Genome evolution related to γ-hexachlorocyclohexane metabolic function in the soil microbial population.

γ-Hexachlorocyclohexane (γ-HCH)-degrading strain, Sphingobium sp. TA15, was newly isolated from an experimental field soil from which the archetypal γ-HCH-degrading strain, S. japonicum UT26, was isolated previously. Comparison of the complete genome sequences of these 2 strains revealed that TA15 shares the same basic genome backbone with UT26, but also has the variable regions that are presumed to have changed either from UT26 or from a putative common ancestor. Organization and localization of lin genes of TA15 were different from those of UT26. It was inferred that transposition of IS6100 had played a crucial role in these genome rearrangements. The accumulation of toxic dead-end products in TA15 was lower than in UT26, suggesting that TA15 utilizes γ-HCH more effectively than UT26. These results suggested that genome evolution related to the γ-HCH metabolic function in the soil microbial population is ongoing.

Noviherbaspirillum denitrificans sp. nov., a denitrifying bacterium isolated from rice paddy soil and Noviherbaspirillum autotrophicum sp. nov., a denitrifying, facultatively autotrophic bacterium isolated from rice paddy soil and proposal to reclassify Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.

Thirty-nine denitrifying bacterial strains closely related to one another, represented by strains TSA40T and TSA66T, were isolated from rice paddy soils. Strains TSA40T and TSA66T were Gram-stain-negative, slightly curved rod-shaped, and motile by means of polar flagella. They were able to reduce nitrate, nitrite and nitrous oxide, but unable to fix atmospheric N2. While strain TSA66T was able to grow autotrophically by H2-dependent denitrification, strain TSA40T could not. Phylogenetic analysis suggested that they belong to the family Oxalobacteraceae, the order Burkholderiales in the class Betaproteobacteria. Major components in the fatty acids (C16 : 0, C17 : 0 cyclo, C18 : 1ω7c and summed feature 3) and quinone (Q-8) also supported the affiliation of strains TSA40T and TSA66T to the family Oxalobacteraceae. Based on 16S rRNA gene sequence comparisons, strains TSA40T and TSA66T showed the greatest degree of similarity to Herbaspirillum massiliense JC206T, Noviherbaspirillum malthae CC-AFH3T, Noviherbaspirillum humi U15T, Herbaspirillum seropedicae Z67T and Paucimonas lemoignei LMG 2207T, and lower similarities to the members of other genera. Average nucleotide identity values between the genomes of strain TSA40T, TSA66T and H. massiliense JC206T were 75-77 %, which was lower than the threshold value for species discrimination (95-96 %). Based on the 16S rRNA gene sequence analysis in combination with physiological, chemotaxonomic and genomic properties, strains TSA40T (=JCM 17722T=ATCC TSD-69T) and TSA66T (=JCM 17723T=DSM 25787T) are the type strains of two novel species within the genus Noviherbaspirillum, for which the names Noviherbaspirillum denitrificans sp. nov. and Noviherbaspirillum autotrophicum sp. nov. are proposed, respectively. We also propose the reclassification of Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.

Dictyobacter aurantiacus gen. nov., sp. nov., a member of the family Ktedonobacteraceae, isolated from soil, and emended description of the genus Thermosporothrix.

A mesophilic, Gram-stain-positive, spore-forming bacterium that formed branched mycelia was isolated from paddy soil in Gunung Salak (Mount Salak), West Java, Indonesia. This strain, designated S-27T, grew at temperatures between 20 and 37 °C; the optimum growth temperature was 25 to 30 °C, and no growth was observed at 15 or 45 °C. The pH range for growth was pH 3.5 to 8.6; the optimum pH was 6.0, and no growth was observed at pH 3.0 or 9.2. Strain S-27T was able to hydrolyse polysaccharides such as starch, cellulose and xylan. The G+C content of the DNA of strain S-27T was 55.7 mol%. The major fatty acids were iso-C17 : 0 and C16 : 1 2-OH, and the major menaquinone was MK-9 (H2). The cell wall of strain S-27T contained d-glutamic acid, glycine, l-alanine, d-alanine, l-ornithine and ß-alanine in a molar ratio of 1.0 : 1.6 : 1.4 : 0.6 : 0.9 : 1.1. The polar lipids consisted of phosphatidylglycerol, phosphatidylinositol and two glycolipids. The major cell-wall sugar was arabinose. Detailed phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S-27T belongs to the order Ktedonobacterales and is most closely related to Ktedonobacter racemifer SOSP1-21T (89.6 % sequence identity). On the basis of its chemotaxonomic and phenotypic features and phylogenetic position, we concluded that strain S-27T represents a novel genus and species, for which we propose the name Dictyobacter aurantiacus gen. nov., sp. nov. The type strain of Dictyobacter aurantiacus is strain S-27T (=NBRC 109595T=InaCC B312T). Emendation of the description of the genus Thermosporothrix is also provided.

Complete genome sequence of Bacillus subtilis NA05 (=NBRC 116153), a phosphate-solubilizing bacterium isolated from crop field soil.

We report the complete genome sequence of the phosphate-solubilizing bacterium Bacillus subtilis NA05 (=NBRC 116153), consisting of a circular chromosome of ~3.8 M bp and two circular plasmids. The data presented here provide further insight into the genetic and functional potential of B. subtilis and the mechanism of phosphate solubilization.

Brevifollis gellanilyticus gen. nov., sp. nov., a gellan-gum-degrading bacterium of the phylum Verrucomicrobia.

The taxonomic properties of strain DC2c-G4(T), a Gram-staining-negative, ovoid, gellan-gum-degrading bacterial isolate, were examined. Phylogenetic analysis based on 16S rRNA gene sequences identified this isolate as a member of the phylum Verrucomicrobia and closest to the genus Prosthecobacter. The 16S rRNA gene sequence similarities between this isolate and any of the type strains of species of the genus Prosthecobacter were less than 95 %. In addition, the absence of a single prostheca and the predominant menaquinone MK-7(H2) supported the differentiation of this isolate from the genus Prosthecobacter. Here, we propose Brevifollis gellanilyticus gen. nov., sp. nov. to accommodate the isolate. The type strain of the type species is DC2c-G4(T) (= NBRC 108608(T) = CIP 110457(T)).

Roseimicrobium gellanilyticum gen. nov., sp. nov., a new member of the class Verrucomicrobiae.

The taxonomic properties of strain DC2a-G7(T), a Gram-negative, ovoid to rod-shaped, gellan gum-lysing bacterium, were examined. The 16S rRNA gene sequence similarity showed that DC2a-G7(T) is a member of the phylum Verrucomicrobia and the closest type strain of a species with a validly published name is Verrucomicrobium spinosum DSM 4136(T), with a sequence similarity of 91.2%. In addition to this similarity value lower than 95%, the absence of prostheca, the orangey-red colony colour and the compositions of the major menaquinones and polar lipids also supported the differentiation of this bacterium from the genus Verrucomicrobium. Here, we propose the name Roseimicrobium gellanilyticum gen. nov., sp. nov. for the isolate. The type strain of Roseimicrobium gellanilyticum is DC2a-G7(T) (=NBRC 108606(T)=DSM 25532(T)).

Complete genome sequence of the denitrifying and N2O-reducing bacterium Azoarcus sp. strain KH32C.

We report the finished and annotated genome sequence of a denitrifying and N(2)O-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.

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 N(2)O 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.

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria.

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.

Eukaryotic Microbial Communities in Japanese Arable Andisols Investigated by Amplicon Sequencing of 18S rRNA Genes.

Compared with the well-studied soil prokaryotic communities, little is known about soil eukaryotic communities. Here, we investigated the eukaryotic community structures in 43 arable soils using amplicon sequencing of 18S rRNA genes. Major taxonomic groups, such as Fungi, Holozoa, and Stramenopiles, were detected in all samples.

Dissimilatory Nitrate Reduction to Ammonium and Responsible Microbes in Japanese Rice Paddy Soil.

Nitrification-denitrification processes in the nitrogen cycle have been extensively examined in rice paddy soils. Nitrate is generally depleted in the reduced soil layer below the thin oxidized layer at the surface, and this may be attributed to high denitrification activity. In the present study, we investigated dissimilatory nitrate reduction to ammonium (DNRA), which competes with denitrification for nitrate, in order to challenge the conventional view of nitrogen cycling in paddy soils. We performed paddy soil microcosm experiments using 15N tracer analyses to assess DNRA and denitrification rates and conducted clone library analyses of transcripts of nitrite reductase genes (nrfA, nirS, and nirK) in order to identify the microbial populations carrying out these processes. The results obtained showed that DNRA occurred to a similar extent to denitrification and appeared to be enhanced by a nitrate limitation relative to organic carbon. We also demonstrated that different microbial taxa were responsible for these distinct processes. Based on these results and previous field observations, nitrate produced by nitrification within the surface oxidized layer may be reduced not only to gaseous N2 via denitrification, but also to NH4+ via DNRA, within the reduced layer. The present results also indicate that DNRA reduces N loss through denitrification and nitrate leaching and provides ammonium to rice roots in rice paddy fields.

Distinct Community Composition of Previously Uncharacterized Denitrifying Bacteria and Fungi across Different Land-Use Types.

Recent studies demonstrated that phylogenetically more diverse and abundant bacteria and fungi than previously considered are responsible for denitrification in terrestrial environments. We herein examined the effects of land-use types on the community composition of those denitrifying microbes based on their nitrite reductase gene (nirK and nirS) sequences. These genes can be phylogenetically grouped into several clusters. We used cluster-specific PCR primers to amplify nirK and nirS belonging to each cluster because the most widely used primers only amplify genes belonging to a single cluster. We found that the dominant taxa as well as overall community composition of denitrifying bacteria and fungi, regardless of the cluster they belonged to, differed according to the land-use type. We also identified distinguishing taxa based on individual land-use types, the distribution of which has not previously been characterized, such as denitrifying bacteria or fungi dominant in forest soils, Rhodanobacter having nirK, Penicillium having nirK, and Bradyrhizobium having nirS. These results suggest that land-use management affects the ecological constraints and consequences of denitrification in terrestrial environments through the assembly of distinct communities of denitrifiers.

Microbial populations responsive to denitrification-inducing conditions in rice paddy soil, as revealed by comparative 16S rRNA gene analysis.

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.

Community composition of soil bacteria nearly a decade after a fire in a tropical rainforest in East Kalimantan, Indonesia.

Soil bacterial community compositions in burnt and unburnt areas in a tropical rainforest in East Kalimantan, Indonesia, were investigated 8 and 9 years after a fire by denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene. Three study sites were set in the forest area devoid of fire damage (control), and in the lightly damaged and heavily damaged forest areas. Succession of aboveground vegetation in the two damaged areas had clearly proceeded after the fire, but the vegetation types still differed from the unburnt area at the time of this study. Community composition of total soil bacteria was similar among the three areas, and so was that of actinobacteria. However, the composition of ammonia oxidizing bacteria clearly differed depending on the presence or absence of past fire damage. These results indicate that even nearly a decade after the forest fire, impacts of the fire remained on the community composition of ammonia oxidizing bacteria, but not apparently on those of dominant bacteria and actinobacteria.

Prokaryotic Community Structure of Long-Term Fertilization Field Andisols in Central Japan.

Long-term fertilization experiments are a useful way to elucidate the impacts of fertilization on soil ecosystems. Here, we report the prokaryotic community structure in experimental field soil after 80 years of successive fertilization. Our 16S rRNA gene sequencing detected 20,996 amplicon sequence variants, including major phyla such as Proteobacteria, Acidobacteria, and Actinobacteria.

Presence of previously undescribed bacterial taxa in non-axenic Chlorella cultures.

We determined the bacterial community profile in non-axenic cultures of Chlorella (Chlorophyceae, Chlorophyta) isolated from soil. The bacterial composition at the phylum level was different from that of whole soil bacteria, but it was similar to that reported for non-axenic cultures of marine microalgae such as diatoms (Bacillariophyceae, Heterokontophyta). Expected novel bacteria, i.e. those which do not have close relatives among described species, were maintained in the cultures, and these bacteria were chiefly composed of members of the phylum Bacteroidetes. They may have been 'as-yet-uncultured' but in practice unintentionally been cultured in microalgal cultures. They could serve as good bioresources in various fields of biological and ecological studies.

Phosphorus-mineralizing Communities Reflect Nutrient-Rich Characteristics in Japanese Arable Andisols.

Elucidating the soil phosphorus cycle driven by soil microbes is a vital question in soil microbial ecology. The Japanese arable Andisols, occupying half of the Japanese cropland, are known for their high phosphorus sorption capacity. However, limited information is currently available on microbially driven phosphorus mineralization in arable Andisols. We herein report that the phosphorus-mineralizing community in the Japanese arable Andisols showed characteristic distribution and composition patterns, from those in other types of soils. We performed a chemical analysis and microbial community analysis of 43 arable Andisols along the Japanese archipelago. Soil phosphomonoesterase activities measured at pH 11 were approximately 70% of those at pH 6.5, which indicates that alkaline phosphatase contributes to phosphorus cycling, although most soil samples were acidic. Functional gene predictions based on 16S rRNA gene sequencing indicated that the alkaline phosphatase gene phoD was more abundant than other alkaline phosphatase genes and, thus, plays major roles. Hence, amplicon sequencing targeting phoD was performed and the results obtained showed that alphaproteobacterial phoD was dominant. This is in contrast to previously reported phoD compositions in other soils and may be attributed to the nutrient conditions in arable Andisols, which favor copiotrophic Alphaproteobacteria. Furthermore, the composition of phoD correlated with soil pH and bioavailable phosphorus concentrations rather than carbon or nitrogen concentrations. These results were partly different from previous findings, varying in the soil types and geographic ranges of sampling sites. Collectively, the present results indicate that the phosphorus-mineralizing community in the Japanese arable Andisols is regulated differently from those in other soil types.

Nitrogen supply rate regulates microbial resource allocation for synthesis of nitrogen-acquiring enzymes.

Although microorganisms will preferentially allocate resources to synthesis of nitrogen (N)-acquiring enzymes when soil N availability is low according to the resource allocation model for extracellular enzyme synthesis, a robust link between microbial N-acquiring enzyme activity and soil N concentration has not been reported. To verify this link, we measured several indices of soil N availability and enzyme activity of four N-acquiring enzymes [N-acetyl-ß-glucosaminidase (NAG), protease (PR), urease (UR), and L-asparaginase (LA)] and a carbon (C)-acquiring enzyme [ß-D-glucosidase (BG)] in arable and forest soils. Although the ratios of NAG/BG and PR/BG were not significantly related with indices of soil N availability, ratios of LA/BG and UR/BG were strongly and negatively related with potentially mineralizable N estimated by aerobic incubation but not with pools of labile inorganic N and organic N. These results suggest that microorganisms might allocate their resources to LA and UR synthesis in response to N supply rate rather than the size of the easily available N pools. It was also suggested that the underlying mechanism for synthesis was different between these N-acquiring enzymes in soil microorganisms: microbial LA and UR were primarily synthesized to acquire N, whereas NAG and PR syntheses were regulated not only by N availability but also by other factors.

Presence of Cu-Type (NirK) and cd1-Type (NirS) Nitrite Reductase Genes in the Denitrifying Bacterium Bradyrhizobium nitroreducens sp. nov.

Nitrite reductase is a key enzyme for denitrification. There are two types of nitrite reductases: copper-containing NirK and cytochrome cd1-containing NirS. Most denitrifiers possess either nirK or nirS, although a few strains been reported to possess both genes. We herein report the presence of nirK and nirS in the soil-denitrifying bacterium Bradyrhizobium sp. strain TSA1T. Both nirK and nirS were identified and actively transcribed under denitrification conditions. Based on physiological, chemotaxonomic, and genomic properties, strain TSA1T (=JCM 18858T=KCTC 62391T) represents a novel species within the genus Bradyrhizobium, for which we propose the name Bradyrhizobium nitroreducens sp. nov.

Predominant but Previously-overlooked Prokaryotic Drivers of Reductive Nitrogen Transformation in Paddy Soils, Revealed by Metatranscriptomics.

Waterlogged paddy soils possess anoxic zones in which microbes actively induce reductive nitrogen transformation (RNT). In the present study, a shotgun RNA sequencing analysis (metatranscriptomics) of paddy soil samples revealed that most RNT gene transcripts in paddy soils were derived from Deltaproteobacteria, particularly the genera Anaeromyxobacter and Geobacter. Despite the frequent detection of the rRNA of these microbes in paddy soils, their RNT-associated genes have rarely been identified in previous PCR-based studies. This metatranscriptomic analysis provides novel insights into the diversity of RNT microbes present in paddy soils and the ecological function of Deltaproteobacteria predominating in these soils.