Department of Environmental Science and Engineering, Huaqiao University, Xiamen, 361021, China.
Instrumental Analysis Center of Huaqiao University, Huaqiao University, Xiamen, 361021, China.
Chemosphere. 2023 Dec;345:140558. doi: 10.1016/j.chemosphere.2023.140558. Epub 2023 Oct 26.
Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox), a widely used organoarsenical feed additive, can enter soils and be further biotransformed into various arsenic species that pose human health and ecological risks. However, the pathway and molecular mechanism of Rox biotransformation by soil microbes are not well studied. Therefore, in this study, we isolated a Rox-transforming bacterium from manure-fertilized soil and identified it as Pseudomonas chlororaphis through morphological analysis and 16S rRNA gene sequencing. Pseudomonas chlororaphis was able to biotransform Rox to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), arsenate [As(V)], arsenite [As(III)], and dimethylarsenate [DMAs(V)]. The complete genome of Pseudomonas chlororaphis was sequenced. PcmdaB, encoding a nitroreductase, and PcnhoA, encoding an acetyltransferase, were identified in the genome of Pseudomonas chlororaphis. Expression of PcmdaB and PcnhoA in E. coli Rosetta was shown to confer Rox(III) and 3-AHPAA(III) resistance through Rox nitroreduction and 3-AHPAA acetylation, respectively. The PcMdaB and PcNhoA enzymes were further purified and functionally characterized in vitro. The kinetic data of both PcMdaB and PcNhoA were well fit to the Michaelis-Menten equation, and nitroreduction catalyzed by PcMdaB is the rate-limiting step for Rox transformation. Our results provide new insights into the environmental risk assessment and bioremediation of Rox(V)-contaminated soils.
罗硝唑（3-硝基-4-羟基苯胂酸，Rox）是一种广泛使用的有机胂饲料添加剂，可进入土壤并进一步生物转化为各种砷物种，对人类健康和生态构成风险。然而，土壤微生物对 Rox 生物转化的途径和分子机制尚未得到很好的研究。因此，在这项研究中，我们从粪肥施肥的土壤中分离出一株 Rox 转化菌，并通过形态分析和 16S rRNA 基因测序鉴定为假单胞菌chlororaphis。假单胞菌chlororaphis 能够将 Rox 生物转化为 3-氨基-4-羟基苯胂酸（3-AHPAA）、N-乙酰-4-羟基-m-苯胂酸（N-AHPAA）、砷酸盐[As（V）]、亚砷酸盐[As（III）]和二甲基砷酸盐[DMAs（V）]。假单胞菌chlororaphis 的完整基因组被测序。在假单胞菌chlororaphis 的基因组中鉴定出编码硝基还原酶的 PcmdaB 和编码乙酰转移酶的 PcnhoA。在大肠杆菌 Rosetta 中表达 PcmdaB 和 PcnhoA 分别通过 Rox 硝基还原和 3-AHPAA 乙酰化赋予 Rox（III）和 3-AHPAA（III）抗性。进一步在体外纯化和功能表征了 PcMdaB 和 PcNhoA 酶。PcMdaB 和 PcNhoA 的动力学数据均很好地符合米氏方程，并且 PcMdaB 催化的硝基还原是 Rox 转化的限速步骤。我们的结果为 Rox（V）污染土壤的环境风险评估和生物修复提供了新的见解。
