Alternative method for genetic transformation of Pasteurella multocida X73 using a hyaluronidase-producing Staphylococcus aureus strain.
The hyaluronic acid-rich capsules make affected bacteria relatively retractile to transformation. In this work, native hyaluronidase produced in situ by Staphylococcus aureus, was investigated to replace the pure enzyme in bacterial capsule depolymerization and its subsequent genetic transformation. The encapsulated Pasteurella multocida X73 was cultivated as a mixed culture with a hyaluronidase-producing strain of S. aureus. Then different concentrations (50 ng and 200 ng) of the Pasteurella-Escherichia coli shuttle vector pPBA1100 were electro-transformed into 40 microl of the mentioned culture. Recombinants were selected on nutrient agar supplemented with 50 microg/ml of kanamycine (Kan). The effects of a capsule-weakening agent on electroporation rates were also evaluated. The use of this technique has allowed us to attain transformation frequencies 10(5) times higher than a previously available protocol using pure enzyme. A control experiment involving a non-hyaluronidase-producing strain S. aureus substantiated the effect of the staphylococcal enzyme. The proposed alternative method could be also used for genetic transformation for other capsulated bacteria, which is not possible without pure hyaluronidase.
Abdelhak
Darbouche
D
Institute of Biology, Department of Microbiology, University Khenchela, Route de El Hamma, Khenchela, Algeria. adarbouche@hotmail.com
D016667:Bacterial Capsules / Q000187:drug effects; D018014:Gene Transfer Techniques; D006821:Hyaluronoglucosaminidase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D016979:Pasteurella multocida / Q000187:drug effects* / Q000235:genetics*; D013211:Staphylococcus aureus / Q000201:enzymology*; D014169:Transformation, Bacterial
D006821:Hyaluronoglucosaminidase