Allocation of Carbon from an Arbuscular Mycorrhizal Fungus, Gigaspora margarita, to Its Gram-Negative and Positive Endobacteria Revealed by High-Resolution Secondary Ion Mass Spectrometry.

Arbuscular mycorrhizal fungi are obligate symbionts of land plants; furthermore, some of the species harbor endobacteria. Although the molecular approach increased our knowledge of the diversity and origin of the endosymbiosis and its metabolic possibilities, experiments to address the functions of the fungal host have been limited. In this study, a C flow of the fungus to the bacteria was investigated. Onion seedlings colonized with Gigaspora margarita, possessing Candidatus Glomeribacter gigasporarum (CaGg, Gram-negative, resides in vacuole) and Candidatus Moeniiplasma glomeromycotorum (CaMg, Gram-positive, resides in the cytoplasm,) were labelled with 13CO2. The 13C localization within the mycorrhiza was analyzed using high-resolution secondary ion mass spectrometry (SIMS). Correlative TEM-SIMS analysis of the fungal cells revealed that the 13C/12C ratio of CaGg was the lowest among CaMg and mitochondria and was the highest in the cytoplasm. By contrast, the plant cells, mitochondria, plastids, and fungal cytoplasm, which are contributors to the host, showed significantly higher 13C enrichment than the host cytoplasm. The C allocation patterns implied that CaMg has a greater impact than CaGg on G. margarita, but both seemed to be less burdensome to the host fungus in terms of C cost.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4 +/15NH4 + Analyses via Sequential Conversion to N2O.

The importance of understanding the fate of nitrate (NO3-), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3- for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3- transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3- added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3- via persulfate oxidation. Third, the NO3- is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography-mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Hybrid Nitrous Oxide Production from a Partial Nitrifying Bioreactor: Hydroxylamine Interactions with Nitrite.

The goal of this study was to elucidate the mechanisms of nitrous oxide (N2O) production from a bioreactor for partial nitrification (PN). Ammonia-oxidizing bacteria (AOB) enriched from a sequencing batch reactor (SBR) were subjected to N2O production pathway tests. The N2O pathway test was initiated by supplying an inorganic medium to ensure an initial NH4+-N concentration of 160 mg-N/L, followed by 15NO2- (20 mg-N/L) and dual 15NH2OH (each 17 mg-N/L) spikings to quantify isotopologs of gaseous N2O (44N2O, 45N2O, and 46N2O). N2O production was boosted by 15NH2OH spiking, causing exponential increases in mRNA transcription levels of AOB functional genes encoding hydroxylamine oxidoreductase (haoA), nitrite reductase (nirK), and nitric oxide reductase (norB) genes. Predominant production of 45N2O among N2O isotopologs (46% of total produced N2O) indicated that coupling of 15NH2OH with 14NO2- produced N2O via N-nitrosation hybrid reaction as a predominant pathway. Abiotic hybrid N2O production was also observed in the absence of the AOB-enriched biomass, indicating multiple pathways for N2O production in a PN bioreactor. The additional N2O pathway test, where 15NH4+ was spiked into 400 mg-N/L of NO2- concentration, confirmed that the hybrid N2O production was a dominant pathway, accounting for approximately 51% of the total N2O production.

Insecticide applications to soil contribute to the development of Burkholderia mediating insecticide resistance in stinkbugs.

Some soil Burkholderia strains are capable of degrading the organophosphorus insecticide, fenitrothion, and establish symbiosis with stinkbugs, making the host insects fenitrothion-resistant. However, the ecology of the symbiotic degrading Burkholderia adapting to fenitrothion in the free-living environment is unknown. We hypothesized that fenitrothion applications affect the dynamics of fenitrothion-degrading Burkholderia, thereby controlling the transmission of symbiotic degrading Burkholderia from the soil to stinkbugs. We investigated changes in the density and diversity of culturable Burkholderia (i.e. symbiotic and nonsymbiotic fenitrothion degraders and nondegraders) in fenitrothion-treated soil using microcosms. During the incubation with five applications of pesticide, the density of the degraders increased from less than the detection limit to around 10(6)/g of soil. The number of dominant species among the degraders declined with the increasing density of degraders; eventually, one species predominated. This process can be explained according to the competitive exclusion principle using V(max) and K(m) values for fenitrothion metabolism by the degraders. We performed a phylogenetic analysis of representative strains isolated from the microcosms and evaluated their ability to establish symbiosis with the stinkbug Riptortus pedestris. The strains that established symbiosis with R. pedestris were assigned to a cluster including symbionts commonly isolated from stinkbugs. The strains outside the cluster could not necessarily associate with the host. The degraders in the cluster predominated during the initial phase of degrader dynamics in the soil. Therefore, only a few applications of fenitrothion could allow symbiotic degraders to associate with their hosts and may cause the emergence of symbiont-mediated insecticide resistance.

Occurrence and potential activity of denitrifiers and methanogens in groundwater at 140 m depth in Pliocene diatomaceous mudstone of northern Japan.

Anaerobic microbial activity has a major influence on the subsurface environment. We investigated the denitrification and methanogenesis in anoxic groundwater at a depth of 140 m in two boreholes drilled in a sedimentary geological setting, where the redox potential fluctuated. The average maximum potential denitrification rates, measured under anaerobic conditions in the two boreholes using an (15) N tracer, were 0.060 and 0.085 nmol (30) N2  mL(-1)  h(-1) . The deduced NirS amino acid sequences obtained from in situ samples were similar to those of isolates belonging to the α-, ß-, and γ-Proteobacteria, and the Firmicutes (72-100% similarity). Based on the nirS gene, the same operational taxonomic unit dominated incubated samples from each borehole. Methanogenesis candidates were detected by 16S rRNA gene analysis, but no sequence was detected using primers for the functional methanogenesis gene mcrA. Although the stable isotope signatures suggested that some of the dissolved methane was of biogenic origin, no potential for methane production was evident during the incubations. The groundwater at 140 m depth did not contain oxygen, had an Eh ranging from -144 to 6.8 mV, and was found to be a potential field for denitrification.

N(2)O emission from degraded soybean nodules depends on denitrification by Bradyrhizobium japonicum and other microbes in the rhizosphere.

A model system developed to produce N(2)O emissions from degrading soybean nodules in the laboratory was used to clarify the mechanism of N(2)O emission from soybean fields. Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110 (ΔnosZ, lacking N(2)O reductase) were grown in aseptic jars. After 30 days, shoot decapitation (D, to promote nodule degradation), soil addition (S, to supply soil microbes), or both (DS) were applied. N(2)O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N(2)O from the soybean rhizosphere. The N(2)O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N(2)O emission. A (15)N tracer experiment indicated that N(2)O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N(2)O emission was compared between a nirK mutant (ΔnirKΔnosZ, lacking nitrite reductase) and ΔnosZ. The N(2)O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N(2)O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N(2)O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N(2)O from nodule N via their denitrification processes (N(2)O source), whereas nosZ-competent B. japonicum exclusively takes up N(2)O (N(2)O sink). Net N(2)O flux from soybean rhizosphere is likely determined by the balance of N(2)O source and sink.

Population dynamics of Crenarchaeota and Euryarchaeota in the mixing front of river and marine waters.

A transect from the Tomoe River Mouth through Shimizu Port to Suruga Bay, Japan, was examined between 2005 and 2009 to reveal the population dynamics of Crenarchaeota and Euryarchaeota in an estuary environment. Crenarchaeota tended to increase in abundance in waters deeper than 100 m compared with Euryarchaeota, and comprised 11% of total direct counts. Archaeal abundance was highest in the Tomoe River Mouth, with a strong negative correlation between surface euryarchaeal abundance and salinity (P<0.001). The diversity index for the phylotypic archaeal community in the mouth was three times higher than that at sites St1-1m and St1-10m in the estuary, and OTUs represented most of the OTU groups at the sites. Three of the seven total OTUs, which comprised 83.6% of the 140 sequenced clones in the estuary, were related to the OTUs in the mouth with similarities higher than 97%. A significant proportion of the archaeal community appears to be derived from the Tomoe River. The two dominant phylotypes of the archaeal community in Shimizu Port, belonging to MGI and MGII, occurred ubiquitously.

Complementary cooperation between two syntrophic bacteria in pesticide degradation.

Interactions between microbial species, including competition and mutualism, influence the abundance and distribution of the related species. For example, metabolic cooperation among multiple bacteria plays a major role in the maintenance of consortia. This study aims to clarify how two bacterial species coexist in a syntrophic association involving the degradation of the pesticide fenitrothion. To elucidate essential mechanisms for maintaining a syntrophic association, we employed a mathematical model based on an experimental study, because experiment cannot elucidate various conditions for two bacterial coexistence. We isolated fenitrothion-degrading Sphingomonas sp. TFEE and its metabolite of 3-methyl-4-nitrophenol (3M4N)-degrading Burkholderia sp. MN1 from a fenitrothion-treated soil microcosm. Neither bacterium can completely degrade fenitrothion alone, but they can utilize the second intermediate, methylhydroquinone (MHQ). Burkholderia sp. MN1 excretes a portion of MHQ during the degradation of 3M4N, from which Sphingomonas sp. TFEE carries out degradation to obtain carbon and energy. Based on experimental findings, we developed mathematical models that represent the syntrophic association involving the two bacteria. We found that the two bacteria are characterized by the mutualistic degradation of fenitrothion. Dynamics of two bacteria are determined by the degree of cooperation between two bacteria (i.e., supply of 3M4N by Sphingomonas sp. TFEE and excretion of MHQ by Burkholderia sp. MN1) and the initial population sizes. The syntrophic association mediates the coexistence of the two bacteria under the possibility of resource competition for MHQ, and robustly facilitates the maintenance of ecosystem function in terms of degrading xenobiotics. Thus, the mathematical analysis and numerical computations based on the experiment indicate the key mechanisms for coexistence of Sphingomonas sp. TFEE and Burkholderia sp. MN1 in syntrophic association involving fenitrothion degradation.

Unique distribution of deep groundwater bacteria constrained by geological setting.

We collected groundwater samples at depths of up to 482 m from three boreholes drilled into sedimentary rock within two formations in Hokkaido, Japan. The prokaryotic community in each subsurface groundwater sample was analysed by microscopic counts and cloning-sequencing the 16S rRNA genes. On total direct counts, there were between 4.61 × 10(4) and 5.06 × 10(6) prokaryote cells ml(-1) in the samples, which is similar to the numbers observed at the marine subsurface. However, the vertical distribution of the prokaryotes did not show a simple decrease in abundance with increasing depth. A high abundance of cells with significant amounts of RNA was identified in the domain Bacteria using fluorescence in situ hybridization, with a high frequency of dividing cells at the transition zone between the two sedimentary rock formations. Cloning-sequencing analysis showed the predominance of γ-Proteobacteria at this transition zone at 281-312 m. The horizontal heterogeneity of the microbial distribution in the subsurface environment was also demonstrated by a relatively high density of members of the domain Archaea in borehole HDB-4, drilled only 1.5 km northeast of HDB-6 and in the same formation.

Denitrification activity and relevant bacteria revealed by nitrite reductase gene fragments in soil of temperate mixed forest.

Denitrification activity and bacterial community constituents were investigated in both well-drained and poorly drained soils of a temperate forest in central Japan by (15)N tracer experiments and a cloning-sequencing approach. Denitrification activity was much higher in wet soil than in dry soil, based on (15)N(15)N ((30)N(2)) and (15)N(15)NO ((46)N(2)O) production. Labeled nitrate ((15)NO(3)(-)) was immediately reduced to (30)N(2) in wet soil, whereas it was only reduced to (46)N(2)O in dry soil. Thus, the wet soil at the lower end of the catchment is a functional site for the scavenging for NO(3)(-) and N(2)O. Nitrite reductase gene (nirK and nirS) fragments from these soils were PCR amplified, cloned, and sequenced. Both nirK and nirS fragments were detected in the wet soil, whereas only nirK fragments were detected in the dry soil. All the nirK and nirS clones showed less than 90% similarity to known clones. Numerous operational taxonomic units for nirK and nirS were found in the wet soil. Considerable diversification within the largest clade on the nirK phylogenetic tree, which contained no known sequence, was observed in wet soil. Thus, a wet soil environment can provide both the habitat and conditions for the expression of denitrification activity.