Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD ofPseudomonas aeruginosa

Abstract

A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenemspecific outer membrane porin protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD‐sufficiency by a cloned 6‐kb EcoRI‐PstI fragment of DNA from the region of chromosome of the wild‐type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N‐terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD), predicted a hydrophobic protein of MT 41 592. Using the above‐mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1‐kb BamHI‐KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal sequence.

Detection of metalloβ-lactamase genes in heteroresistant Pseudomonas aeruginosa isolated from clinical isolates in Egypt

“…Future studies should be done to make effective combinations of the previously mentioned antibiotics to accelerate the recovery and shorten the duration of the illness period. To detect the imipenem heteroresistant strains, they used the double disk synergy test and SYBGreen RT-PCR for ef lux pump (Huang et al, 1992;Pirnay et al, 2002), in their study, they con irmed twenty imipenem heteroresistant strains and demonstrated that the imipenem resistant mechanism of P.aeruginosa is compromised with the high MexAB expression of ef lux pump, P.aeruginosa can go away from the bacterial membrane and resistance occur (Huang et al, 1992;Pirnay et al, 2002). They made PCR to detect the metallo β-lactamases, but not detected in the twenty heteroresistant ones.…”

Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity

“…This unique structural feature is reflected in its channel function that protein D2 forms a channel of about 400 pico Siemens in 1 M NaC1 as reconstituted in lipid bilayers and flickers frequently [4]. The amino acid sequence deduced from the nucleotide sequence of the cloned oprD gene showed typical features of the porin proteins [5,6]. Furthermore, clinical and laboratory isolates of P. aeruginosa resistant to imipenem, one of the 13-lactam antibiotics, were found to lack protein D2 exclusively [7 9].…”