Digital PCR is a technique that quantifies target genes based on the absence or presence of the targets in PCR amplicons. The present study exami-ned 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 ana-lysis 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.

The present study proposes a categorization of bacteria that leak from activated sludge processes to secondary treated water (STW). Bacterial populations in primary treated water (PTW), activated sludge (AS), STW, and the 0.2‍ ‍μm-filtrate of STW (FSTW) in a full-scale wastewater treatment plant with two treatment trains were observed for a period of one year using a 16S rRNA ana­lysis approach. The taxonomic groups detected were categorized as different “leak types” based on the read occupancies in PTW, AS, STW, and FSTW, where a leak type indicates the likelihood of a taxonomic group to leak to STW. Five leak types were introduced: “LTE”, “LTE-I”, “LTEF”, “LTF”, and “NLT”, with “LT” for leak type, “E” for high read occupancy in STW or the effluent of secondary settling tanks, “I” for high read occupancy in PTW or influent to the AS process, “F” for high read occupancy in FSTW, and “NLT” for a smaller likelihood to leak. Representative taxonomic groups for each leak type were Neisseria and ABY1 for “LTE” Parcubacteria for “LTEF”, Campylobacterota for “LTE-I”, and Saccharimonadia, Bdellovibrionota, and some lineages in Comamonadaceae for “LTF”. Although some taxonomic groups, such as Comamonadaceae, included different leak types, the categorization assigned to each taxonomic group was mostly consistent between the two treatment trains. The categorization scheme proposed herein may become a useful key for understanding the characteristics of bacteria that appear in AS and STW.

We herein exami­ned the inoculation effects of indigenous Bradyrhizobium diazoefficiens isolates on the growth and yield of adzuki beans and their competition with other bradyrhizobia using pot and field experiments. In the pot experiment, shoot nitrogen content was significantly higher following inoculations with AMP1 and Bd (a mixture of AN9 and AN20) than with the control. Furthermore, a correlation was observed between shoot nitrogen content and shoot dry weight. In the field experiment, the inoculating isolates did not significantly affect growth or yield. However, an interaction effect was observed in pod numbers and yield, suggesting that the effects of inoculation varied depending on the cultivar and inoculating isolate. In the correlation ana­lysis, pod number correlated with node number and nodule number. Similarly, yield correlated with shoot length, node number, nodule number, and pod number. Regarding competition between inoculated isolates and other strains, B. elkanii was dominant in pot and field experiments. To enhance the yield of adzuki bean through inoculations, it is necessary to overcome competition from indigenous B. elkanii and increase the occupancy rate of B. diazoefficiens isolates.

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.

This study investigated the effects of elevated hydrostatic pressure on methane production and gene expression in a hydrogenotrophic methanogen isolated from subseafloor sediments at biogenic gas hydrate sites, with a focus on the implications of CO2 availability. Using high-pressure cultivation, the methane production rate decreased by 15% at 25 MPa, while a transcriptomic anal-ysis revealed the marked up-regulation of methyl-coenzyme M reductase and ATP synthase. These results suggest that methanogens compensate for pressure-driven constraints on CO2 utilization by increasing the expression of key methanogenic enzymes, underscoring the overlooked role of CO2 in deep biosphere microbial processes.

Microcystins (MCs) produced by Microcystis aeruginosa are harmful to animal and human health, and there is currently no effective method for their removal. Therefore, the development of biological approaches that inhibit cyanobacteria and remove MCs is needed. We identified strain MB1, confirmed as Morchella, using morphological and mole-cular evolution methods. To assess the impact of strain MB1 on M. aeruginosa, we conducted an experiment in which we inoculated M. aeruginosa with Morchella strain MB1. After their co-cultivation for 4‍ ‍d, the inoculation with 0.9696‍ ‍g MB1 completely inhibited and removed M. aeruginosa while concurrently removing up to 95% of the MC content. Moreover, within 3‍ ‍d of their co-cultivation, MB1 removed more than 50% of nitrogen and phosphorus from the M. aeruginosa solution. Therefore, the development of effective biological techniques for MC removal is paramount in safeguarding both the environment and human well-being. We herein successfully isolated MB1 from its natural habitat. This strain effectively inhibited and removed M. aeruginosa and also reduced the content of nitrogen and phosphorus in the M. aeruginosa solution. Most importantly, it exhibited a robust capability to eliminate MCs. The present results offer a new method and technical reference for mitigating harmful algal blooms.

With the explosion of available genomic information, comparative genomics has become a central approach to understanding microbial ecology and evolution. We developed DiGAlign (https://www.genome.jp/digalign/), a web server that provides versatile functionality for comparative genomics with an intuitive interface. It allows the user to perform the highly customizable visualization of a synteny map by simply uploading nucleotide sequences of interest, ranging from a specific region to the whole genome landscape of microorganisms and viruses. DiGAlign will serve a wide range of biological researchers, particularly experimental biologists, with multifaceted features that allow the rapid characterization of genomic sequences of interest and the generation of a publication-ready figure.

Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they potentially differ from the genetically defined "true Pseudomonas putida" because many were classified as P. putida based on their phenotypic and metabolic characteristics. A phylogenetic ana-lysis based on the concatenated sequences of the 16S rRNA and rpoD genes revealed that 46 strains of P. putida deposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain produces N-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibited a ppuI-rsaL-ppuR quorum-sensing system that controls biofilm formation and motility. The P. putida type strain JCM 13063T and six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063T and defined as true P. putida. When orthologous genes in the whole-genome sequences of true P. putida strains were screened, PP4_28660 from P. putida NBRC 14164T (=JCM 13063T) was present in all true P. putida genome sequences. The internal region of PP4_28660 was successfully amplified from all true P. putida strains using the specific primers designed in this study.

Roseateles depolymerans is an obligately aerobic bacterium that produces a photosynthetic apparatus only under the scarcity of carbon substrates. We herein examined changes in the transcriptomes of R. depolymerans cells to clarify the expression of photosynthesis genes and their upstream regulatory factors under carbon starvation. Transcriptomes 0, 1, and 6‍ ‍h after the depletion of a carbon substrate indicated that transcripts showing the greatest variations (a 500-fold increase [6 h/0 h]) were light-harvesting proteins (PufA and PufB). Moreover, loci with more than 50-fold increases (6 h/0‍ ‍h) were fully related to the photosynthetic gene cluster. Among 13 sigma factor genes, the transcripts of a sigma 70 family sigma factor related to RpoH (SP70) increased along photosynthesis genes under starvation; therefore, a knockout experiment of SP70 was performed. ΔSP70 mutants were found to lack photosynthetic pigments (carotenoids and bacteriochlo-rophyll a) regardless of carbon starvation. We also examined the effects of heat stress on ΔSP70 mutants, and found that SP70 was also related to heat stress tolerance, similar to other RpoH sigma factors (while heat stress did not trigger photosystem production). The deficient accumulation of photosynthetic pigments and the heat stress tolerance of ΔSP70 mutants were both complemented by the introduction of an intact SP70 gene. Furthermore, the transcription of photosynthetic gene operons (puf, puh, and bch) was markedly reduced in the ΔSP70 mutant. The RpoH homologue SP70 was concluded to be a sigma factor that is essential for the transcription of photosynthetic gene operons in R. depolymerans.

Microorganisms develop into communities in nearly every environmental niche, which is typically replete with micrometer-scale gaps and features. In each of these habitats, microorganisms adapt to and are affected by their physical environment. Conventional culture methods use glass bottom dishes or millimeter-scale flow cells, which poorly mimic the complexity of natural micrometer-scale environments; therefore, the limitations associated with the creation of microbe-scale environments with granularity hinder the ability to examine their ecological behavior. Microfluidics is a tool that is increasingly being used to study microorganisms because it enables the manipulation of micrometer-scale flows while simultaneously facilitating real-time and live-cell imaging. In this review, we discuss several insights into the behavior of bacteria and fungi that were gained through the adoption of microfluidics to control complex micrometer-scale environments. We also discuss the potential of the increased adoption of this tool.