Mutational analysis and biochemical characterization of the Burkholderia thailandensis DW503 quorum-sensing network

Abstract

Numerous gram-negative bacteria communicate and regulate gene expression through a cell density-responsive mechanism termed quorum sensing (QS), which involves the synthesis and perception of diffusible N-acyl-homoserine lactones (AHL). In this study we genetically and physiologically characterized the Burkholderia thailandensis DW503 QS network. In silico analysis of the B. thailandensis genome revealed the presence of at least three AHL synthases (AHS) and five transcriptional regulators belonging to the LuxIR family of proteins. Mass spectrometry demonstrated that wild-type B. thailandensis synthesizes N-hexanoyl-homoserine lactone (C6-HSL), N-octanoyl-homoserine lactone (C8-HSL), and N-decanoyl-homoserine lactone (C10-HSL). Mutation of the btaI1 (luxI) AHS gene prevented accumulation of C8-HSL in culture supernatants, enhanced beta-hemolysis of sheep erythrocytes, increased lipase production, and altered colony morphology on swarming and twitching motility plates. Disruption of the btaI3 (luxI) AHS prevented biosynthesis of C6-HSL and increased lipase production and beta-hemolysis, whereas mutagenesis of the btaI2 (luxI) allele eliminated C10-HSL accumulation and reduced lipase production. Complementation of the btaI1 and btaI3 mutants fully restored the synthesis of C8-HSL and C6-HSL to parental levels. In contrast, mutagenesis of the btaR1, btaR3, btaR4, and btaR5 (luxR) transcriptional regulators had no effect on AHL accumulation, enhanced lipase production, and resulted in extensive beta-hemolysis on sheep blood agar plates. Furthermore, interruption of the btaI1, btaR1, and btaR3 genes altered colony morphology on twitching and swarming motility plates and induced pigmentation. Additionally, phenotypic microarray analysis indicated that QS in B. thailandensis both positively and negatively affects the metabolism of numerous substrates, including citric acid, formic acid, glucose 6-phosphate, capric acid, gamma-hydroxybutyric acid, and d-arabinose. These results demonstrate that mutagenesis of the B. thailandensis QS system affects various cellular processes, including lipase production, swarming and twitching motility, beta-hemolysis of sheep erythrocytes, and carbon metabolism and/or transport.

FIG. 1.

Synthesis of the AHL molecules produced by B. thailandensis and each luxI mutant. AHL extracts were spotted (10 μl) onto C₁₈ reversed-phase TLC plates and visualized by using an A. tumefaciens A136 overlay. Lane 1, wild-type B. thailandensis; lane 2, BTRJ1 (btaI1); lane 3, BTRJ2 (btaI2); lane 4, BTRJ3 (btaI3); lane 5, BTRJ9 (pBHR1-btaI1); and lane 6, BTRJ10 (pBHR1-btaI3).

FIG. 2.

Structural analysis of the AHLs biosynthesized by B. thailandensis. (A) Synthetic C₆-HSL; (B) synthetic C₈-HSL; (C) synthetic C₁₀-HSL; (D to F) AHLs (C₆-HSL, C₈-HSL, and C₁₀-HSL, respectively) extracted from an overnight culture of B. thailandensis.

FIG. 3.

Mutations in the B. thailandensis QS system result in hyper-beta-hemolysis of sheep erythrocytes and altered colony morphologies on twitching and swarming motility plates. Experiments were performed in triplicate, as described in Materials and Methods. Panel 1 shows beta-hemolytic activity, while panels 2 and 3 show the results of twitching and swarming motility assays, respectively. DW503 represents wild-type B. thailandensis, btaI corresponded to luxI mutants, and btaR was disrupted luxR homologues.