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1.  Seasonal Transition of Active Bacterial and Archaeal Communities in Relation to Water Management in Paddy Soils 
Microbes and Environments  2013;28(3):370-380.
Paddy soils have an environment in which waterlogging and drainage occur during the rice growing season. Fingerprinting analysis based on soil RNA indicated that active microbial populations changed in response to water management conditions, although the fundamental microbial community was stable as assessed by DNA-based fingerprinting analysis. Comparative clone library analysis based on bacterial and archaeal 16S rRNAs (5,277 and 5,436 clones, respectively) revealed stable and variable members under waterlogged or drained conditions. Clones related to the class Deltaproteobacteria and phylum Euryarchaeota were most frequently obtained from the samples collected under both waterlogged and drained conditions. Clones related to syntrophic hydrogen-producing bacteria, hydrogenotrophic methanogenic archaea, rice cluster III, V, and IV, and uncultured crenarchaeotal group 1.2 appeared in greater proportion in the samples collected under waterlogged conditions than in those collected under drained conditions, while clones belonging to rice cluster VI related to ammonia-oxidizing archaea (AOA) appeared at higher frequency in the samples collected under drained conditions than in those collected under waterlogged conditions. These results suggested that hydrogenotrophic methanogenesis may become active under waterlogged conditions, whereas ammonia oxidation may progress by rice cluster VI becoming active under drained conditions in the paddy field.
doi:10.1264/jsme2.ME13030
PMCID: PMC4070958  PMID: 24005888
Paddy soil; Soil microbial diversity; Clone libraries
2.  Identification and Phylogenetic Characterization of Cobalamin Biosynthetic Genes of Ensifer adhaerens 
Microbes and Environments  2012;28(1):153-155.
Ensifer adhaerens CSBa was screened as a cobalamin producer. The draft genome sequence revealed that the strain possesses 22 cobalamin biosynthetic genes (cob genes). The cob gene arrangement on the genome of E. adhaerens CSBa was similar to that of other Ensifer species, and most similar to that of Pseudomonas denitrificans SC510. The cobN sequence phylogeny was generally congruent with that of the 16S rRNA gene, and it is suggeted that E. adhaerens CSBa might have inherited the cob genes from common ancestors of the Ensifer species. It was also suggested that the cob genes can be laterally transferred.
doi:10.1264/jsme2.ME12069
PMCID: PMC4070679  PMID: 23257908
cobalamin biosynthetic genes; cob; Ensifer; Sinorhizobium; phylogeny
3.  Identification and isolation of active N2O reducers in rice paddy soil 
The ISME Journal  2011;5(12):1936-1945.
Dissolved N2O is occasionally detected in surface and ground water in rice paddy fields, whereas little or no N2O is emitted to the atmosphere above these fields. This indicates the occurrence of N2O reduction in rice paddy fields; however, identity of the N2O reducers is largely unknown. In this study, we employed both culture-dependent and culture-independent approaches to identify N2O reducers in rice paddy soil. In a soil microcosm, N2O and succinate were added as the electron acceptor and donor, respectively, for N2O reduction. For the stable isotope probing (SIP) experiment, 13C-labeled succinate was used to identify succinate-assimilating microbes under N2O-reducing conditions. DNA was extracted 24 h after incubation, and heavy and light DNA fractions were separated by density gradient ultracentrifugation. Denaturing gradient gel electrophoresis and clone library analysis targeting the 16S rRNA and the N2O reductase gene were performed. For culture-dependent analysis, the microbes that elongated under N2O-reducing conditions in the presence of cell-division inhibitors were individually captured by a micromanipulator and transferred to a low-nutrient medium. The N2O-reducing ability of these strains was examined by gas chromatography/mass spectrometry. Results of the SIP analysis suggested that Burkholderiales and Rhodospirillales bacteria dominated the population under N2O-reducing conditions, in contrast to the control sample (soil incubated with only 13C-succinate). Results of the single-cell isolation technique also indicated that the majority of the N2O-reducing strains belonged to the genera Herbaspirillum (Burkholderiales) and Azospirillum (Rhodospirillales). In addition, Herbaspirillum strains reduced N2O faster than Azospirillum strains. These results suggest that Herbaspirillum spp. may have an important role in N2O reduction in rice paddy soils.
doi:10.1038/ismej.2011.69
PMCID: PMC3223309  PMID: 21677691
denitrification; Herbaspirillum; nitrous oxide; rice paddy soil; single-cell isolation; stable isotope probing
4.  Identification of Active Denitrifiers in Rice Paddy Soil by DNA- and RNA-Based Analyses 
Microbes and Environments  2012;27(4):456-461.
Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil.
doi:10.1264/jsme2.ME12076
PMCID: PMC4103554  PMID: 22972387
denitrification; nirS; nirK; nosZ
5.  Advantages of functional single-cell isolation method over standard agar plate dilution method as a tool for studying denitrifying bacteria in rice paddy soil 
AMB Express  2012;2:50.
We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211–1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution (APD) methods and compared in this study. The proportion of denitrifying bacteria in the total isolates was more than 6-fold larger with FSC isolation (57.1%) compared with the APD method (9.2%). Denitrifying bacteria belonging to Alphaproteobacteria and Bacilli were commonly isolated using both methods, whereas those belonging to Betaproteobacteria, which had been found to be active in the denitrification-inductive paddy soil, were isolated only with the FSC method. On the other hand, Actinobacteria were only isolated using the APD method. The mean potential denitrification activity of the FSC isolates was higher than that of the APD isolates. Overall, FSC isolation was confirmed to be an excellent method for studying denitrifying bacteria compared with the standard agar plate dilution method.
doi:10.1186/2191-0855-2-50
PMCID: PMC3488030  PMID: 22985609
16S rRNA gene; Denitrifying bacteria; Functional single-cell isolation; Phylogenetic analysis; Rice paddy soil
6.  Complete Genome Sequence of the Denitrifying and N2O-Reducing Bacterium Azoarcus sp. Strain KH32C 
Journal of Bacteriology  2012;194(5):1255.
We report the finished and annotated genome sequence of a denitrifying and N2O-reducing betaproteobacterium, Azoarcus sp. strain KH32C. The genome is composed of one chromosome and one megaplasmid and contains genes for plant-microbe interactions and the gene clusters for aromatic-compound degradations.
doi:10.1128/JB.06618-11
PMCID: PMC3294784  PMID: 22328754
7.  Isolation of Oligotrophic Denitrifiers Carrying Previously Uncharacterized Functional Gene Sequences▿ †  
Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N2O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.
doi:10.1128/AEM.02189-10
PMCID: PMC3019733  PMID: 21075882
9.  Microbial Populations Responsive to Denitrification-Inducing Conditions in Rice Paddy Soil, as Revealed by Comparative 16S rRNA Gene Analysis▿ †  
Applied and Environmental Microbiology  2009;75(22):7070-7078.
Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum Firmicutes, including a unique Symbiobacterium clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders Burkholderiales and Rhodocyclales, were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus Herbaspirillum and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to Herbaspirillum are potential key players in nitrate respiration and denitrification under the conditions used.
doi:10.1128/AEM.01481-09
PMCID: PMC2786546  PMID: 19767468

Results 1-9 (9)