Molecular studies of Pseudomonas exotoxin A gene.

A 2.7-kilobase DNA fragment carrying the entire exotoxin A (ETA) structural gene was divided into three nonoverlapping probes. Two probes covering the ETA structural gene were used in colony hybridization experiments to determine whether sequences homologous to the ETA gene could be detected in genera other than Pseudomonas or in Pseudomonas species other than Pseudomonas aeruginosa. The majority of strains examined other than the P. aeruginosa strains failed to react in the colony hybridization assays. Some Pseudomonas spp. other than P. aeruginosa and some Bordetella spp. did react in colony hybridization assays with the probes. However, additional studies in which we used Southern hybridization methods indicated that these reactions were apparently nonspecific and that the ETA gene is limited to P. aeruginosa. Studies in which we used all three ETA-related probes in Southern hybridization experiments to analyze the ETA gene and surrounding sequences in P. aeruginosa strains isolated from diverse sources revealed the following: (i) the incidence of the ETA gene in P. aeruginosa is approximately equal to 95%; (ii) there are strains which have been isolated from human infections that do not carry the ETA structural gene; (iii) there is a maximum of one copy of the ETA gene per genome in any given strain; (iv) sequences within and 4 to 5 kilobases downstream of the ETA structural gene appear to be well conserved in different strains of P. aeruginosa; and (v) in contrast, sequences immediately upstream of the ETA structural gene are considerably rearranged from strain to strain. A multicopy plasmid carrying the entire cloned ETA gene was transferred to a tox- P. aeruginosa strain. This strain synthesized and secreted mature, full-length ETA, but the amount produced was small considering the multicopy nature of the plasmid. Synthesis of toxin in this strain was only minimally affected by iron. Our data suggest that the synthesis of ETA is positively regulated. Finally, we found that the presence of the ETA gene is independent of the ability of P. aeruginosa to produce several other recognized virulence factors, supporting the concept of the multifactorial nature of P. aeruginosa virulence.