Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZ␣ gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-P BAD -I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved ⌬asd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI-and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.Burkholderia pseudomallei is the etiologic agent of melioidosis (3, 35). While the bacterium and disease are typically endemic to tropical and subtropical regions of the world (5), historical precedent for use in bioweapon development programs, low infectious doses, high morbidity and mortality, and arduous therapy caused B. pseudomallei to be listed as a category B select agent by the Centers for Disease Control and Prevention. In the United States, transport, possession, and use of select agents is regulated by strict federal guidelines. These guidelines restrict the use of antibiotic resistance markers in research to those that do not compromise the use of the respective drugs in humans, veterinary medicine, or agriculture (27). The paucity of selection markers approved for this bacterium has led to development of genetic manipulation strategies that allow the isolation of unmarked mutants. These include fragment mutagenesis, where a linear DNA fragment containing the mutation, assembled in vitro by PCR, is transferred to the host strain and selection for the antibiotic resistance encoded by the fragment results in gene replacement in the homologous region of the chromosome (4, 32). When the...

In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus

“…The labeling and isolation of bioluminescent equine uterine strains were conducted with established protocols for Pseudomonas species (38). The reporter delivery plasmid, pUC18T-mini-Tn7T-GM-lux (39), was introduced into P. aeruginosa equine isolates by triparental mating using E. coli mobilizer strain RHO3 (40) and helper plasmid pTNS3 (41). Exconjugants were selected on LB medium with 100 g/ml gentamicin.…”

Colony Morphology Variation of Burkholderia pseudomallei Is Associated with Antigenic Variation and O-Polysaccharide Modification

“…PCR primers were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast) ( Table 2). Mutagenesis and complementation was performed using pEXKm5-based allele replacement, as described previously (22,23). The absence of a 236-bp oriT fragment of the pEXKm5 backbone was examined in all mutants.…”

“…Five B. pseudomallei mutants, including three LPS mutants defective in wbiA (BPSL2680), wbiD (BPSL2677), or oacA (BPSL1936) and two strains of capsule mutants defective in wcbB (BPSL2808), were constructed using a fragment mutagenesis method, as described previously (22,23). LPS mutants were constructed in B. pseudomallei K96243 (nonmucoid), and capsule mutants were constructed in B. pseudomallei 4095a (nonmucoid) and 4095c (mucoid), which are isogenic pairs from the same patient (Table 1).…”

Cross-Species Comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei Quorum-Sensing Regulons

“…The bpsI2 (BP1026B_II1251) and bpsI3 (BP1026B_II1673) deletions were constructed by using the dual-plasmid method of Lopez et al (32) and standard molecular biology procedures with E. coli DH10B as a cloning vehicle. To create gene deletion vectors, we used overlap extension PCR to generate approximately 1,000 bp of DNA flanking each gene with genomic DNA from B. pseudomallei Bp82 as a template.…”