DETECTION OF RTX TOXIN GENES IN GRAM-NEGATIVE BACTERIA WITH A SET OF SPECIFIC PROBES

The family of RTX (RTX representing repeats in the structural toxin) t oxins is composed of several protein toxins with a characteristic nona peptide glycine-rich repeat motif. Most of its members were shown to h ave cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The pro bes include Darts of apxIA, apxIIA, and apxIIIA from Actinobaeillus pl europneumoniae, cyaA from Bordetella pertussis, frpA from Neisseria me ningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escheri chia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA fr om Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gr am-negative bacteria were investigated for the presence of RTX toxin g enes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial speci es for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method wer e optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreove r, it is useful for the detection of already-known or new RTX toxin ge nes in bacteria of medical importance.