Cloning And Characterization Of P-Nitrophenol Degradation Related Genes From Pseudomonas Putida DLL-E4

The strain Pseudomonas putida DLL-E4 degrading methyl parathion (MP) wasisolated from soil polluted by MP for long term, and it can use p-Nitrophenol (PNP) as solecarbon and nitrogen sources. By transposon tagging method, several mutants that lost theability to degrade PNP was screened. Besides PNP degrading characteristic, MT54 andMT18 have some other traits. MT54 was a temperature-sensitive mutant which coulddegrade PNP and hydroquinone at 30℃, while lost the ability of degradation PNP but stillcan degrade hydroquinone at 37℃. M18 cannot degrade hydroquinone and homogentisate.Flanking sequences of the transposon were amplified with SEFA-PCR, the PCRproducts were sequenced and the homology search results by BLAST showed that themutated gene of MT54 was a cryptic gene. A heat shock protein gene hscA was located atthe downstream of this ORF. The polarity effect of Tn5 insertion caused the inactivation ofthe heat shock protein which might play roles in the accurate folding of p-Nitrophenoldenitroase, and subsequently caused temperature-sensitive degradation of PNP by MT54.The flanking sequences of Tn5 in M18 showing a significant level of homology (up to92％) to homogentisate 1,2-dioxygenase gene (hmgA) of Pseudomonasputida KT2440. Thestructure of homogentisate and hydroquinone are similar to each other. Gene hmgA wasamplified using DLL-E4 genomic DNA as template and then cloned into the suicide vectorpEX19Gm for recombination exchange of inserting mutated genomic hmgA. pEX19Gmwith complete hmgA gene was introduced into the genome of M18 by conjugation and therecombinant was designated as Int18. Int18 restored the degradation ability of Phe, whereas, it still cannot degrade PNP and hydroquinone.The reported hydroxyquinol 1,2-dioxygenase genes were aligned using Clustalxsoftware and degenerated primers were designed according to the conserved sequence andcodon bias of Pseudomonas. A 550bp length segment was amplified from DLL-E4 genomicDNA, and sequence analysis showed a significant level of homology (67％) tohydroxyquinol 1,2-dioxygenase of Burkholderia ambifaria AMMD (GeneBank accessionNo. EAO48077), but just a homology of 9％to hydroquinone 1,2-dioxygenase. Gene pnpCwas cloned into the suicide vector pJQ200SK for knocking out of the pnpC in DLL-E4, andthe resultant strain DLL-dpnpC lost the ability to degrade hydroquinone, and the feedbackcontrol of hydroquinone bring on the consequence that DLL-dpnpC can just degrade PNPslightly, about 5％in 21 hours.