Gene Cloning, Mutant Screening And Chemotaxis Of Organic Pollutant Degrading Strains

Abstract: This thesis includes three parts:Part 1: Pseudomonas putida strain WBC-3 grows on methyl-parathion (MP) as a sole carbon, nitrogen and energy source. A 3.7kb fragment of its plasmid which contains the complete methyl-parathion hydrolase (mph,GenBank accession No. AY251554) has been cloned. The gene is 98% similar in the amino acid level with another reported mpd gene (GenBank accession No. AF338729). mph was cloned for function identification. The clone W3M193 gets the ability to transform MP to a yellow compound. The degrading activities of the two strains to methyl-parathion, ethyl-parathion and fenitrothion were tested respectively after cell-lysing. The result shows that the MPH from the two strains share the same function and similar activities, which indicate mph was the methyl-parathion hydrolase gene.Part 2: Pseudomonas sp.ZWL73 grows on 4-chloronitrobenzene (4CNB) as a sole carbon, nitrogen and energy source and its degradation of 4CNB is plasmid-encoded. To find the 4CNB-degrading genes of the strain, mutants were generated by plasposon insertion, and two 4CNB-degrading mutants were screened. Depended on the conditional origin of replication in the inserted fragment, the flanking of the two mutants were cloned and sequenced. The flanking in one clone is probably from the degrading strain's chromosome, which contains partial hslU gene, complete gene of a conserved hypothetical protein, and partial encoding sequences of PHA (poly-3-hydroxyalkanoate) polymerase 1. The other clone maybe contains partial gene of the chromate transport protein.Part 3: Pseudomonas sp. strain ZWL73 and its plasmid transconjugant ZP8 (Pseudomonas putida PaW340 harboring the 4CNB degradative plasmid) was tested, in a chemotaxis assay, for the ability to sense and respond to 4CNB and several other aromatic compounds that may be related to the 4CNB degradation. Both strains were found to be chemotactic to 13 compounds including 4CNB, most of which can also be used as growth substrates for strain ZWL73. The results indicated a correlation between chemotaxis of strain ZWL73 toward 4CNB as well as its intermediates and their degradation/transformation. The same chemotactic pattern towards these compounds between strain ZWL73 and its transconjugant ZP8 suggested the chemotaxis of ZWL73 to 4CNB was controlled by the very plasmid which determines the degradation of 4CNB.