Functional analysis of <i>purM</i> in <i>Burkholderia cenocepacia</i> using a trimethoprim-selectable allelic exchange and mini-Tn7 complementation approach

<i>Burkholderia cenocepacia</i>, a member of the <i>Burkholderia cepacia</i> 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 <i>purM</i> gene for functional analysis because it is essential for growth under purine-limited conditions, intracellular survival, and virulence in the closely related species, <i>Burkholderia pseudomallei</i>. We adapted the non-replicative, trimethoprim-resistance (TMP^r) vector pEDL1005 for markerless gene deletion in <i>B. cenocepacia</i> using an optimized <i>sacB</i>-based counterselection strategy, enabling in-frame deletion of <i>purM</i>, which encodes phosphoribosylaminoimidazole synthetase, in both the reference strain K56-2 and the clinical isolate SCBC075. Notably, efficient <i>sacB</i>-based counterselection required strain-specific sucrose concentrations (20% for K56-2 and 25% for SCBC075). To restore <i>purM</i> function, we employed a mini-Tn7-based complementation system (pUC18T-mini-Tn7T-TMP) for site-specific chromosomal insertion downstream of one of the four <i>glmS</i> homologs identified in this bacterium. A PCR-based method verified insertion 18-20 bp downstream of each target locus. Functional analysis revealed that <i>ΔpurM</i> mutants were strictly adenine auxotrophic, confirming the essential role of <i>purM</i> in <i>de novo</i> purine biosynthesis, consistent with findings in <i>B. pseudomallei</i>. However, unlike in <i>B. pseudomallei</i>, the Δ<i>purM</i> 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 <i>B. cenocepacia</i>, expanding the molecular toolkit for this pathogen and enabling future studies of gene function, metabolism, and pathogenesis.IMPORTANCEGenetic manipulation of <i>Burkholderia cenocepacia</i>, 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 <i>sacB</i>-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 <i>glmS</i>. Using these systems, we functionally characterized the <i>purM</i> 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.