Screening Of Pseudomonas Aeruginosa PAO1 Genes Associated With Biodegradation Of Phenanthrene, Naphthalene And Functional Investigation Of The Operon PA0334-0335 In Pseudomonas Aeruginosa PAO1

The aromatic compounds, especially the polycyclic aromatic hydrocarbons (PAHs), had draw widespread attention because of its carcinogenic and mutagenic effects, which caused great threaten to the nature ecology and environment. Phenanthrene and naphthalene as two representative carcinogenic polycyclic aromatic hydrocarbon contain three and two fused benzene rings respectively. The dominant way of mineralization of PAHs was microbial degradation and mostly starins of Pseudomonas sp. had the ability to degrade PAHs, what's more, Pseudomonas aeruginosa PAO1 which was from this species has a higher rate of degradation of PAHs, so it was chosen as our research object.Our group successfully constructed transposon mutant library of Pseudomonas aeruginosa PAO1, and the establishment of naphthalene and phenanthrene degradation gene related libraries. Based the previous study, this study further screened phenanthrene or naphthalene degradation related genes,then mutants which is poor growth at minidal medium with phenanthrene or naphthalene as the only carbon source and energy were selected. One mutant which was inserted by transposon in PA0334 was studied in further. PA0334 encoding protein was predicted belonging to MFS family based on the database. Because the MFS may be involved in the transport of carbon,and then affecting the degradation of naphthalene or phenanthrene.Transposon insertion mutant E9/PAO1 (0334m) hardly grow after expose to naphthalene or phenanthrene.To investigate the reason, PAO1(0334m) was complemented.The results indicated the operon PA0334-0335's destoration lead to PAO1(0334m)'s hardly growing,but not losing the ability of degradation of naphthalene and phenanthrene.In order to study the function of PA0334-0335 operon, the knockout mutant PAO1(?0334-0335) was constructed. By bioinformatics analysis, we know that PA0334 encodes a efflux pump belonging to the MFS family. To simulate a low-carbon source of degradation of the environment, the growth station of PAO1(?0334-0335) and PAO1 on amino acid,naphthalene,phenanthrene,glycerol and glucose as the sole carbon source and energy is measured after starvation treatment. It is speculated that operon PA0334-0335 encoding protein may participate in the transfer and metabolism of carbon.As a result of MFS transporter's substrate diversity, the mutant has resistance to other toxicants like Chl. The tolerance to high osmotic of operon PA0334-0335's deletion mutant had reduced significantly. Further studies showed that PAO1 (?0334-0335) colonies become smaller, but no significant change in the growth rate. Acorrding to the literature reports, this small colony variants related to many factors, such as biofilm formation, motility changes and other reasons. Biofilm formation can promote the degradation of phenanthrene or naphthalene and a small colony variants are also associated with it. Inconsistent with an earlier study, the small colony variants (SCVs) decreased biofilm production,but it's not a SCVs incentives. The motility of PAO1(?0334-0335) declined significantly explain the reasons for SCVs in further.This study focused on PA0334-0335 studied gene function, for a better understanding of the degradation of phenanthrene or naphthalene and molecular mechanisms involved in physiological functions related processes provide new clues in Pseudomonas aeruginosa.