Global analysis of disulfide bond proteins in Pseudomonas aeruginosa exposed to hydrogen peroxide and gamma rays.
OBJECTIVE
To isolate and characterise the oxidative stress-responsible disulfide bond proteins (DSBP) from Pseudomonas aeruginosa, under hydrogen peroxide (H(2)O(2)) and gamma ray-induced stress conditions.


METHODS
Oxidative stress in P. aeruginosa was induced by H(2)O(2) or (60)Co gamma-rays. DSBP of P. aeruginosa were isolated by using a thiol affinity purification method and liquid chromatography-mass spectrometry (LC-MS/MS). Induction of DSBP and their transcriptional levels were analysed by Western blot and quantitative-polymerase chain reaction (q-PCR), respectively.


RESULTS
We isolated and identified 40 DSBP in P. aeruginosa. A comparison of DSBP patterns under different stress conditions, including exposure to 0.5 mM H(2)O(2) and 30 Gy of gamma rays, was carried out. We selected 13 DSBP based on their increased intensity under oxidative stress and investigated their expression levels using q-PCR. Of these 13 proteins, P. aeruginosa (PA) 0126 and PA0277 transcriptional levels were strongly induced (4- to 9-fold) by gamma ray. A corresponding induction in the translational levels of PA0126 was confirmed by Western blot analysis. PA0126 was identified is a hypothetical protein with 206 amino acids and contained three cysteine residues. A shift in molecular weight of PA0126 under oxidised/reduced state indicated its homo-oligomeric structure under non-reducing condition.


CONCLUSIONS
PA0126, a hypothetical protein with disulfide bonds was identified as a key responder to oxidative stress along with other known DSBP of P. aeruginosa. We determined that all DSBP underwent a redox cycling. Furthermore, our results give deeper insight into the relationship between oxidative stress and DSBP in P. aeruginosa.
An
Byung Chull
BC
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeollabuk-do, Korea.
Lee
Seung Sik
SS
Lee
Eun Mi
EM
Wi
Seung Gon
SG
Park
Woojun
W
Chung
Byung Yeoup
BY
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004220:Disulfides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004781:Environmental Exposure / Q000009:adverse effects; D005720:Gamma Rays / Q000009:adverse effects*; D006861:Hydrogen Peroxide / Q000633:toxicity*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*
D001426:Bacterial Proteins; D004220:Disulfides; D006861:Hydrogen Peroxide