Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei

Phage lambda-Red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the gamma-proteobacteria. Procedures are described here to easily knock out (KO) and pull out (POPO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counterselectable and selectable markers (sacB, pheS, gat and the arabinose-utilization operon) and lambda-Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (POPO) PCR fragments are generated with flanking 40- to 45-bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubation of the PCR product with cells expressing lambda-Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes similar to 10 d and is advantageous over previously published protocols: (i) smaller PCR products reduce primer numbers and amplification steps, (ii) POPO fragments suitable for downstream manipulation in Escherichia coli are obtained and (iii) chromosomal KO increases flexibility for downstream processing.