Xanthomonas albilineans 細菌素基因選殖及 Xanthomonascampestris pv. glycines 細菌素基因表現之探討

Abstract

Bacteriocin is known to be produced by bacteria and has bactericidal activity against the closely related species. Two Xanthomonas species, Xanthomonas albilineans (Xa) and Xanthomonas campestris pv. glycines YR32 (XcgYR32), have been identified in this laboratory and have antimicrobial activity toward several tested Xanthomonas strains. Moreover, bacteriocin produced by Xa had been purified from cultural broth by polyethylene glycol 6000 precipitation and gel filtration chromatography and characterized as a phage tail-like bacteriocin. An alternative purification procedure using sucrose gradient centrifugation was applied in this study and the components of the purified protein complex were then analyzed by two-dimensional gel electrophoresis. A total of nine protein spots were isolated, digested by trypsin and analyzed by LC/MS/MS. Results of the BLAST analysis revealed that most of these proteins are closely related to prophage proteins of Pseudomonas syringae pv. syringae B728a. The genes corresponding to the Xa bacteriocin were identified from the constructed Xa partial genomic library. DNA sequence analyses revealed that a total of 14 ORFs could be identified in the cloned 5.8-kb and 4.6-kb DNA fragments from the constructed libraries. These ORFs also show high sequence similarity to the Mu phage of Pseudomonas syringae pv. syringae B728a. In the second part of this study, the bacteriocin genes, glyA and glyB, of XcgYR32 were subcloned into various pET vectors and expressed in several E. coli strains. A very low amount of soluble GlyAB-His with bactericidal activity could be obtained from BL21(DE3) at a lower IPTG concentration and induction temperature. The GlyAB-His protein was purified through Ni2+-NTA column and Hiprep 16/60 Sephacryl S-300 column chromatography. A total of 50 μg purified GlyAB-His could be obtained from 100 ml of culture and with a specific activity of 212 x 100 AU/ml. The purified GlyAB-His was stable at temperature up to 70℃ for one hour and resistant to trypsin and chymotrypsin digestion. To promote the stability of GlyA and GlyB, chimeric proteins GlyA5 to A7 were constructed. Results showed that a very high level expression of chimeric A5 and A6 proteins could be achieved, however, most of the expressed proteins were insoluble and the chimeric protein expressed from A7 construct was found to be soluble and have bactericidal activity.