Identification of an RTX determinant of Burkholderia cenocepacia J2315 by subtractive hybridization

Abstract

This study utilized suppressive subtractive hybridization between the clinical isolate Burkholderia cenocepacia J2315 and the closely related environmental isolate Burkholderia cepacia ATCC 25416(T) to isolate DNA fragments specific to B. cenocepacia J2315. Analysis of the resulting pools of B. cenocepacia-specific DNAs identified several fragments that may be part of putative virulence factors. Further in silico analysis of a single fragment indicated that it was internal to a gene of which the predicted product had characteristics of repeat in toxin (RTX)-like proteins and high similarity to proteins in other human or plant pathogens. In conjunction with this finding, phenotypic traits associated with known RTX proteins were assessed. A haemagglutinating activity of B. cenocepacia J2315 was identified that was absent in B. cepacia ATCC 25416(T). The expression of this activity appeared to be growth phase-dependent. Analysis of the gene presence and haemagglutinating activity across the species of the B. cepacia complex showed that both were common to the ET12 lineage of B. cenocepacia, but were absent in the other species examined. Haemagglutinating activity was limited to isolates with the RTX-like gene. Expression studies utilizing quantitative PCR demonstrated an association between onset of haemagglutinating activity and increased expression of the gene, which suggests that the putative RTX determinant encodes a haemagglutinating activity.