Functional analysis of quorum sensing-mediated pathogenicity in Burkholderia contaminans SK875 using transposon mutagenesis.

Burkholderia contaminans SK875, a member of Burkholderia cepacia complex (Bcc), are known to cause lung infections in cystic fibrosis patients. To gain deeper insights into its quorum sensing (QS)-mediated pathogenicity, we employed a transposon (Tn) insertion-based random mutagenesis approach. A Tn mutant library comprising of 15,000 transconjugants was generated through conjugation between wild-type (WT) recipient B. contaminans SK875 and the donor E. coli BW20767 carrying pRL27 plasmid. From this library, 26 mutants were initially screened using the reporter strain Agrobacterium tumefaciens NT1, identified as blue-colored indicator colonies. These mutants were further analyzed for phenotypic variations related to autoinducer (AI) production, morphological changes, motility, biofilm formation, protease activity, and virulence in Caenorhabditis elegans. The Tn insertion sites in the mutants were sequenced and aligned with the reference genome of B. contaminans SK875 (PRJNA439184). Sequence analysis revealed the Tn5 insertion in genes encoding Ribonuclease P protein, a hypothetical protein, gamma-glutamyltranspeptidase 1, GCN5-related N-acetyltransferase (DUF1311), cytochrome C oxidase assembly protein, glutamyl-Q tRNA synthetase, AFG1-like ATPase, chorismate synthase, and aldehyde oxidase. Compared to wild-type (WT) strain B. contaminans SK875, the mutants (SK1917, SK1925, SK1926, SK1927, SK1935) exhibited attenuated AI production, impaired swimming and swarming motility, reduced biofilm formation and protease activity, and decreased virulence in C. elegans. We suggest that these genes are likely involved in the QS-dependent pathogenicity of B. contaminans. This study also introduces a visual color-screening method for identifying novel gene functions related to QS-dependent pathogenicity in Burkholderia species.