New Vector for Efficient Allelic Replacement in Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria
Maryvonne Arnaud(Centre National de la Recherche Scientifique), Arnaud Chastanet(Centre National de la Recherche Scientifique), Michel Débarbouillé(Centre National de la Recherche Scientifique)
Cited by 950Open Access
Abstract
A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.