Here we report the cell surface display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion by employing the N- and C-terminal domains of ice nucleation protein (INPNC) as an anchoring motif. An E. coli-Pseudomonas shuttle vector, pNOG33, coding for INPNC-OPH-GFP was constructed for targeting the fusion onto the cell surface of p-nitrophenol (PNP)-degrading P. putida JS444. The surface localization of INPNC-OPH-GFP was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH-GFP was demonstrated by OPH assays and fluorescence measurements. Surface display of macromolecular OPH-GFP fusion (63 kDa) neither inhibited cell growth nor affected cell viability. These results suggest that INP is an useful tool for the presentation of heterologous proteins on cell surfaces of indigenous microbes. The engineered P. putida JS444 degraded organophosphates (OPs) as well as PNP rapidly and could be easily monitored by fluorescence. Parathion (100 mg kg⁻¹) could be degraded completely within 15 days in soil inoculated with the engineered strain. These merits make this engineered strain an ideal biocatalyst for in situ bioremediation of OP-contaminated soil.

Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences and BIOLOG test, a soil bacterium was identified as Bacillus sp. DM-1. Using either growing cells or a cell-free extract, it transformed parathion and methyl parathion to amino derivatives by reducing the nitro group. Pesticide transformation by a cell-free extract was specifically inhibited by three nitroreductase inhibitors, indicating the presence of nitroreductase activity. The nitroreductase activity was NAD(P)H-dependent, O(2)-insensitive, and exhibited the substrate specificity for parathion and methyl parathion. Reductive transformation significantly decreased the toxicity of pesticides.

An autofluorescent whole-cell biocatalyst capable of simultaneously degrading organophosphates (OPs) and γ-hexachlorocyclohexane (γ-HCH) was constructed by display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion on the cell surface of a γ-HCH-degrading Sphingobium japonicum UT26 using the truncated ice nucleation protein (INPNC) as an anchoring motif. The surface localization of INPNC-OPH-GFP fusion was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Surface display of macromolecular OPH-GFP fusion (63 kDa) neither inhibits cell growth nor affects cell viability. In sterile and nonsterile soil samples, a mixture of parathion (100 mg kg(-1)) and γ-HCH (10 mg kg(-1)) could be degraded completely within 15 days when inoculated with the engineered UT26, and the strain could be easily monitored by fluorescence during bioremediation. These results indicate that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of environments contaminated with multiple pesticides.