Screening Of Pseudomonas Aeruginosa FabI Inhibitors And Study On Biodegration Of 2-MIB

This paper including two parts:screening of Pseudomonas aeruginosa enoyl-ACP reductase potential inhibitors and isolation, identification and BAC library construction of a 2-methylisoborneol biodegradation bacterium strain.By structure-based virtual screening research, five Chinese medicine monomers, luteolin, genistein, polydatin, imperatorin and crucumin, were identified as potential FabI inhibitors.In present study, fabI gene encoding the FabI of Pseudomonas aeruginosa ATCC 15442 (PaFabI) was amplified and inserted in to pQE30 expression vector. The recombinant DNA was transformed into E. coli M15 competent cells. Enoyl-ACP reductase of E.coli (EcFabI) and PaFabI could catalyze the reactions in vitro. By the reaction system, the inhibit activities of the five potential inhibitors were assayed. Luteolin was the the most effective EcFabI inhibitor followed by imperatorin, polydatin, crucumin and genistein. For PaFabI, luteolin could inhibit the reaction potently, polydatin next, genistein and crucumin had tenuous inhibit ability. An accidental result was imperatorin promoted the reaction of PaFabl.In bacteriostatic test, curcumin exhibited the antibacterial activity with a minimum inhibition concentration (MIC90) value 0.2μmol/L against E. coli MG1655 whereas it couldn’t inhibit the growth of E. coli CY1655 effectively. On the other hand, crucumin had tenuous antibacterial activity against the growth of P. aeruginosa ATCC 15442 and the MIC90 was 5μmol/L. Luteolin inhibited the P aeruginosa ATCC 15442 with a MIC90 1.0μmol/L.Enzyme reaction kinetics assay showed luteolin was a non-competitive inhibitor to EcFabI and PaFabI with the Ki 7.05 nmol/L and 0.40 nmol/L. Crucumin was also a non-competitive inhibitor to EcFabI whose Ki was 14.96 nmol/L. Polydatin was confirmed as a competitive inhibitor to PaFabI with at 0.40 nmol/L Ki.2-Methylisoborneol (2-MIB) is produced by a variety of bacteria including actinomycetes and cyanobacteria. When present, it gives a camphoraceous earthy off-flavor and smell to aquaculture water or drinking water. Conventional water treatment process were not very effective on removal of 2-MIB in water. An obvious solution to the 2-MIB problem might be to remove the chemical by treatment with 2-MIB degrading bacteria.In this study,2-MIB with 98% purity was synthesised from d-camphor. A bacterium strain was isolated from the sewage samples which were collected from various areas in Wuhan. The bacterium can degrade 89.7% 2-MIB. Growth curves of the bacterium strain in different culture conditions were measured. The sequence of 16SrDNA showed it was Enterobacter cloacae. A BAC library with 810 transformants was constructed. The foreign fragments were about 40 kb. This BAC library would be a platform for the analysis of 2-MIB biodegradation mechanism.