Use of suppression-subtractive hybridization to identify genes in the Burkholderia cepacia complex that are unique to Burkholderia cenocepacia

Abstract

We have previously shown differences in virulence between species of the Burkholderia cepacia complex using the alfalfa infection model and the rat agar bead chronic infection model. Burkholderia cenocepacia strains were more virulent in these two infection models than Burkholderia multivorans and Burkholderia stabilis strains. In order to identify genes that may account for the increased virulence of B. cenocepacia, suppression-subtractive hybridization was performed between B. cenocepacia K56-2 and B. multivorans C5393 and between B. cenocepacia K56-2 and B. stabilis LMG14294. Genes identified included DNA modification/phage-related/insertion sequences and genes involved in cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, as well as genes of unknown function. Several of these genes were present in the ET12 lineage of B. cenocepacia but not in other members of the B. cepacia complex. Virulence studies in a chronic lung infection model determined that the hypothetical YfjI protein, which is unique to the ET12 clone, contributes to lung pathology. Other genes specific to B. cenocepacia and/or the ET12 lineage were shown to play a role in biofilm formation and swarming or swimming motility.

FIG. 1.

Virulence studies in a chronic lung infection model. A. Means ± standard deviations of CFU recovered 14 days postinfection from the lungs of four to five rats per bacterial strain. B. Means ± standard deviations of the percent of the lung with inflammatory exudates. Four or five rats were analyzed per group at 14 day postinfection. SB002 is significantly different than K56-2 (P < 0.001) by ANOVA.

FIG. 2.

Biofilm formation. Strains were grown in LB broth for 24 h at 37°C on a rocking platform in a 96-well polystyrene microtiter plate with a pegged lid. Biofilms formed on polystyrene pegs were stained with 1% crystal violet. Values shown are the means ± standard deviations for four pegs. This experiment was repeated at least three times with similar results. 36B4 is significantly different than K56-2 (*, P < 0.05) by ANOVA. SB002, SB006, 32D2, and 34D8 are significantly different than K56-2 (**, P < 0.001) by ANOVA.