Anchorage of GFP fusion on the cell surface of Pseudomonas putida

• Published in: Biodegradation (Dordrecht) Vol. 22; no. 1; pp. 51 - 61
• Main Authors: Yuan, Yulan, Yang, Chao, Song, Cunjiang, Jiang, Hong, Mulchandani, Ashok, Qiao, Chuanling
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.02.2011; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Aquatic Pollution; Aryldialkylphosphatase - genetics; Aryldialkylphosphatase - metabolism; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biodegradation of pollutants; Biodegradation, Environmental; Biological and medical sciences; Biomedical and Life Sciences; Bioremediation; Biotechnology; Cell Membrane - genetics; Cell Membrane - metabolism; Cells; Degradation; E coli; Environment and pollution; Enzymes; Escherichia coli; Fluorescence; Fractionation; Fundamental and applied biological sciences. Psychology; Gene Expression; Geochemistry; green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Hydrolases; Ice nucleation protein; Industrial applications and implications. Economical aspects; Life Sciences; Microbiology; Microorganisms; Nitrophenols - metabolism; Organophosphates; Organophosphorus hydrolase; Original Paper; Parathion; Position (location); Protein Engineering; Protein Transport; Proteins; Pseudomonas - enzymology; Pseudomonas putida; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Soil (material); Soil contamination; Soil Science & Conservation; Strain; Surface display; Terrestrial Pollution; Vector space; Waste Management/Waste Technology; Waste Water Technology; Water Management; Water Pollution Control; Organophosphorus hydrolase; Surface display; Ice nucleation protein; Green fluorescent protein; Pseudomonadales; Biodegradation; Enzyme; Ice nucleation efficiency; Esterases; Phosphoric triester hydrolases; Bacteria; Hydrolases; Pseudomonadaceae; Aryldialkylphosphatase; Pseudomonas putida
• ISSN: 0923-9820; 1572-9729
• DOI: 10.1007/s10532-010-9375-7

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.