Polymyxa Paenibacillus Antibacterial Substances In The Separation, Purification And Salmonella Toxicity Factor Promoter Library Construction And Applications

Staphylococcus aureus is an opportunistic pathogen, which has become resistant to many antibiotics in recent years. It is urgently needed to develope novel anti-Staphylococcus agents. Prevously, a Paenibacillus polymyxa J strain has been isolated in our lab and found to secrete unknown anti-Staphylococcus agents. It's a Gram-positive bacterium, belonging to the genus Paenibacillus. Because it can produce many kinds of useful secondary metabolites, such as antibacterial and antifungal compounds, Intensive studies have been carried out with P. polymyxa in recent years and various strains have been used for antibiotics discovery and for plant diseases control.In this study, the unknown anti-Staphylococcus agent from P. polymyxa J has been investigated in detail. The compound was very effective against methicillin resistant staphylococcus aureus, and was isolated through silica-gel chromatography and preparative HPLC (High Performance Liquid Chromatography). The identity of the compound was characterized by MS (mass spectrometry) and NMR (Nuclear Magnetic Resonance). The results indicate that this anti-Staphylococcus agent is a non-peptide compound with molecular mass of 414.5 Da.It is always difficult to track the activities of antibiotic in natural settings due to its low concentration, which poses a problem during separation and purification. To resolve this problem, a virulence factors-luxCDABE reporter library of Salmonella typhimurium was constructed. This promoter-library can be both used as a tool for drug screening but also used for investigation of S. typhimurium pathogenesis. This system has the following advantages for antibiotic detection:high sensitive, high throughput, real-time and reproducible.Forty sevenvirulence factor genes were selected from Salmonella typhimurium LT2. The promoters of these genes were amplified by PCR and cloned onto a plasmid vector pCS26. The vector contains a promoterless luciferase reporter gene cluster luxCDABE. The promoter-lux plasmids were then transferred back to strain LT2 by electroporation.This promoter-lux reporter library was used to detect low concentrations of antibiotic compounds from the Paenibacillus polymyxa J. A 1000-fold dilution of the anti-Staphylococcus compound collected from column chromatography could still decrease the luminescence of four salmonella strains in the library. The single-peak fraction collected from HPLC contained very low concentration of active compound. It was hard to detect this anti-Staphylococcus agent by using conventional disc-difussion assay. However, using this promoter-lux based method sensitive luminous change was observed, indicating such a detecting system has its advantages.