Differential modification of the Pseudomonas aeruginosa PAO1 outer membrane under hydrogen peroxide and gamma ray

Complete text of publication follows. Objective: Pseudomonas aeruginosa PAO1 is causes opportunistic infections in humans. Studies with animals suggest that an adaptive mechanism is important for the ability of P. aeruginosa PAO1. The adaptive mechanism is protective mechanism against oxidative stress. This mechanism is aimed at preventing by reactive oxygen species. Reactive oxygen species can induce and modulate a variety of biological responses including gene expression. Materials and Methods: Pseudomonas aeruginosa PAO1(a wild-type strain) was grown aerobically with vigorous shaking at 30 deg C in LB broth (Difco). When the optical cells density at 600 nm reached 0.4 that exposed to 0.5-50 mM H2O2 for 30 min and 30-100 Gy Gamma irradiation (60Co, ca.150 TBq of capacity, AECL) for 30 min. For the recovery, the cultures were immediately exchanged fresh media and incubation for 30 min. then, cells were prefixed with 2.5% glutaraldehyde for 30 min at 4 deg C. After two washes by centrifugation at 15,000 X g for 5 min each, the cells were postfixed with 1% osmium tetroxide for 16 h at 24 deg C. The sample was dehydrated with absolute ethanol, stained with 2% uracyl acetate, embedded in Epon resin. Thin sections were stained with lead citrate and uranyl acetate and observed with a electron microscope. Expression level of candidate genes were analyzed using real-time PCR. The amplification program was consist of one cycle at 94 deg C for 30 sec, followed 40 cycles of 94 deg C (5 sec) - 60 deg C (31 sec). Results and Conclusion: In the present study, we have observed differential membrane damage to P. aeruginosa PAO1 cells when exposed to different oxidative stresses such as hydrogen peroxide 0.5-50 mM for 30 min and gamma radiation 30-100 Gy for 30 min using TEM. In order to understand its behaviour, we isolated 3 genes which are related to membrane maintaining. Its transcription level was identified using Real-Time PCR. Each gene was differently expressed under various stresses.