New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria

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.

FIG. 1.

Physical map of pE194^ts::pBR322 and its derivative pMAD. (A) Construction of the pMAD plasmid. DNA fragments used to construct pMAD are indicated: mcs, multiple cloning site; pclpB, DNA fragment from S. aureus containing the clpB promoter region; bgaB, DNA fragment containing the promoterless bgaB gene encoding a thermostable β-galactosidase (8). Asterisks indicate restriction sites which have been lost during the construction steps. (B) Map of the pMAD plasmid. Unique restriction sites are indicated.

FIG. 2.

Schematic representation of a two-step procedure used to obtain gene replacement recombination. Areas labeled A and B represent DNA sequences located upstream and downstream from vraF and vraG genes. The crossed lines indicate crossover events. The integration of pMAD via homologous sequences can take place in area A or B. The cointegrate undergoes a second recombination event, regenerating the pMAD plasmid. Depending on whether the second recombination event occurs between the two homologous sequences in area A or B, the spectinomycin resistance marker will either remain in the chromosome (area B) or be excised along with the plasmid (area A). Gene replacement occurs only if the second recombination event occurs in area B, as shown.

FIG. 3.

L. monocytogenes LO28 colonies following transformation with the pMADΔclpB plasmid. Cells were cultivated at 30°C for 6 h, followed by incubation at 39°C for 3 h; dilutions were plated on TSA X-Gal plates in the absence of antibiotics. White colonies have undergone the excision and loss of the plasmid vector, whereas blue colonies retain a copy of the plasmid integrated in the chromosome.