FIG. 1.

Schematic of allele exchange procedures. For plasmid-based allelic exchange, a PCR-assembled chromosomal segment containing a deletion of orfY with flanking orfX and orfZ sequences is cloned into an appropriate vector, e.g., pEXKm5. The nonreplicative plasmid is delivered to the host strain by conjugation (or electroporation), followed by kanamycin resistance selection. This step results in integration of the allelic replacement construct into the chromosome by homologous recombination between cloned and chromosomal sequences and can be visualized by the appearance of blue colonies on Km- and X-Gluc-containing medium. The two different merodiploid resolution strategies enabled by pEXKm5 are illustrated. For I-SceI-catalyzed resolution (illustrated on the left side), the merodiploid is transformed with the I-SceI expression construct, which results in double-stranded cleavage of the chromosome and release of most of the plasmid backbone. This event can be monitored by the appearance of white colonies on X-Gluc-containing medium. Repair of the double-stranded break by homologous flanking repeat sequences leads to formation of a wild-type strain (event denoted by the circled number 1) or a mutant strain (event denoted by the circled number 2). The two events are distinguished by phenotypic analyses and/or PCR. In a final step that is not illustrated in this figure, purification of colonies with the desired mutant genotype/phenotype at 42°C leads to loss of the pBADSce expression vector in 100% of the colonies. For sacB-mediated counterselection (illustrated on the right side), the merodiploid strain is plated on medium containing sucrose. This counterselection will either result in a wild-type strain (event denoted by Δ1) or in a mutant strain (event denoted by Δ2). These events can be monitored by the appearance of white colonies on X-Gluc- and sucrose-containing medium and are distinguished by phenotypic analyses and/or PCR. Abbreviations: gusA, Escherichia coli glucuronidase-encoding gene; ori, pMB9-derived narrow-range origin of replication; sacB, Bacillus subtilis levansucrase-encoding gene optimized for expression and localization in B. pseudomallei (9).