Abstract
Aerobic microbial degradation of p-nitrophenol (PNP) has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative bacteria, whereas in Gram-positive PNP degraders it proceed via ‘Benzenetriol (BT) pathway’. These pathways are characterized by the ring cleavage of HQ and BT as terminal aromatic intermediates respectively. Earlier reports on PNP degradation have indicated these pathways to be mutually exclusive. We report involvement of both ‘HQ’ and ‘BT’ ring cleavage pathways in PNP degradation by Burkholderia sp. strain SJ98. Genetic characterization of an ~41 Kb DNA fragment harboring PNP degradation gene cluster cloned and sequenced from strain SJ98 showed presence of multiple orfs including pnpC and pnpD which corresponded to previously characterized ‘benzenetriol-dioxygenase (BtD)’ and ‘maleylacetate reductase (MaR)’ respectively. This gene cluster also showed presence of pnpE1 and pnpE2, which shared strong sequence identity to cognate sub-units of ‘hydroquinone dioxygenase’ (HqD). Heterologous expression and biochemical characterization ascertained the identity of PnpE1 and PnpE2. In in vitro assay reconstituted heterotetrameric complex of PnpE1 and PnpE2 catalyzed transformation of hydroquinone (HQ) into corresponding hydroxymuconic semialdehyde (HMS) in a substrate specific manner. Together, these results clearly establish branching of PNP degradation in strain SJ98. We propose that strain SJ98 presents a useful model system for future studies on evolution of microbial degradation of PNP.
Highlights
P-Nitrophenol (PNP) is one of the most thoroughly studied toxic environmental pollutants; it has been widely used in industries for chemical synthesis of dyes and plastics, resulting in high levels of PNP contaminations (Bhushan et al 2000; Spain 1995)
The sequence analyses and annotation of this fragment indicated that the organization of pnp gene cluster in strain SJ98 is very similar to that of the nph gene cluster of Arthrobacter sp. strain JS443 (Perry and Zylstra 2007)
The only striking difference is the presence of two orfs within pnp cluster of strain SJ98 that share strong sequence similarity with genes encode for cognate subunits of hydroquinone dioxygenase’ (HqD)
Summary
P-Nitrophenol (PNP) is one of the most thoroughly studied toxic environmental pollutants; it has been widely used in industries for chemical synthesis of dyes and plastics, resulting in high levels of PNP contaminations (Bhushan et al 2000; Spain 1995). A number of studies have shown biochemical characterization and elucidation of the catabolic pathway for PNP degradation. Results presented in these studies indicated that aerobic PNP degradation could substrate in the degradation of 4-hydroxybenzoate and pnitrophenol by Candida parapsilosis CBS604 and Pseudomonas aeruginosa HS-D38 respectively (Figure 1A) (Eppink et al 2000; Zheng et al 2009). Strain WBC-3 could degraded 4-NC, wherein degradation selectively proceeds via formation of BT (Wei et al 2010a) Based on these reports, it could be argued that microbial degradation of PNP proceeds via either BT or HQ branch of the pathway. Heterologous expression and biochemical characterization of these genes ascertained their identity
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