Abstract
Several strains have been reported to grow on 3-methyl-4-nitrophenol (3M4NP), the primary breakdown product of the excessively used insecticide fenitrothion. However, the microbial degradation of 3M4NP at molecular and biochemical levels remains unknown. Here, methyl-1,4-benzoquinone (MBQ) and methylhydroquinone (MHQ), rather than catechol proposed previously, were identified as the intermediates before ring cleavage during 3M4NP degradation by Burkholderia sp. strain SJ98. Real-time quantitative PCR analysis indicated that the pnpABA1CDEF cluster involved in para-nitrophenol (PNP) and 2-chloro-4-nitrophenol (2C4NP) catabolism was also likely responsible for 3M4NP degradation in this strain. Purified PNP 4-monooxygenase (PnpA) is able to catalyze the monooxygenation of 3M4NP to MBQ and exhibited an apparent Km value of 20.3 ± 2.54 μM for 3M4NP, and pnpA is absolutely necessary for the catabolism of 3M4NP by gene knock-out and complementation. PnpB, a 1,4-benzoquinone reductase catalyzes the reduction of MBQ to MHQ, and also found to enhance PnpA activity in vitro in the conversion of 3M4NP to MBQ. By sequential catalysis assays, PnpCD, PnpE, and PnpF were likely involved in the lower pathway of 3M4NP catabolism. Although NpcCD, NpcE, and NpcF are able to catalyze the sequential conversion of MHQ in vitro, these enzymes are unlikely involved in 3M4NP catabolism because their coding genes were not upregulated by 3M4NP induction in vivo. These results revealed that the enzymes involved in PNP and 2C4NP catabolism were also responsible for 3M4NP degradation in strain SJ98. This fills a gap in our understanding of the microbial degradation of 3M4NP at molecular and biochemical levels and also provides another example to illustrate the adaptive flexibility in microbial catabolism for structurally similar compounds.
Highlights
As a representative among the organophosphorus pesticides, fenitrothion (O,O-dimethylO-pnitro-m-tolyl phosphorothioate), is a highly toxic chemical and extensively used to control major insect pests (Hayatsu et al, 2000; Zhang et al, 2006; Itoh et al, 2014), especially in developing countries
The microbial degradation of 3M4NP was predominantly reported in Burkholderia spp., such as strains SJ98, SH-1 (Kim et al, 2007), NF100 (Hayatsu et al, 2000), FDS-1 (Zhang et al, 2006), and RKJ800 (Arora and Jain, 2012)
The intermediates of 3M4NP catabolism were identified by high performance liquid chromatography (HPLC) analysis after the confirmation of strain SJ98 as a 3M4NP utilizer
Summary
As a representative among the organophosphorus pesticides, fenitrothion (O,O-dimethylO-pnitro-m-tolyl phosphorothioate), is a highly toxic chemical and extensively used to control major insect pests (Hayatsu et al, 2000; Zhang et al, 2006; Itoh et al, 2014), especially in developing countries. 3M4NP is a analog of the priority environmental pollutant PNP and 2C4NP These nitrophenols compounds are widely used for manufacturing of drugs, dyes, pesticides, herbicides, and fungicides (Arora et al, 2012, 2014). The microbial degradation of PNP (Spain and Gibson, 1991; Jain et al, 1994; Kadiyala and Spain, 1998; Kitagawa et al, 2004; Takeo et al, 2008; Zhang et al, 2009; Liu et al, 2010; Shen et al, 2010; Wei et al, 2010) and 2C4NP (Ghosh et al, 2010; Arora and Jain, 2011, 2012; Pandey et al, 2011; Min et al, 2014, 2016) has been extensively investigated. SJ98 (Min et al, 2014) and the enzymes encoded by pnpA1A2BC were proved to be responsible for the hydroxyquinol pathway of 2C4NP catabolism in Grampositive Rhodococcus imtechensis RKJ300 (Min et al, 2016)
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