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Endohyphal bacteria (EHB), dwelling within fungal hyphae, markedly affect the growth and metabolic potential of their hosts. To date, two EHB belonging to the family Burkholderiaceae have been isolated and characterized as new taxa, Burkholderia rhizoxinica (HKI 454T) and Mycoavidus cysteinexigens (B1-EBT), in Japan. Metagenome sequencing was recently reported for Mortierella elongata AG77 together with its endosymbiont M. cysteinexigens (Mc-AG77) from a soil/litter sample in the USA. In the present study, we elucidated the complete genome sequence of B1-EBT and compared it with those of Mc-AG77 and HKI 454T. The genomes of B1-EBT and Mc-AG77 contained a higher level of prophage sequences and were markedly smaller than that of HKI 454T. Although the B1-EBT and Mc-AG77 genomes lacked the chitinolytic enzyme genes responsible for invasion into fungal cells, they contained several predicted toxin-antitoxin systems including an insecticidal toxin complex and PIN domain imposing an addiction-like mechanism essential for endohyphal growth control during host colonization. Despite the different host fungi, the alignment of amino acid sequences showed that the HKI 454T genome consisted of 1,265 (32.6%) and 1,221 (31.5%) orthologous coding sequences (CDSs) with those of B1-EBT and Mc-AG77, respectively. This comparative study of three phylogenetically associated endosymbionts has provided insights into their origin and evolution, and suggests the later bacterial invasion and adaptation of B1-EBT to its host metabolism.

Endohyphal bacteria (EHB), dwelling within fungal hyphae, markedly affect the growth and metabolic potential of their hosts. To date, two EHB belonging to the family Burkholderiaceae have been isolated and characterized as new taxa, Burkholderia rhizoxinica (HKI 454T) and Mycoavidus cysteinexigens (B1-EBT), in Japan. Metagenome sequencing was recently reported for Mortierella elongata AG77 together with its endosymbiont M. cysteinexigens (Mc-AG77) from a soil/litter sample in the USA. In the present study, we elucidated the complete genome sequence of B1-EBT and compared it with those of Mc-AG77 and HKI 454T. The genomes of B1-EBT and Mc-AG77 contained a higher level of prophage sequences and were markedly smaller than that of HKI 454T. Although the B1-EBT and Mc-AG77 genomes lacked the chitinolytic enzyme genes responsible for invasion into fungal cells, they contained several predicted toxin-antitoxin systems including an insecticidal toxin complex and PIN domain imposing an addiction-like mechanism essential for endohyphal growth control during host colonization. Despite the different host fungi, the alignment of amino acid sequences showed that the HKI 454T genome consisted of 1,265 (32.6%) and 1,221 (31.5%) orthologous coding sequences (CDSs) with those of B1-EBT and Mc-AG77, respectively. This comparative study of three phylogenetically associated endosymbionts has provided insights into their origin and evolution, and suggests the later bacterial invasion and adaptation of B1-EBT to its host metabolism.

The 2000 eruption of Mount Oyama on the island of Miyake (Miyake-jima) created a unique opportunity to study the early ecosystem development on newly exposed terrestrial substrates. In this study, bacterial and fungal communities on 9- and 11-year-old volcanic deposits at poorly to fully vegetation-recovered sites in Miyake-jima, Japan, were characterized by conventional culture-based methods and pyrosequencing of 16S rRNA and 18S rRNA genes. Despite the differences in the vegetation cover, the upper volcanic deposit layer samples displayed low among-site variation for chemical properties (pH, total organic carbon, and total nitrogen) and microbial population densities (total direct count and culturable count). Statistical analyses of pyrosequencing data revealed that the microbial communities of volcanic deposit samples were phylogenetically diverse, in spite of very low-carbon environmental conditions, and their diversity was comparable to that in the lower soil layer (buried soil) samples. Comparing with the microbial communities in buried soil, the volcanic deposit communities were characterized by the presence of Betaproteobacteria and Gammaproteobacteria as the main bacterial class, Deinococcus- Thermus as the minor bacterial phyla, and Ascomycota as the major fungal phyla. Multivariate analysis revealed that several bacterial families and fungal classes correlated positively or negatively with plant species.

Microbial colonization on new terrestrial substrates represents the initiation of new soil ecosystem formation. In this study, we analyzed early bacterial communities growing on volcanic ash deposits derived from the 2000 Mount Oyama eruption on the island of Miyake (Miyake-jima), Japan. A site was established in an unvegetated area near the summit and investigated over a 6-year period from 2003 to 2009. Collected samples were acidic (pH 3.0-3.6), did not utilize any organic substrates in ECO microplate assays (Biolog), and harbored around 106 cells (g dry weight)(-1) of autotrophic Fe(II) oxidizers by most-probable-number (MPN) counts. Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, and the Leptospirillum groups I, II and III were found to be abundant in the deposits by clone library analysis of bacterial 16S rRNA genes. The numerical dominance of Acidithiobacillus ferrooxidans was also supported by analysis of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Comparing the 16S rRNA gene clone libraries from samples differing in age, shifts in Fe(II)-oxidizing populations seemed to occur with deposit aging. The detection of known 16S rRNA gene sequences from Fe(III)-reducing acidophiles promoted us to propose the acidity-driven iron cycle for the early microbial ecosystem on the deposit.

An anammox assay involving a ¹⁵N tracer and gas chromatography-mass spectrometry revealed that the potential anammox activity accounted for 1 to 5% of total N₂ production in a ravine paddy field, Japan. Among four 4-cm-deep layers, the top layer showed the highest activity. Clone libraries showed that the DNA in the top layer contained sequences related to those of Candidatus 'Brocadia fulgida', Ca. 'B. anammoxidans', and Ca. 'Kuenenia stuttgartiensis'. These results suggest that a specific population of anammox bacteria was present in paddy soils, although a small part of dinitrogen gas was emitted from the soil via anammox.

This study aimed to characterize soil fungal communities in upland rice fields managed with tillage/non-tillage and winter cover-cropping (hairy vetch and cereal rye) practices, using PCR-based molecular methods. The study plots were maintained as upland fields for 5 years and the soils sampled in the second and fifth years were analyzed using T-RFLP (terminal restriction fragment length polymorphism) profiling and clone libraries with the internal transcribed spacer (ITS) region and domain 1 (D1) of the fungal large-subunit (fLSU) rRNA (D1(fLSU)) as the target DNA sequence. From the 2nd-year-sample, 372 cloned sequences of fungal ITS-D1(fLSU) were obtained and clustered into 80 nonredundant fungal OTUs (operational taxonomic units) in 4 fungal phyla. The T-RFLP profiling was performed with the 2nd- and 5th-year-samples and the major T-RFs (terminal restriction fragments) were identified using a theoretical fragment analysis of the ITS-D1(fLSU) clones. These molecular analyses showed that the fungal community was influenced more strongly by the cover-cropping than tillage practices. Moreover, the non-tilled, cover-cropped soil was characterized by a predominance of Cryptococcus sp. in the phylum Basidiomycota. We provided a genetic database of the fungal ITS-D1(fLSU)s in the differently managed soils of upland rice fields.

The genus Leptospirillum is known to dominate acid mine drainage and bioleaching systems. In this paper, we describe the isolation of iron-oxidizing bacteria closely related to Leptospirillum ferrooxidans from an acidic volcanic ash deposit on the island of Miyake (Miyake-jima), Japan. We further show the nitrogenase activity (acetylene reduction) for one (strain C2-1) of the isolates. The deposit harbored 1.2×10(8) total direct count (g dry weight)(-1) and 7.1×10(5) (most-probable-number, MPN) (g dry weight)(-1) of iron-oxidizer. A ferrous iron-limited, aerobic chemostat culture using Leptospirillum HH medium (pH 1.8) was performed to select and isolate the Leptospirillum group. Nine isolates were regarded as pure cultures based on uniform colony morphology on Fe(2+)-containing silica gel plates and absence of growth on 100-fold diluted nutrient broth plates. Six strains examined further shared 99.9-100% identity in 16S rRNA gene sequence with each other. Homology-based searches showed that all of the strains belonged to the Leptospirillum ferrooxidans clade. Strain C2-1 grown in ammonium sulfate-free Leptospirillum HH medium (pH 1.8) showed an initial rate of acetylene reduction of 58 μmol h(-1) (g cell carbon)(-1).

We have assayed rates of atmospheric CO and hydrogen uptake, maximum potential CO uptake and the major phylogenetic composition of CO-oxidizing bacterial communities for a variety of volcanic deposits on Miyake-jima, Japan. These deposits represented different ages and stages of plant succession, ranging from unvegetated scoria deposited in 1983 to forest soils on deposits >800 yr old. Atmospheric CO and hydrogen uptake rates varied from -2.0±1.8-6.3±0.1 mg CO m(-2) d(-1) and 0.0±0.4-2.0±0.2 mg H(2) m(-2) d(-1), respectively, and were similar to or greater than values reported for sites on Kilauea volcano, Hawaii, USA. At one of the forested sites, CO was emitted to the atmosphere, while two vegetated sites did not consume atmospheric hydrogen, an unusual observation. Although maximum potential CO uptake rates were also comparable to values for Kilauea, the relationship between these rates and organic carbon contents of scoria or soil indicated that CO oxidizers were relatively more abundant in Miyake-jima deposits. Phylogenetic analyses based on the large sub-unit gene for carbon monoxide dehydrogenase (coxL) indicated that many novel lineages were present on Miyake-jima, that CO-oxidizing Proteobacteria were prevalent in vegetated sites and that community structure appeared to vary more than composition among sites.

A soil column was percolated with Triton X-100 (octylphenol polyethoxylates; OPEOn) under aerobic conditions for 300 days and OPEOn-degrading bacteria were isolated and characterized phylogenetically. Populations of total culturable bacteria and OPEOn (1000 mg L-1)-resistant bacteria increased 10 times and 46 times, respectively, during the percolation. 16S rRNA gene sequencing of 128 OPEOn-resistant isolates revealed an increase in the population level of Alphaproteobacteria and decreases in levels of Betaproteobacteria and Actinobacteria with the exposure to the compound. Four OPEOn-degrading strains were isolated and characterized as being related to Bradyrhizobium liaoningense (3 strains) and Afipia sp. (1 strain).

Miyagi University of Education, Aramaki-aza-Aoba, Aoba-ku, Sendai 980–0845, Japan(Received October 7, 2006—Accepted November 29, 2006)A PCR primer set was designed to amplify the 18S rRNA gene from the phylum Ciliophora (ciliates). Thepair amplified DNA from 5 organisms representing the major taxonomic groups of ciliates but not from 13 non-ciliate organisms. To test the ability of the primers to amplify ciliate rRNA genes from environmental samples, aclone library was constructed from an agricultural soil sample and analyzed. The rRNA gene sequences of allclones were affiliated with the Ciliophora. This newly developed PCR primer set is able to amplify ciliate rRNAgenes from soil, and may be useful for the detection and identification of ciliates.

Thallium (Tl) is a heavy metal found in trace amounts in the earth’s crust and has been studied to a much lesser degree than other heavy metals. Since the discovery of high-temperature superconducting components in the Tl-Ca-Ba-Cu-O system, Tl has attracted greater attention as a potential major pollution source of the future. In this study, the response of soil culturable bacterial communities to the addition of Tl was examined with fresh samples of non-polluted garden and arable soils. A preserved air-dried sample from heavy metal-polluted river sediment was also used as a reference. Soil suspension experiments were performed to homogenize the soils to evenly distribute both the microbial populations and Tl and reduce spatial variability. From soil suspensions loaded with Tl at 0.24 to 0.98 mM, bacteria tolerant to 0.49 mM Tl (100 mg Tl l �1 ) were isolated and characterized phylogenetically. The background level of culturable Tl-tolerant bacterial populations was 0.0003–0.013% for the non-polluted soils. The Tl-tolerant fraction increased rapidly in response to Tl loading and dominated the bacterial community, which might be attributed mainly to an immediate effect due to the death of Tl-sensitive microorganisms and leaking of nutrients from cell lysis of sensitive species supporting the growth of primarily Tl-tolerant heterotrophs. Such Tl-tolerant culturable bacteria were isolated and classified into 9 groups of Alphaproteobacteria, those from the arable soil were identified as Pseudomonas spp., and those from the contaminated river sediment were identified as Bacillus niacini.

Microscopic and molecular analyses showed the presence of endobacteria inside the mycelia of four out of twelve nitrous oxide (N(2)O)-producing fungal isolates identified as Mortierella elongata. The 16S rRNA gene was successfully amplified with DNA extracted directly from the endobacterium-containing fungal strains and all sequences were related to that of Candidatus Glomeribacter gigasporarum in the family Burkholderiaceae. Bacterial endotoxin was detected in the endobacterium-positive fungal strains but only trace levels were found in endobacterium-negative strains. No significant relationship was found between the fungal N(2)O-producing activity and the presence of endobacteria.

Twenty seven soil bacterial isolates able to grow on cinnamic acid or ferulic acid as a sole carbon and energy source were examined for the ability to utilize other phenylpropanoids and phylogenetic interrelationships. A 16S rRNA gene sequence analysis revealed that 26 of the isolates were closely related to Pseudomonas putida biovar A and classified into 6 OTUs: cinnamic acid utilization-positive (OTUs, CN-1 to CN-3) and -negative groups (FR-1 to FR-3). P. putida NBRC 14164(T) and JCM 13061 belonged to FR-3. A probable reclassification of P. putida was discussed in relation to the utilization of phenylpropanoids.

The role of microbes in the early development of ecosystems on new volcanic materials seems to be crucial to primary plant succession but is not well characterized. Here we analyzed the bacterial community colonizing 22-year-old volcanic deposits of the Miyake-jima Island (Japan) using culture-based and 16S rRNA gene clone library methods. The majority of 91 bacterial isolates were placed phylogenetically in two clusters (A and B) of the Betaproteobacteria. Cluster A (82% of isolates) was related to the genus Limnobacter and Cluster B (9%) was affiliated with the Herbaspirillum clade. The clone library analysis supported the predominance of Cluster B rather than Cluster A. Strain KP1-50 of Cluster B was able to grow on a mineral medium under an atmosphere of H(2), O(2), and CO(2) (85:5:10), and characterized by its large-subunit gene of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) and nitrogenase reductase gene (nifH). In contrast, strains of Cluster A did not grow chemolithoautotrophically with H(2), O(2), and CO(2) but increased their cell biomass with the addition of thiosulfate to the succinate medium, suggesting the use of thiosulfate as an energy source. From phenotypic characterization, it was suggested that the Cluster A and B strains were novel species in the genus Limnobacter and Herbaspirillum, respectively.

The relationships between soil microbial properties and nitrous oxide emission were examined in upland soil under different tillage systems [no tillage (NT), rotary and plow tillage] and cover crop systems (fallow, cereal rye, and hairy vetch) in 2004 and 2005. Microbiological analyses included the determination of soil ergosterol as an indicator of fungal biomass, bacterial plate counting, and MPN estimations of ammonia oxidizers and denitrifiers. The combined practice of NT with rye-cover crop treatment increased fungal biomass but not bacterial populations in 0-10 cm deep soils. Such increase in fungal biomass was not found in 10-20 cm and 20-30 cm deep cover-cropped NT soil. The combined practice of NT with rye-cover cropping resulted in higher in situ N(2)O emission rates compared with rotary- and plow-till treatments. N(2)O flux was positively correlated with soil ergosterol content but not with denitrifier MPN and other soil chemical properties. These results suggested a significant contribution of fungi to N(2)O emission in cover-cropped NT soils.