Masahito Hayatsu

Various players in the nitrogen cycle: Diversity and functions of the microorganisms involved in nitrification and denitrification

Abstract Microorganisms play important roles in the nitrogen cycles of various ecosystems. Research has revealed that a greater diversity of microorganisms is involved in the nitrogen cycle than previously understood. It is becoming clear that denitrifying fungi, nitrifying archaea, anammox bacteria, aerobic denitrifying bacteria and heterotrophic nitrifying microorganisms are key players in the nitrogen cycle. Studies have revealed a major contribution by fungi in the production of N2O and N2 in grasslands, semiarid regions and forest soils. Some fungi can grow under various O2 conditions by using three types of energy-yielding metabolism: O2 respiration, denitrification (nitrite respiration) and ammonia fermentation. The amoA-like gene copies of Crenarchaeota were shown to be more abundant in soils than in autotrophic ammonia-oxidizing bacteria, and the gene was expressed at higher levels in soil to which ammonia was added. There are some contradictory findings, however, regarding archaeal and bacterial nitrification. Anammox bacteria have been shown to be widely distributed and to play an important role in both artificial and natural environments. The contribution of heterotrophic microorganisms to nitrification has been recognized in soil, and the biochemical mechanisms of several bacteria are becoming clear. A wide variety of bacteria have been found to be able to carry out aerobic denitrification and to be distributed across diverse environments. Using molecular biological techniques for soil bacteria, Nitrosospira species of clusters 2, 3 and 4 have been shown to be the dominant group in soils. Genome analyses of autotrophic nitrifying bacteria are providing new insights into their ecology and functions in soils.

Symbiont-mediated insecticide resistance

Development of insecticide resistance has been a serious concern worldwide, whose mechanisms have been attributed to evolutionary changes in pest insect genomes such as alteration of drug target sites, up-regulation of degrading enzymes, and enhancement of drug excretion. Here, we report a previously unknown mechanism of insecticide resistance: Infection with an insecticide-degrading bacterial symbiont immediately establishes insecticide resistance in pest insects. The bean bug Riptortus pedestris and allied stinkbugs harbor mutualistic gut symbiotic bacteria of the genus Burkholderia, which are acquired by nymphal insects from environmental soil every generation. In agricultural fields, fenitrothion-degrading Burkolderia strains are present at very low densities. We demonstrated that the fenitrothion-degrading Burkholderia strains establish a specific and beneficial symbiosis with the stinkbugs and confer a resistance of the host insects against fenitrothion. Experimental applications of fenitrothion to field soils drastically enriched fenitrothion-degrading bacteria from undetectable levels to >80% of total culturable bacterial counts in the field soils, and >90% of stinkbugs reared with the enriched soil established symbiosis with fenitrothion-degrading Burkholderia. In a Japanese island where fenitrothion has been constantly applied to sugarcane fields, we identified a stinkbug population wherein the insects live on sugarcane and ≈8% of them host fenitrothion-degrading Burkholderia. Our finding suggests the possibility that the symbiont-mediated insecticide resistance may develop even in the absence of pest insects, quickly establish within a single insect generation, and potentially move around horizontally between different pest insects and other organisms.

Involvement of Two Plasmids in Fenitrothion Degradation by Burkholderia sp. Strain NF100

ABSTRACT A bacterium capable of utilizing fenitrothion (O,O-dimethylO-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.

Aluminium kinetics in the tea plant using 27Al and 19F NMR

Abstract To clarify aluminium kinetics in the tea plant, 27 Al and 19 F NMR spectroscopy was applied to the analysis of intact tissues. Although most of t

Nucleotide sequence and genetic structure of a novel carbaryl hydrolase gene (cehA) from Rhizobium sp. strain AC100.

ABSTRACT Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.

The lowest limit of pH for nitrification in tea soil and isolation of an acidophilic ammonia oxidizing bacterium

Abstract Investigation to determine the lowest limit of soil pH for nitrification were carried out in the laboratory using acid tea soils with pH range of 3.3 to 5.1. A significant linear correlation was observed between the soil pH value and nitrification rate which was measured by the soil incubation method. It was indicated that the lowest limit of the pH for nitrification was around pH 2.9. Two types of morphologically distinct strains of chemoautotrophic ammonia-oxidizing bacteria were isolated from four tested soils. One of them which was acidophilic was found to grow in a pH range from 3.5 to 7.0 in pure culture. The optimum pH for growth and ammonia oxidation of the acidophilic strain was pH 5.0. Acidophilic ammonia-oxidizing bacteria presumably play an important role in nitrification in acid tea soils.

Autotrophic nitrification in acid tea soils

Abstract Investigations to analyze the nature of nitrification in acid soils were carried out in the laboratory using 2 soil types from a tea field. The optimum temperature for the nitrification activity in these soils was 25°C and the activity was inhibited above 35°C, suggesting that the nitrifying bacteria became adapted to the soil conditions. The optimum NH4 +-N concentrations for nitrification ranged between 20 and 200 mg N/100 g of soil and the activity decreased at a concentration above 300 mg N. The effect of acidity on nitrification was studied by using soil samples amended with various amounts of CaCO3. Although the pattern of NO2¯ production changed, the rate of ammonia oxidation was not influenced by the addition of CaCO3. The nitrification activity was completely inhibited by the addition of nitrapyrin or acetylene. These results suggest that acid-tolerant or acidophilic autotrophic nitrification occurs in acid tea soils in Japan.

Nitrous Oxide Flux from a Tea Field Amended with a Large Amount of Nitrogen Fertilizer and Soil Environmental Factors Controlling the Flux(Environment)

Abstract Nitrous oxide (N2O) is one of the main greenhouse gases, and accurate estimation of the N2O flux from fertilized arable land is required. It is known that acidic tea field soil displays a higher N2O production activity than neutral arable soil and that tea fields could be a major source of N2O. Therefore, N2O fluxes from four plots (Std, 2N, 2Ca and −Ca plots) in a tea field that had been subjected to different conditions of fertilizer management were measured using the closed chamber method over a period of two years, and the relationships between the N2O flux and soil environmental factors were analyzed. The amounts of nitrogen fertilizer and liming material (dolomite) applied to the Std plot were 600 kg N ha−1 y−1 and 1,500 kg ha−1 y−1, respectively. The amount of nitrogen fertilizer applied to the 2N plot was two-times larger than that applied to the Std plot and corresponded to the conventionallevel in Japanese tea fields. The soil was acidified due to heavy nitrogen fertilization in the 2N plot. The 2Ca plot was amended with two-times the amount of liming material of the Std plot and in the −Ca plot no liming material was applied. There were significant differences among the N2O fluxes from the plots, and the highest value of N2O flux was 8.785 mg N m−2 h−1 in the 2N plot. Annual emission rate and emission factor of N2O in the 2N plot were 25.22 kg N2O-N ha−1 and 2.10%, respectively. Both long-term heavy nitrogen fertilization and subsequent soil acidification possibly enhanced the N2O flux from the tea field. The N2O flux from the tea field showed temporal variations, namely the N2O flux was relatively low from December to March possibly due to the low soil temperature and it increased gradually after March as the soil temperature increased over 10°C. The N2O flux reached the first major peak in July, decreased transiently in August presumably due to the drying of soil, increased again and reached the second peak in September or October, and then decreased after November. Multiple linear regression analysis of the relationships between the N2O flux and soil environmental factors indicated that the N2O production activity was significantly related to the N2O flux from the tea field. The N2O production activity showed temporal variations corresponding to the temporal variations of the N2O flux.

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.

Insect’s intestinal organ for symbiont sorting

Significance In general, animals have a mouth for feeding, an anus for defecation, and a gut connecting them for digestion and absorption. However, we discovered that the stinkbug’s gut is functionally disconnected in the middle by a previously unrecognized organ for symbiont sorting, which blocks food fluid and nonsymbiotic bacteria but selectively allows passing of a specific bacterial symbiont. Though very tiny and inconspicuous, the organ governs the configuration and specificity of stinkbug gut symbiosis, wherein the posterior gut region is devoid of food flow, populated by a specific bacterial symbiont, and transformed into an isolated organ for symbiosis. Mutant analyses showed that the symbiont’s flagellar motility is needed for passing the host organ, highlighting intricate host–symbiont interactions underpinning the symbiont sorting process. Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host–symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ’s ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host’s control and symbiont’s motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.