Microbes and Environments最新文献

Symbiotic N₂-fixing bradyrhizobia nodulate various leguminous plants and possess a large symbiosis island (SI) encoding symbiotic functions in their genomes. We obtained 30 rhizobial isolates from root nodules of the tribe Desmodieae of native leguminous plants in northern Japan. Based on their 16S rRNA gene sequences, most isolates (24/30=80%) phylogenetically belonged to Bradyrhizobium. Two isolates (LCT1 and LCT2) from Lespedeza cuneata were placed phylogenetically with Bradyrhizobium diazoefficiens USDA110^T, a well-studied soybean (Glycine max [L.] Merr.) symbiont. Genomic comparisons revealed different SIs in the Met-tRNA and Val-tRNA genes on the respective genomes. In contrast, core genomic regions outside the SI regions showed strong collinearity between strains LCT2 and USDA110. Phenotypically, LCT2 formed N₂-fixing root nodules on L. cuneata, an original host plant, but not on soybean, whereas USDA110 formed N₂-fixing root nodules on soybean, but not on L. cuneata. Therefore, the SI variants were expected to contain the genes responsible for this host specificity. Genes relevant to the type III secretion system (T3SS) showed less homology between LCT2 and USDA110 than nod genes encoding Nod factor biosynthesis. Host plant inoculations with T3SS mutants suggested the involvement of T3SS effectors in differential host specificity. Therefore, the acquisition of distinct SI variants may confer strong host specificity through symbiotic interactions between Bradyrhizobium and host legumes. We discuss the possible pathway of symbiotic bradyrhizobial evolution and its application to the mitigation of greenhouse gas emissions.

We exami-ned the effects of soil moisture changes on soybean growth, yield, and the structure of soybean-nodulating bradyrhizobial communities in cultivars with different Rj genotypes. The experiment was conducted using cultivation pots with soybean cultivars Bragg (non-Rj), CNS (Rj₂Rj₃), D-51 (Rj₃), and Fukuyutaka (Rj₄). Test strains included Bradyrhizobium diazoefficiens USDA 110^T, B. japonicum USDA 6^T and USDA 123, and B. elkanii USDA 31. Cultivation pots were built with 15-cm ridges, and three soil moisture conditions were generated by varying the presence and placement of drainage holes on the pots. Declining soil moisture significantly reduced shoot length, shoot dry weight, root dry weight, root length, nodule number, pod number, pod dry weight, and seed number. An occupancy anal-ysis showed that USDA 110 dominated Fukuyutaka only; across treatments, it was the most abundant under high soil moisture, but significantly declined with reductions in soil moisture, where USDA 31 became dominant. A non-metric multidimensional scaling anal-ysis revealed shifts in community compositions in response to soil moisture and cultivar. Collectively, these results indicate that soybean growth, yield, and symbiosis with bradyrhizobia are strongly affected by soil moisture and also that these effects vary among cultivars.

A genome anal-ysis is essential for identifying valuable microbial resources for future applications. In the present study, we exami-ned potential CO₂-fixing microorganisms based on the presence of the Calvin-Benson-Bassham (CBB) cycle using 6,262 bacterial and 487 archaeal genomes from available cultures in the Japan Collection of Microorganisms (JCM), a well-established culture collection, in October 2023. A total of 306 strains (147 genera, eight phyla) carried CBB cycle genes, and a literature survey showed that 74 genera had experimental evidence of autotrophic growth while 73 lacked supporting information. A phylogenetic anal-ysis of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcL) identified diverse forms (IA, IB, IC, IE, I+α, II, and III) with distinct metabolic associations; IA was associated with sulfur species oxidation and formed IC with hydrogen oxidation. Genome-based metabolic predictions identified the potential for CO₂ fixation in numerous species lacking experimental evidence. Our anal-yses indicate that members of Actinomycetota harboring IE RbcL were generally associated with hydrogen oxidation, possibly by using oxygen or nitrate as an electron acceptor. Additionally, 12 species in Pseudomonadota contained photosystem II reaction center proteins (PufL and PufM), suggesting phototrophic capabilities. However, the prediction of electron donors failed in some of these species. They may use the CBB cycle to regulate the intracellular redox balance under photoheterotrophic growth. The present results reveal unrecognized autotrophic potential in JCM strains and broaden our knowledge of the diversity of CO₂-fixing microorganisms. Experimental validation will clarify their roles in the global carbon cycle and their potential for biotechnological applications towards environmental sustainability.

Bradyrhizobium ottawaense has prospects as an environmentally friendly inoculant for soybean farming because of its higher N₂O reductase (N₂OR) activity than that of B. diazoefficiens. To examine high N₂O-reducing B. ottawaense, we performed a PCR anal-ysis of nosZ genes in 8,640 soybean nodules from 68 fields in Japan. Of 384 PCR-positive nodules, we obtained 90 isolates of bradyrhizobia with B. ottawaense-type nosZ, derived exclusively from 18 fields in Gunma and Osaka prefectures. Of 77 monophyletic isolates, 73 had significantly higher N₂OR activity than B. diazoefficiens USDA110. Another 13 isolates‍ ‍from Osaka were phylogenetically placed outside of the B. ottawaense clade with B. liaoningense or B. betae, 8 of which also exhibited significantly higher N₂OR activity than B. diazoefficiens USDA110. An anal-ysis of nopP gene sequences revealed amino acid sequence variations in the NopP effector protein among these high N₂O-reducing isolates, with the NopP_USDA122 type being one of the variations identified. The NopP-mediated symbiotic incompatibility of soybean host plants may eliminate nodulation by indigenous bradyrhizobia and facilitate inoculant nodulation to reduce N₂O emissions. Therefore, 90 isolates and their observed NopP types are‍ ‍potentially important resources for N₂O mitigation. Furthermore, the dense geographical map of Bradyrhizobium species based on Internal Transcribed Spacer-Restriction Fragment Length Polymorphisms (ITS-RFLP) of the 16S-23S rRNA gene from 8,640 nodules revealed the recent northward expansion of B. elkanii to central Japan potentially due to global warming. This change in indigenous soybean bradyrhizobia is important for application strategies of bradyrhizobial inoculants under field conditions.

The biological reduction of N₂O, a potent greenhouse gas, is crucial for environmental sustainability. We developed an automated system for continuous N₂O monitoring in the gas phase of a flask containing an anaerobic bradyrhizobial culture, and then exami-ned the kinetic parameters of bacterial N₂O reduction. The maximum reaction rate (V_max) was approximately 61-fold higher for Bradyrhizobium ottawaense SG09 (1,471‍ ‍nmol h^-1 10⁹ cells^-1) than for B. diazoefficiens USDA110 (24‍ ‍nmol h^-1 10⁹ cells^-1). Our kinetics anal-ysis confirmed that SG09 maintained higher N₂O-reducing activity than USDA110 even at the atmospheric concentration of N₂O (0.34 ppm).

Lettuce black root rot caused by Berkeleyomyces rouxiae occurs during the hot season in Japan, whereas black root rot in other crops often develops during cooler seasons. The present study investigated the relationship between temperature and symptom severity in lettuce and other plant species. Inoculation tests conducted with different isolates revealed that symptoms on lettuce were the most severe at 25°C, whereas those on cotton, okra, and cowpea were the most severe at 15-20°C. These results align with the seasonal occurrence of lettuce black root rot in Japan. The present study provides valuable insights for predicting and managing this disease.

Giant viruses are distinguished not only by their large particle size, but also by their extensive genomes, often reaching megabase levels. Many sequences within these genomes are considered to have been introduced by hosts, surrounding organisms, or other viruses. Since the natural hosts of many giant viruses remain unidentified, analyzing sequences potentially derived from other organisms may aid in clarifying their hosts. In the present study, we identified eukaryote-homologous sequences by isolating those not shared among viruses, an aspect previously overlooked. Our primary focus was on pandoravirus, which, with a genome size of ~2 Mb, is the largest among giant viruses. We obtained 375 BLAST hits with an average sequence identity of ~90%. Among the 102 detected species, those with higher hits included Mus musculus, Lampetra planeri, Melanogrammus aeglefinus, Lampetra fluviatilis, Scylla paramamosain, Cardiocondyla obscurior, Monodelphis domestica, Vespula pensylvanica, Micromonas pusilla, Physcomitrium patens, and Peromyscus californicus. Similar anal-yses of Cedratvirus and Pithovirus, which share an amphora-shaped particle structure with pandoraviruses, yielded fewer data (48 and 5 hits, respectively), with no common taxa at the order level. Thirteen BLAST hits exceeded 100 bp, including conserved non-coding elements (CNEs) in fish and other taxa, along with sequences of unknown functions. These results indicate the presence of short regions with sequence similarity in non-shared sequences, although direct host identification proved difficult.

Insertion sequences (ISs) are major drivers of genomic plasticity in rhizobia, frequently promoting local recombination events. To quantitatively compare the stability of genomic regions inside and outside of the symbiosis island, we engineered Bradyrhizobium diazoefficiens USDA122 mutants carrying a sacB/aadA counter-selectable cassette at four distinct loci-three on symbiosis island A (SymA) and one in the core genome. During 5 days of saprophytic growth, cassette deletion occurred at frequencies of up to 1.77×10^-3 within SymA, whereas the deletion rate in core genomic regions was markedly lower (3.29×10^-6). Within SymA, cassettes inserted adjacent to the nif and rhc clusters, where IS copies with the same orientation were enriched, were lost more frequently than those placed in other SymA regions, indicating marked intra-island variability in genomic stability. Similar yet overall lower deletion frequencies were observed in B. diazoefficiens USDA110. These results demonstrate that SymA contains genomic loci with greater susceptibility to IS-mediated rearrangements and also that such recombination events may contribute to the diversification of Bradyrhizobium symbiosis islands. Based on our comparative IS mapping in B. japonicum and B. ottawaense, we discuss the potential for the IS-mediated deletion of genome regions harboring nod genes.

Electric field-assisted (EFA) technology has been extensively employed in the realm of biodegradation. Candida tropicalis, with a high capability for degrading thiophene, has been successfully screened through electric field-assisted screening (EFAS). The present study investigated the cell density distribution of C. tropicalis at various locations within an electric field. Under an electric field, cells migrated and accumulated toward the cathode plate. Under a loading electric field intensity of 0.6 V cm^-1, the concentration of cells peaked near the cathode plate and remained stable with a loading time of 10 min. Furthermore, the thiophene degradation efficiency of strain CZ60, which was screened under optimal loading voltage and time conditions, reached 91.4% after a 4-h reaction. These results establish a solid theoretical foundation for utilizing EFAS to identify biodegradable microorganisms, with potential implications for environmental remediation strategies.

Gene mutations are a fundamental survival strategy in bacteria and often accompany cell growth. We herein demonstrate that Pseudomonas aeruginosa recovered growth under anoxic conditions through a mutation in an essential regulatory gene required for nitrate respiration, overcoming initial growth limitations. Although the open-ocean isolate P. aeruginosa Ocean-1187 possesses denitrification-related genes, it was initially unable to grow under nitrate-respiring conditions due to a defect in the transcriptional regulator NarL, part of the NarX-NarL two-component system that controls the expression of denitrification genes. The growth of this strain was limited by the defective NarL; however, a prolonged incubation under anoxic conditions led to the emergence of spontaneous mutants that regained the ability to grow. Whole-genome sequencing and functional complementation assays revealed that a single amino acid substitution in NarL was sufficient to restore nitrate respiration. Additional variants independently isolated from separate cultures also carried substitutions at the same residue, underscoring its functional importance. The present study shows that a single amino acid substitution in an essential regulatory protein restored metabolic function. These results offer novel insights into the adaptive strategies of bacteria, highlighting the emergence of critical mutations even under restricted growth conditions.