event during the process of evolution in P. megaterium. The amino acid sequences of AiiB in Group 1 and 3 strains were almost identical, and Escherichia coli harboring aiiB from Groups 1 and 3 exhibited high AHL-degrading activity. Although the AHL-degrading activity of Group 3 strains was markedly weaker than that of Group 1 strains, they degraded AHL in a long-term incubation. Based on the present results, Group 1 and 3 strains, the genomes of which contain aiiB, may reduce potato maceration activity under the control of AHL-mediated quorum sensing in P. carotovorum subsp. carotovorum NBRC 12380.

In traditional indigo dyeing, water-insoluble indigo is anaerobically converted into soluble leuco-indigo via microbial reduction in alkaline dye suspensions, allowing its use as a fabric dye. Although various indigo-reducing bacteria have been isolated to date, culture-independent microbial community ana­lyses have suggested that bacteria belonging to uncultured clades also contribute to indigo reduction. Therefore, we aimed to isolate previously overlooked indigo-reducing bacteria using an unconventional culture method. We conducted enrichment cultures and single-colony isolation using a medium supplemented with sukumo, an indigo dye source derived from the composted leaves of indigo-containing plants, as the sole energy, carbon, and nitrogen sources. We isolated a previously uncultured bacterium belonging to the family Tissierellaceae, which had been predicted as a major indigo reducer in various indigo dyeing processes solely based on microbial community ana­lyses. The insoluble indigo-reducing activity of the Tissierellaceae isolate, strain TU-1 was significantly higher than that of known indigo-reducing bacteria. The addition of the culture supernatant of strain TU-1 enhanced the reduction of indigo powder by other indigo-reducing bacteria, with similar stimulatory effects to those of the insoluble electron mediator, anthraquinone. These results indicate that strain TU-1 possesses a high capacity for secreting electron mediators, conferring a significant reduction capacity for insoluble indigo. Further investigations, including the discovery of additional unknown indigo-reducing bacteria and the identification of the mediators they produce, will provide a more detailed understanding of the mechanisms underlying indigo reduction in practical dyeing processes.

A sulfate-reducing bacterium was isolated from the anode surface of a microbial fuel cell (MFC) producing a high current density. 16S rRNA gene ana­lyses showed that the isolate was affiliated with the genus Nitratidesulfovibrio, and the strain was named HK-II. When Nitratidesulfovibrio sp. strain HK-II was incubated anaerobically under sulfate-reducing conditions with Fe(III) citrate, a black precipitate formed. The resulting black precipitate was investigated using multidisciplinary methods. An X-ray diffraction (XRD) ana­lysis revealed that the black precipitate was mainly composed of mackinawite. A cyclic voltammetry ana­lysis showed clear redox peaks, and biogenic mackinawite possessed rechargeable properties. The XRD ana­lysis also showed that the form of the rechargeable biogenic mineral induced by strain HK-II (RBM-II) was changed by discharge and recharge treatments. Field-emission transmission electron microscopy revealed that lepidocrocite and amorphous iron oxide formed from mackinawite under discharged conditions, and the three mineral types were intermingled via charge and discharge cycles. Physicochemical parameters regularly changed under the treatments, suggesting that discharge occurred via iron oxidation followed by sulfur reduction and vice versa. These results indicate that sulfur dynamics are important key processes in charge and discharge mechanisms. MFCs equipped with lactate, strain HK-II, and an anode containing RBM-II consumed lactate under open-circuit conditions, after which MFCs generated a higher current density under reclosed-circuit conditions. These results demonstrate that RBM-II is a rechargeable material that enables the capture of electrons produced by bacterial cells and is useful for enhancing the performance of MFCs.