Yumi Shimomura

Complete genome sequence of Bradyrhizobium sp. S23321: insights into symbiosis evolution in soil oligotrophs.

Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

Environmental Factors Shaping the Community Structure of Ammonia-Oxidizing Bacteria and Archaea in Sugarcane Field Soil

The effects of environmental factors such as pH and nutrient content on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in soil has been extensively studied using experimental fields. However, how these environmental factors intricately influence the community structure of AOB and AOA in soil from farmers’ fields is unclear. In the present study, the abundance and diversity of AOB and AOA in soils collected from farmers’ sugarcane fields were investigated using quantitative PCR and barcoded pyrosequencing targeting the ammonia monooxygenase alpha subunit (amoA) gene. The abundances of AOB and AOA amoA genes were estimated to be in the range of 1.8 × 105–9.2 × 106 and 1.7 × 106–5.3 × 107 gene copies g dry soil−1, respectively. The abundance of both AOB and AOA positively correlated with the potential nitrification rate. The dominant sequence reads of AOB and AOA were placed in Nitrosospira-related and Nitrososphaera-related clusters in all soils, respectively, which varied at the level of their sub-clusters in each soil. The relationship between these ammonia-oxidizing community structures and soil pH was shown to be significant by the Mantel test. The relative abundances of the OTU1 of Nitrosospira cluster 3 and Nitrososphaera subcluster 7.1 negatively correlated with soil pH. These results indicated that soil pH was the most important factor shaping the AOB and AOA community structures, and that certain subclusters of AOB and AOA adapted to and dominated the acidic soil of agricultural sugarcane fields.

Comparison among amoA Primers Suited for Quantification and Diversity Analyses of Ammonia-Oxidizing Bacteria in Soil

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment.

An acid-tolerant ammonia-oxidizing γ-proteobacterium from soil

Nitrification, the microbial oxidation of ammonia to nitrate via nitrite, occurs in a wide range of acidic soils. However, the ammonia-oxidizing bacteria (AOB) that have been isolated from soil to date are acid-sensitive. Here we report the isolation and characterization of an acid-adapted AOB from an acidic agricultural soil. The isolated AOB, strain TAO100, is classified within the Gammaproteobacteria based on phylogenetic characteristics. TAO100 can grow in the pH range of 5–7.5 and survive in highly acidic conditions until pH 2 by forming cell aggregates. Whereas all known gammaproteobacterial AOB (γ-AOB) species, which have been isolated from marine and saline aquatic environments, are halophiles, TAO100 is not phenotypically halophilic. Thus, TAO100 represents the first soil-originated and non-halophilic γ-AOB. The TAO100 genome is considerably smaller than those of other γ-AOB and lacks several genes associated with salt tolerance which are unnecessary for survival in soil. The ammonia monooxygenase subunit A gene of TAO100 and its transcript are higher in abundance than those of ammonia-oxidizing archaea and betaproteobacterial AOB in the strongly acidic soil. These results indicate that TAO100 plays an important role in the nitrification of acidic soils. Based on these results, we propose TAO100 as a novel species of a new genus, Candidatus Nitrosoglobus terrae.

Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens

Agricultural soil is the largest source of nitrous oxide (N2O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N2O emissions often increase during decomposition of the root nodules. Our previous study showed that N2O reductase can be used to mitigate N2O emission from soybean fields during nodule decomposition by inoculation with nosZ++ strains [mutants with increased N2O reductase (N2OR) activity] of Bradyrhizobium diazoefficiens. Here, we show that N2O emission can be reduced at the field scale by inoculation with a mixed culture of indigenous nosZ+ strains of B. diazoefficiens USDA110 group isolated from Japanese agricultural fields. Our results also suggested that nodule nitrogen is the main source of N2O production during nodule decomposition. Isolating nosZ+ strains from local soybean fields would be more applicable and feasible for many soybean-producing countries than generating mutants.

Responses of denitrifying bacterial communities to short-term waterlogging of soils

Agricultural soil is often subjected to waterlogging after heavy rainfalls, resulting in sharp and explosive increases in the emission of nitrous oxide (N2O), an important greenhouse gas primarily released from agricultural soil ecosystems. Previous studies on waterlogged soil examined the abundance of denitrifiers but not the composition of denitrifier communities in soil. Also, the PCR primers used in those studies could only detect partial groups of denitrifiers. Here, we performed pyrosequencing analyses with the aid of recently developed PCR primers exhibiting high coverage for three denitrification genes, nirK, nirS, and nosZ to examine the effect of short-term waterlogging on denitrifier communities in soil. We found that microbial communities harboring denitrification genes in the top 5 cm of soil distributed according to soil depth, water-soluble carbon, and nitrate nitrogen. Short-term waterlogging scarcely affected abundance, richness, or the alpha-diversities of microbial communities harboring nirK, nirS, and nosZ genes, but significantly affected their composition, particularly in microbial communities at soil depths of 0 to 1 cm. Our results indicated that the composition of denitrifying microbial communities but not the abundance of denitrifiers in soil was responsive to short-term waterlogging of an agricultural soil ecosystem.

Effect of dicyandiamide and polymer coated urea applications on N2O, NO and CH4 fluxes from Andosol and Fluvisol fields

Abstract Soil type influences the effectiveness of enhanced-efficiency fertilizers in reducing nitrous oxide (N2O) and nitric oxide (NO) emissions, although the effect has not been well studied. We measured N2O, NO and methane (CH4) fluxes after the application of enhanced-efficiency fertilizers and conventional fertilizer (urea) in two contrasting soils, an Andosol and a Fluvisol, in lysimeter fields. Brassica rapa var. perviridis L.H.Bailey (komatsuna) was cultivated for 1.5 months in spring and in autumn. A nitrification inhibitor, dicyandiamide (DCD), and polymer coated urea (CU) were tested in the spring and autumn experiments, respectively. In spring, DCD was effective in reducing N2O and NO emissions in the Andosol but not in the Fluvisol, compared with urea. Nitrification was likely to be a more important production process for N2O and NO in the Andosol than in the Fluvisol. This difference in N2O and NO production processes was inferred to be the main reason why DCD effectively reduced N2O and NO emissions only in the Andosol. Yield-scaled N2O emission for DCD was lower by 63% than for urea in the Andosol, but no difference was observed in the Fluvisol. In autumn in the Andosol, CU increased N2O emission compared with urea, but no difference was observed for NO emissions. In the Fluvisol, CU was not effective in reducing N2O and NO emissions. CH4 uptake from the Andosol was significantly higher than that from the Fluvisol. Fertilizer type had no effect on cumulative CH4 uptake in either soil. Our results showed that the effectiveness of DCD and CU in reducing N2O and NO emissions varied with soil because the main production processes of these gases varied with soil.

A 2,4-dichlorophenoxyacetic acid degradation plasmid pM7012 discloses distribution of an unclassified megaplasmid group across bacterial species.

Analysis of the complete nucleotide sequence of plasmid pM7012 from 2,4-dichlorophenoxyacetic-acid (2,4-D)-degrading bacterium Burkholderia sp. M701 revealed that the plasmid had 582 142 bp, with 541 putative protein-coding sequences and 39 putative tRNA genes for the transport of the standard 20 aa. pM7012 contains sequences homologous to the regions involved in conjugal transfer and plasmid maintenance found in plasmids byi_2p from Burkholderia sp. YI23 and pBVIE01 from Burkholderia sp. G4. No relaxase gene was found in any of these plasmids, although genes for a type IV secretion system and type IV coupling proteins were identified. Plasmids with no relaxase gene have been classified as non-mobile plasmids. However, nucleotide sequences with a high level of similarity to the genes for plasmid transfer, plasmid maintenance, 2,4-D degradation and arsenic resistance contained on pM7012 were also detected in eight other megaplasmids (~600 or 900 kb) found in seven Burkholderia strains and a strain of Cupriavidus, which were isolated as 2,4-D-degrading bacteria in Japan and the United States. These results suggested that the 2,4-D degradation megaplasmids related to pM7012 are mobile and distributed across various bacterial species worldwide, and that the plasmid group could be distinguished from known mobile plasmid groups.