Functional analysis of purM in Burkholderia cenocepacia using a trimethoprim-selectable allelic exchange and mini-Tn7...

Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc) poses a significant threat to immunocompromised individuals, particularly those with cystic fibrosis. Progress in understanding its pathogenesis has been hindered by the availability of efficient genetic tools. We selected the purM gene for functional analysis because it is essential for growth under purine-limited conditions, intracellular survival, and virulence in the closely related species, Burkholderia pseudomallei. We adapted the non-replicative, trimethoprim-resistance (TMP^r) vector pEDL1005 for markerless gene deletion in B. cenocepacia using an optimized sacB-based counterselection strategy, enabling in-frame deletion of purM, which encodes phosphoribosylaminoimidazole synthetase, in both the reference strain K56-2 and the clinical isolate SCBC075. Notably, efficient sacB-based counterselection required strain-specific sucrose concentrations (20% for K56-2 and 25% for SCBC075). To restore purM function, we employed a mini-Tn7-based complementation system (pUC18T-mini-Tn7T-TMP) for site-specific chromosomal insertion downstream of one of the four glmS homologs identified in this bacterium. A PCR-based method verified insertion 18-20 bp downstream of each target locus. Functional analysis revealed that ΔpurM mutants were strictly adenine auxotrophic, confirming the essential role of purM in de novo purine biosynthesis, consistent with findings in B. pseudomallei. However, unlike in B. pseudomallei, the ΔpurM mutant of SCBC075 showed no significant attenuation in intracellular survival within RAW264.7 macrophages. These results validate pEDL1005 and the mini-Tn7 system as effective tools for unmarked gene deletion and chromosomal complementation in B. cenocepacia, expanding the molecular toolkit for this pathogen and enabling future studies of gene function, metabolism, and pathogenesis.IMPORTANCEGenetic manipulation of Burkholderia cenocepacia, a challenging opportunistic pathogen, is essential for elucidating its pathogenesis. However, the available genetic tools remain limited. This study addresses this gap by adapting a pair of trimethoprim-selectable systems: the pEDL1005 vector for markerless gene deletion using strain-specific sacB-based counterselection, and a mini-Tn7-based strategy for site-specific chromosomal complementation. A key advancement is the development of a PCR-based method to verify precise transposon insertion downstream of glmS. Using these systems, we functionally characterized the purM gene, demonstrating that its deletion causes a purine-specific growth defect in minimal media. Together, these tools provide a robust platform for functional studies and enable future research into gene function and pathogenesis in this clinically relevant pathogen.