Cloning and expression in Escherichia coli of a gene encoding nonenzymatic chloramphenicol resistance from Pseudomonas aeruginosa

Abstract

High-level chloramphenicol resistance in Pseudomonas aeruginosa may be due to enzymatic inactivation, ribosomal mutation, or a permeability barrier. We investigated the nonenzymatic resistance mechanism encoded by Tn1696, a transposon found in P. aeruginosa. A 1-megadalton DNA fragment from Tn1696 was cloned which mediated expression of chloramphenicol resistance in Escherichia coli. Comparison of the effects of chloramphenicol on in vitro translation revealed no difference between the susceptible recipient strain and the resistant transformant containing the cloned gene. The rate of chloramphenicol uptake was slower in the resistant strain, suggesting a permeability barrier to the antibiotic. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membranes demonstrated the absence of a 50,000-dalton protein in the resistant strain. DNA homology was evident between Tn1696 and chloramphenicol-resistant isolates of Haemophilus influenzae possessing altered outer membrane permeability. We conclude that chloramphenicol resistance encoded by Tn1696 is due to a permeability barrier and hypothesize that the gene from P. aeruginosa may share a common ancestral origin with these genes from other gram-negative organisms.