Metagenomic study of endophytic bacterial community of sweet potato (Ipomoea batatas) cultivated in different soil and climatic conditions.

The aim of this study was to clarify effects of soil and climatic conditions on community structure of sweet potato bacterial endophytes by applying locked nucleic acid oligonucleotide-PCR clamping technique and metagenomic analysis. For this purpose, the soil samples in three locations were transferred each other and sweet potato nursery plants from the same farm were cultivated for ca. 3 months. After removal of plastid, mitochondria and undefined sequences, the averaged numbers of retained sequences and operational taxonomic units per sample were 20,891 and 846, respectively. Proteobacteria (85.0%), Bacteroidetes (6.6%) and Actinobacteria (6.3%) were the three most dominant phyla, accounting for 97.9% of the reads, and γ-Proteobacteria (66.3%) being the most abundant. Top 10 genera represented 81.2% of the overall reads in which Pseudomonas (31.9-45.0%) being the most predominant. The overall endophytic bacterial communities were similar among the samples which indicated that the soil and the climatic conditions did not considerably affect the entire endophytic community. The original endophytic bacterial community might be kept during the cultivation period.

A thaumatin-like protein, Rj4, controls nodule symbiotic specificity in soybean.

Soybeans exhibit a nitrogen-fixing symbiosis with soil bacteria of the genera Bradyrhizobium and Ensifer/Sinorhizobium in a unique organ, the root nodule. It is well known that nodulation of soybean is controlled by several host genes referred to as Rj (rj) genes. Among these genes, a dominant allele, Rj4, restricts nodulation with specific bacterial strains such as B. elkanii USDA61 and B. japonicum Is-34. These incompatible strains fail to invade the host epidermal cells as revealed by observations using DsRed-labeled bacteria. Here, we describe the molecular identification of the Rj4 gene by using map-based cloning with several mapping populations. The Rj4 gene encoded a thaumatin-like protein (TLP) that belongs to pathogenesis-related (PR) protein family 5. In rj4/rj4 genotype soybeans and wild soybeans, we found six missense mutations and two consecutive amino acid deletions in the rj4 gene as compared with the Rj4 allele. We also found, using hairy root transformation, that the rj4/rj4 genotype soybeans were fully complemented by the expression of the Rj4 gene. Whereas the expression of many TLPs and other PR proteins is induced by biotic/abiotic stress, Rj4 gene expression appears to be constitutive in roots including root nodules.

Genetic diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in the United States.

We investigated the relationship between the genetic diversity of indigenous soybean-nodulating bradyrhizobia and their geographical distribution in the United States using nine soil isolates from eight states. The bradyrhizobia were inoculated on three soybean Rj genotypes (non-Rj, Rj(2)Rj(3), and Rj(4)). We analyzed their genetic diversity and community structure by means of restriction fragment length polymorphisms of PCR amplicons to target the 16S-23S rRNA gene internal transcribed spacer region, using 11 USDA Bradyrhizobium strains as reference strains. We also performed diversity analysis, multidimensional scaling analysis based on the Bray-Curtis index, and polar ordination analysis to describe the structure and geographical distribution of the soybean-nodulating bradyrhizobial community. The major clusters were Bradyrhizobium japonicum Bj123, in the northern United States, and Bradyrhizobium elkanii, in the middle to southern regions. Dominance of bradyrhizobia in a community was generally larger for the cluster belonging to B. elkanii than for the cluster belonging to B. japonicum. The indigenous American soybean-nodulating bradyrhizobial community structure was strongly correlated with latitude. Our results suggest that this community varies geographically.

Effect of Rj genotype and cultivation temperature on the community structure of soybean-nodulating bradyrhizobia.

The nodulation tendency and community structure of indigenous bradyrhizobia on Rj genotype soybean cultivars at cultivation temperatures of 33/28°C, 28/23°C, and 23/18°C for 16/8 h (day/night degrees, hours) were investigated using 780 bradyrhizobial DNA samples from an Andosol with 13 soybean cultivars of four Rj genotypes (non-Rj, Rj(2)Rj(3), Rj(4), and Rj(2)Rj(3)Rj(4)). A dendrogram was constructed based on restriction fragment length polymorphism of the PCR products (PCR-RFLP) of the 16S-23S rRNA gene internal transcribed spacer region. Eleven Bradyrhizobium U.S. Department of Agriculture strains were used as a reference. The dendrogram indicated seven clusters based on similarities among the reference strains. The occupancy rate of the Bj123 cluster decreased with increasing cultivation temperature, whereas the occupancy rates of the Bj110 cluster, Be76 cluster, and Be94 cluster increased with increasing cultivation temperature. In particular, the Rj(2)Rj(3)Rj(4) genotype soybeans were infected with a number of Bj110 clusters, regardless of the increasing cultivation temperature, compared to other Rj genotype soybean cultivars. The ratio of beta diversity to gamma diversity (H'(ß)/H'(γ)), which represents differences in the bradyrhizobial communities by pairwise comparison among cultivation temperature sets within the same soybean cultivar, indicated that the bradyrhizobial communities tended to be different among cultivation temperatures. Multidimensional scaling analysis indicated that the infection of the Bj110 cluster and the Bj123 cluster by host soybean genotype and the cultivation temperature affected the bradyrhizobial communities. These results suggested that the Rj genotypes and cultivation temperatures affected the nodulation tendency and community structures of soybean-nodulating bradyrhizobia.

Rj (rj) genes involved in nitrogen-fixing root nodule formation in soybean.

It has long been known that formation of symbiotic root nodules in soybean (Glycine max (L.) Merr.) is controlled by several host genes referred to as Rj (rj) genes, but molecular cloning of these genes has been hampered by soybean's complicated genome structure and large genome size. Progress in molecular identification of legume genes involved in root nodule symbiosis have been mostly achieved by using two model legumes, Lotus japonicus and Medicago truncatula, that have relatively simple and small genomes and are capable of molecular transfection. However, recent development of resources for soybean molecular genetic research, such as genome sequencing, large EST databases, and high-density linkage maps, have enabled us to isolate several Rj genes. This progress has been achieved in connection with systematic utilization of the information obtained from molecular genetics of the model legumes. In this review, we summarize the current status of knowledge of host-controlled nodulation in soybean based on information from recent studies on Rj genes, and discuss the future research prospects.

Development of an effective method of human DNA extraction from soil as forensic evidence.

In forensic investigations, it is crucial to detect a suspect's DNA from the available evidence. In an outdoor crime scene, the evidence may be mixed with the soil. However, soil is speculated to inhibit DNA extraction from forensic science samples. In the field of soil microbiology, it is necessary to extract DNA directly from soil to analyse its microbial composition. In this study, we investigated whether skim milk used in procedure of DNA extraction from soil samples could be applied to forensic science to enhance human DNA extraction efficiency from soil mixed with forensic evidence, such as blood, buccal cells, and skin cells. The use of additive reagents, skim milk and bovine serum albumin (BSA), are known to blocking reagent. In the selection of additive reagents experiment, about blood sample, using the skim milk and BSA were found to increase the DNA yield. Therefore, we observed extracted DNA yield from blood, buccal cells, and skin cells when skim milk and BSA were used as additive reagents. The DNA recovery rate was high across all samples upon addition of skim milk. However, in STR analysis, a non-specific peak was detected in the extracted DNA in the presence of skim milk, which was not detected in the presence of BSA, indicating its suitability in forensic analysis. Our study suggests that addition of BSA can efficiently aid the extraction of DNA from forensic evidence mixed with soil.

Catalytic biomineralization of fluorescent calcite by the thermophilic bacterium Geobacillus thermoglucosidasius.

The thermophilic Geobacillus bacterium catalyzed the formation of 100-µm hexagonal crystals at 60°C in a hydrogel containing sodium acetate, calcium chloride, and magnesium sulfate. Under fluorescence microscopy, crystals fluoresced upon excitation at 365 ± 5, 480 ± 20, or 545 ± 15 nm. X-ray diffraction indicated that the crystals were magnesium-calcite in calcite-type calcium carbonate.

Interleukin-17A/F1 Deficiency Reduces Antimicrobial Gene Expression and Contributes to Microbiome Alterations in Intestines of Japanese medaka (Oryzias latipes).

In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1ß (IL-1ß), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.

Randomized, double-blind, placebo, and risperidone-controlled study of lurasidone in the treatment of schizophrenia: Results of an inconclusive 6-week trial.

OBJECTIVE: The efficacy and safety of lurasidone in schizophrenia has been demonstrated in multiple controlled trials, primarily in US and European populations. The aim of the current study was To evaluate lurasidone for the treatment of schizophrenia among patients in Japan, Korea, and Taiwan. METHODS: Hospitalized patients (N = 460) with schizophrenia were randomized to 6 weeks of fixed-dose lurasidone 40 mg/d, lurasidone 80 mg/d, risperidone 4 mg/d, or placebo. Efficacy was assessed using the Positive and Negative Syndrome Scale (PANSS) and Clinical Global Impression-Severity (CGI-S). RESULTS: No significant endpoint differences in PANSS total score were found for lurasidone or risperidone vs placebo. Lurasidone was safe and well tolerated, with minimal effects on weight and metabolic parameters. DISCUSSION: The current study was inconclusive regarding the efficacy of lurasidone in schizophrenia but further confirmed its safety and tolerability.

Influence of flooding and soil properties on the genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in the Philippines.

One of the strategies that is commonly used in the Philippines to improve the production of soybean is by inoculation. However, this technique often fails mainly due to the lack of information about the indigenous soybean rhizobia in the Philippines soil. In this study, the diversity of indigenous bradyrhizobia collected from the non-flooded and flooded soil conditions at 11 locations in the country was investigated using a local soybean cultivar as the host plant. The genetic variation among the 424 isolates was detected through Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) treatment and sequence analysis for 16S rRNA gene, 16S-23S rRNA internal transcribed spacer (ITS) region and rpoB housekeeping gene. All the isolates were classified under the Bradyrhizobium species namely B. elkanii, B. diazoefficiens, B. japonicum, B. yuanmingense and a considerable proportion of the isolates were clustered under Bradyrhizobium sp. The isolates which were classified under Bradyrhizobium sp. were thought to be endemic to Philippines soil as evidenced by their nucleotide divergence against the known rhizobia and the historical absence of rhizobia inoculation in the collection sites. The major influence on the distribution and diversity of soybean bradyrhizobia is attributed to the difference in the flooding period, followed by soil properties such as pH, soil type, and nutrient content. As determined, it is proposed that the major micro-symbiont of soybean in the Philippines are B. elkanii for non-flooded soils, then B. diazoefficiens and B. japonicum for flooded soils.

Effect of Flooding and the nosZ Gene in Bradyrhizobia on Bradyrhizobial Community Structure in the Soil.

We investigated the effects of the water status (flooded or non-flooded) and presence of the nosZ gene in bradyrhizobia on the bradyrhizobial community structure in a factorial experiment that examined three temperature levels (20°C, 25°C, and 30°C) and two soil types (andosol and gray lowland soil) using microcosm incubations. All microcosms were inoculated with Bradyrhizobium japonicum USDA6T, B. japonicum USDA123, and B. elkanii USDA76T, which do not possess the nosZ gene, and then half received B. diazoefficiens USDA110Twt (wt for the wild-type) and the other half received B. diazoefficiens USDA110ΔnosZ. USDA110Twt possesses the nosZ gene, which encodes N2O reductase; 110ΔnosZ, a mutant variant, does not. Changes in the community structure after 30- and 60-d incubations were investigated by denaturing-gradient gel electrophoresis and an image analysis. USDA6T and 76T strains slightly increased in non-flooded soil regardless of which USDA110T strain was present. In flooded microcosms with the USDA110Twt strain, USDA110Twt became dominant, whereas in microcosms with the USDA110ΔnosZ, a similar change in the community structure occurred to that in non-flooded microcosms. These results suggest that possession of the nosZ gene confers a competitive advantage to B. diazoefficiens USDA110T in flooded soil. We herein demonstrated that the dominance of B. diazoefficiens USDA110Twt within the soil bradyrhizobial population may be enhanced by periods of flooding or waterlogging systems such as paddy-soybean rotations because it appears to have the ability to thrive in moderately anaerobic soil.

Temperature-Dependent Expression of NodC and Community Structure of Soybean-Nodulating Bradyrhizobia.

In order to assess the physiological responses of bradyrhizobia and competition for the nodulation of soybean at different temperatures, we investigated the expression of the nodC gene at 20, 25, and 30°C and the abilities of bacteria to nodulate soybean in microcosms at day/night cultivation temperatures of 23/18°C, 28/23°C, and 33/28°C for 16/8 h. We tested five Bradyrhizobium USDA strains: B. diazoefficiens USDA 110(T) and 122, B. japonicum USDA 123, and B. elkanii USDA 31 and 76(T). The expression of nodC was up-regulated by increasing culture temperatures in USDA 110(T), 122, 31, and 76(T), but was down-regulated in USDA 123. The proportions of USDA 110(T) and 122 within the community were the greatest at 28/23°C. The population of USDA 31 increased, whereas that of USDA 123 decreased with increasing cultivation temperatures. On the other hand, infection by USDA 76(T) was not detected, and low numbers of USDA 76(T) nodules confirmed its poor nodulation ability. These results indicate that the competitiveness of and infection by USDA 110(T), 122, 123, and 31 for soybean nodulation depend on cultivation temperatures, and suggest that the temperature dependence of nodC expression affects the bradyrhizobial community structure.

Sulphation of acetaminophen by the human cytosolic sulfotransferases: a systematic analysis.

Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo. This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported.

Chrysotile fibers penetrate Escherichia coli cell membrane and cause cell bursting by sliding friction force on agar plates.

A mixture (50 microl) consisting of Escherichia coli cells, chrysotile fibers, and 200 mM NaCl was added to 2% agar plates and spread with a plastic stir stick. An apparatus was developed to generate a sliding friction force on the surface of the plate by applying a fixed vertical reaction force to the stir stick while turning the plate. This operation was defined as chrysotile-exposure. The number of living cells, expressed as colony forming units, was reduced in proportion to the duration of chrysotile-exposure. The decrease in the number of living cells was greater following 120 s of chrysotile-exposure than crocidolite- or amosite-exposure. There was no decrease in the number of living cells exposed to chrysotile on 0.5% agar even after 120 s. The number of living cells after chrysotile-exposure decreased with increasing chrysotile concentration. Leakage of beta-galactosidase from cells increased with increasing duration of chrysotile-exposure. Transmission and scanning electron microscopy observation of E. coli revealed chrysotile penetration of the cell membrane. These results show that chrysotile fibers destroy cells by penetration. The driving force for the chrysotile penetration was the sliding friction force, as the number of living cells following chrysotile-exposure decreased with increasing exposure times, but did not decrease following chrysotile-exposure on a low concentration of agar, which provided cells with low sliding friction force.

Relationship between soil type and N2O reductase genotype (nosZ) of indigenous soybean bradyrhizobia: nosZ-minus populations are dominant in Andosols.

Bradyrhizobium japonicum strains that have the nosZ gene, which encodes N2O reductase, are able to mitigate N2O emissions from soils (15). To examine the distribution of nosZ genotypes among Japanese indigenous soybean bradyrhizobia, we isolated bradyrhizobia from the root nodules of soybean plants inoculated with 32 different soils and analyzed their nosZ and nodC genotypes. The 1556 resultant isolates were classified into the nosZ+/nodC+ genotype (855 isolates) and nosZ-/nodC+ genotype (701 isolates). The 11 soil samples in which nosZ- isolates significantly dominated (P < 0.05; the χ(2) test) were all Andosols (a volcanic ash soil prevalent in agricultural fields in Japan), whereas the 17 soil samples in which nosZ+ isolates significantly dominated were mainly alluvial soils (non-volcanic ash soils). This result was supported by a principal component analysis of environmental factors: the dominance of the nosZ- genotype was positively correlated with total N, total C, and the phosphate absorption coefficient in the soils, which are soil properties typical of Andosols. Internal transcribed spacer sequencing of representative isolates showed that the nosZ+ and nosZ- isolates of B. japonicum fell mainly into the USDA110 (BJ1) and USDA6 (BJ2) groups, respectively. These results demonstrated that the group lacking nosZ was dominant in Andosols, which can be a target soil type for an N2O mitigation strategy in soybean fields. We herein discussed how the nosZ genotypes of soybean bradyrhizobia depended on soil types in terms of N2O respiration selection and genomic determinants for soil adaptation.

Mathematical ecology analysis of geographical distribution of soybean-nodulating Bradyrhizobia in Japan.

We characterized the relationship between the genetic diversity of indigenous soybean-nodulating bradyrhizobia from weakly acidic soils in Japan and their geographical distribution in an ecological study of indigenous soybean rhizobia. We isolated bradyrhizobia from three kinds of Rj-genotype soybeans. Their genetic diversity and community structure were analyzed by PCR-RFLP analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) region with 11 Bradyrhizobium USDA strains as references. We used data from the present study and previous studies to carry out mathematical ecological analyses, multidimensional scaling analysis with the Bray-Curtis index, polar ordination analysis, and multiple regression analyses to characterize the relationship between soybean-nodulating bradyrhizobial community structures and their geographical distribution. The mathematical ecological approaches used in this study demonstrated the presence of ecological niches and suggested the geographical distribution of soybean-nodulating bradyrhizobia to be a function of latitude and the related climate, with clusters in the order Bj123, Bj110, Bj6, and Be76 from north to south in Japan.

Phylogenetic diversity of indigenous cowpea bradyrhizobia from soils in Japan based on sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region.

Cowpea [Vigna unguiculata (L.) Walp.] is an important legume crop and yet its rhizobia have not been well characterized in many areas. In the present study, sequence analysis of the bacterial 16S-23S rRNA internal transcribed spacer (ITS) region was performed to characterize genetically 76 indigenous cowpea rhizobia from five different geographic regions (Okinawa, Miyazaki, Kyoto, Fukushima and Hokkaido) of Japan. The sequence analysis clustered all isolates in the genus Bradyrhizobium. They were conspecific with B. japonicum, B. yuanmingense, B. elkanii and Bradyrhizobium sp., although none of them grouped with B. liaoningense, B. canariense, B. betae or B. iriomotense. B. yuanmingense was only isolated from the southern region (Okinawa) where it achieved the highest frequency of 69%. B. japonicum was predominant at Miyazaki, Fukushima and Hokkaido with more than 60% of the isolates. B. elkanii was mainly recorded in the southern (Okinawa: 31%, Miyazaki: 33%) and middle (Kyoto: 33%) regions. This species was present at a very low frequency in Fukushima and absent in Hokkaido in the northern area. Bradyrhizobium sp. like-strains were absent in the southern part (Okinawa, Miyazaki) but were concentrated either in the middle regions with 67% of Kyoto isolates and 28% of Fukushima isolates, and in the northern region with 40% of the Hokkaido isolates. This study revealed a geographical distribution of cowpea bradyrhizobia which seemed to be related to the differences in the environmental characteristics (soil type and soil pH, temperature, climate, moisture) of the different regions in Japan.

Changes in population occupancy of Bradyrhizobia under different temperature regimes.

To elucidate how temperature affects bradyrhizobial ecology, long-term incubations of Bradyrhizobium japonicum USDA 6(T), 38, and 123 and of Bradyrhizobium elkanii USDA 76(T) were conducted under various temperature conditions. Proliferative traits in liquid culture and population occupancies in soil microcosms were compared. The occupancies of USDA 76(T) and USDA 123 in soil microcosms during long-term incubation changed with the temperature conditions. These results suggest that temperature is an environmental factor affecting the ecology and occupancy of bradyrhizobia in soils.

Phylogenetic diversity and symbiotic effectiveness of root-nodulating bacteria associated with cowpea in the South-West area of Japan.

The phylogenetic diversity of cowpea root-nodulating bacteria in the South-West of Japan was investigated using 60 isolates. Seeds of cowpea were aseptically sown in vermiculite and inoculated with a suspension of Cowpea Soil (CS) or Bean Soil (BS) or without a soil suspension as a control. CS and BS were collected from the Kyushu University's farm (Japan) at sites where cowpea and bean, respectively, have been cultivated previously. Based on an analysis of the 16S rRNA gene and the Internal Transcribed Spacer (ITS) sequence between the 16S and 23S rRNA genes, 56 isolates were assigned to the genus Bradyrhizobium, while one isolate was found to be closely related to the genus Ralstonia. The ITS-based phylogeny showed 53 isolates, 2 isolates, and 1 isolate, to be closely related to B. yuanmingense, B. elkanii and B. japonicum, respectively, suggesting that B. yuanmingense strains predominated in the soils. Among the isolates tested, B. yuanmingense TSC10 and TTC9 exhibited a greater symbiotic activity and could be considered efficient inoculants for cowpea.