Kunihiko Fujii

Different substrate specificities of two triazine hydrolases (TrzNs) from Nocardioides species

Nocardioides sp. strain MTD22 degraded atrazine, ametryn and atraton, as did Arthrobacter aurescens strain TC1 and Nocardioides sp. strain C190. These strains contain trzN, a gene coding for TrzN, triazine hydrolase showing a broad substrate range. However, Nocardioides sp. strain AN3 degraded only atrazine despite containing trzN. These differences in s-triazine degradation are presumed to be due to differences in the amino acid sequences of TrzNs. Consequently, 1371 nucleotides of the trzN coding sequences of strains AN3 and MTD22 were determined. Comparisons of the amino acid sequences of TrzNs indicated that three residues of strain AN3 (Thr(214), His(215) and Gln(241)) were distinct from those of the other three strains (Pro(214), Tyr(215) and Glu(241)). To confirm the relationships between these amino acid sequences and the substrate specificities of TrzNs, wild and chimera trzN genes were constructed and expressed in Escherichia coli cells. Cells expressing wild MTD22 trzN (Pro(214)Tyr(215)Glu(241)) and chimera AN3-MTD22 trzN (Thr(214)His(215)Glu(241)) degraded all s-triazines, but the degradation rate was markedly decreased in AN3-MTD22 trzN. Wild AN3 trzN (Thr(214)His(215)Gln(241)) and chimera MTD22-AN3 trzN (Pro(214)Tyr(215)Gln(241)) degraded only atrazine. These results suggest that the substitution of Glu(241) for Gln(241) significantly decreases enzyme affinity for ametryn and atraton.

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species.

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil–charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.

Enhanced transformation of diphenylarsinic acid in soil under sulfate-reducing conditions

Diphenylarsinic acid (DPAA) is known to be the major contaminant in soils where diphenylchloroarsine and diphenylcyanoarsine were abandoned after World Wars I and II. In this study, experimental model studies were performed to elucidate key factors regulating the transformation of DPAA under anaerobic soil conditions. The elimination of DPAA in Gleysol soils (Qiqihar and Shindori soils) was more rapid than in Mollisol and Regosol soils (Heihe and Ikarashi soils, respectively) during a 5-week incubation. No clear relationship between decreasing rates of DPAA concentrations and soil Eh values was found. The Ikarashi soil showed the slowest decrease in DPAA concentrations among the four soils, but the transformation of DPAA was notably enhanced by addition of exogenous sulfate together with acetate, cellulose or rice straw. Addition of molybdate, a specific inhibitor of sulfate reduction, resulted in the stagnation of DPAA transformation, suggesting that indigenous sulfate reducers play a role in DPAA transformation under anaerobic conditions. Arsenate, phenylarsonic acid, phenylmethylarsinic acid, diphenylmethylarsine oxide and three unknown compounds were detected as metabolites of DPAA. This is the first study to reveal enhancement of DPAA transformation under sulfate-reducing conditions.

Formation of diphenylthioarsinic acid from diphenylarsinic acid under anaerobic sulfate-reducing soil conditions

Diphenylarsinic acid (DPAA) is a toxic phenylarsenical compound often found around sites contaminated with phenylarsenic chemical warfare agents, diphenylcyanoarsine or diphenylchloroarsine, which were buried in soil after the World Wars. This research concerns the elucidation of the chemical structure of an arsenic metabolite transformed from DPAA under anaerobic sulfate-reducing soil conditions. In LC/ICP-MS analysis, the retention time of the metabolite was identical to that of a major phenylarsenical compound synthesized by chemical reaction of DPAA and hydrogen sulfide. Moreover the mass spectra for the two compounds measured using LC/TOF-MS were similar. Subsequent high resolution mass spectral analysis indicated that two major ions at m/z 261 and 279, observed on both mass spectra, were attributable to C12H10AsS and C12H12AsSO, respectively. These findings strongly suggest that the latter ion is the molecular-related ion ([M+H](+)) of diphenylthioarsinic acid (DPTA; (C6H5)2AsS(OH)) and the former ion is its dehydrated fragment. Thus, our results reveal that DPAA can be transformed to DPTA, as a major metabolite, under sulfate-reducing soil conditions. Moreover, formation of diphenyldithioarsinic acid and subsequent dimerization were predicted by the chemical reaction analysis of DPAA with hydrogen sulfide. This is the first report to elucidate the occurrence of DPAA-thionation in an anaerobic soil.