Molecular studies of Pseudomonas exotoxin A gene

Abstract

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-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.

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

“…The reliability and high discriminatory potential of DNA fingerprinting suggest its use in bacteriological epidemiology. In conjunction with sero- [6], phage [13], pyocin typing [14] and the DNA probing of hypervariable genomic sites [15] the genome fingerprinting of P. aeruginosa strains may be employed to investigate outbreaks of nosocomial infection and to aid effective infection control, e.g., in cystic fibrosis, the chronic P. aeruginosa infection of the lungs is primarily responsible for pulmonary deterioration and reduced life expectancy [16]. Using DNA fingerprinting, we were able to detect cross-infection with P. aeruginosa strains among siblings with cystic fibrosis, to monitor the acquisition and genomic variations of strains during the course of the disease, and to follow the spread and transmission of organisms in hospital wards (manuscript in preparation).…”

Virulence factors of Burkholderia cepacia

“…Molecular studies of the Pseudomonas exotoxin A gene by Vasil et al [31] concluded that the production of exotoxin A and the presence of the exotoxin A gene are probably limited to P. aeruginosa and is not found in other Pseudomonas spp. Southern hybridization experiments under low, medium and high stringency conditions with an exotoxin A gene probe failed to produce a positive signal with any of 8 B. cepacia strains tested.…”

Genotyping ofPseudomonas aeruginosasputum and stool isolates from cystic fibrosis patients: evidence for intestinal colonization and spreading into toilets

“…The purified DNA (1-2 jtg) was digested with the restriction endonucleases Bgl II, Sal I and Xho I, electrophoresed through a 0-6% agarose gel and transferred to a nylon membrane (Pall, Dreieich, Federal Republic of Germany) using the Southern method (15). The Escherichia coli plasmid pCMtox (16) was isolated (17) and labelled with biotin-1 1-dUTP (Gibco, Bethesda, USA) by nick translation. In modifications of the original method (13), the whole pCMtox plasmid was used for hybridization.…”