Burkholderia pseudomallei is the causative agent of melioidosis, an overwhelming, rapidly fatal septic infection, and B. thailandensis is a closely related, less virulent species. Both organisms are naturally competent for DNA transformation, and this report describes a procedure exploiting this property for the rapid generation of marked deletion mutations by using PCR products. The method was employed to create 61 mutant strains. Several selectable elements were employed, including elements carrying loxP and FRT recombinase recognition sites to facilitate resistance marker excision. Chromosomal mutations could also be transferred readily between strains by transformation. The availability of simple procedures for creating defined chromosomal mutations and moving them between strains should facilitate genetic analysis of virulence and other traits of these two Burkholderia species.Burkholderia pseudomallei and Burkholderia thailandensis are closely related gram-negative bacteria widely distributed in soils of Southeast Asia and northern Australia (19,21). B. pseudomallei is the causative agent of melioidosis, whereas B. thailandensis is rarely associated with human disease but can kill rodents at high infectious doses (14,22). The B. thailandensis genome is smaller than that of B. pseudomallei (6.7 versus 7.2 Mbp) but encodes homologues of many of the established B. pseudomallei virulence determinants, including type III secretion systems and functions responsible for cellto-cell spread during infection (15,16,20). Phylogenomic comparisons imply that the two species share numerous additional traits as well (10, 23). Thus, B. thailandensis serves as a lowvirulence surrogate for studying numerous physiological and pathogenic characteristics of B. pseudomallei.Progress in the genetic analysis of B. thailandensis and B. pseudomallei has been limited by the lack of a general procedure for creating targeted mutations based on the genome sequences. Standard two-step plasmid-based procedures (18) employing sacB as a counterselective marker have not been generally successful for the two species due to the presence of endogenous sacB genes (although there have been exceptions) (6, 12). A simpler alternative, the direct generation of predefined mutations by transformation of PCR fragments, has greatly facilitated the genetic analysis of several bacterial species (7,11,17). In general, the fragments carry selectable markers flanked by regions of homology oriented such that homologous recombination replaces genomic sequences with the selectable marker. In this study, we developed such a procedure for B. thailandensis and B. pseudomallei which exploits the discovery, presented here, that these bacteria can be rendered naturally competent for DNA transformation.
MATERIALS AND METHODS
Bacterial strains and growth conditions. B. thailandensis E264 (from DonWoods, University of Calgary) and S95019 (Siriraj Hospital collection) and B. pseudomallei 1026a and 1026b (from Sharon Peacock, Wellcome Trust Unit, Bangkok, Thailand) were ...