Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis.
Proteomic analysis of Pseudomonas putida KT2440 cultured in monocyclic aromatic compounds was performed using 2-DE/MS and cleavable isotope-coded affinity tag (ICAT) to determine whether proteins involved in aromatic compound degradation pathways were altered as predicted by genomic analysis (Jiménez et al., Environ Microbiol. 2002, 4, 824-841). Eighty unique proteins were identified by 2-DE/MS or MS/MS analysis from P. putida KT2440 cultured in the presence of six different organic compounds. Benzoate dioxygenase (BenA, BenD) and catechol 1,2-dioxygenase (CatA) were induced by benzoate. Protocatechuate 3,4-dixoygenase (PcaGH) was induced by p-hydroxybenzoate and vanilline. beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway. An additional 110 proteins, including 19 proteins from 2-DE analysis, were identified by cleavable ICAT analysis for benzoate-induced proteomes, which complemented the 2-DE results. Phenylethylamine exposure induced beta-ketoacyl CoA thiolase (PhaD) and ring-opening enzyme (PhaL), both enzymes of the phenylacetate (pha) biodegradation pathway. Phenylalanine induced 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) and homogentisate 1,2-dioxygenase (HmgA), key enzymes in the homogentisate degradation pathway. Alkyl hydroperoxide reductase (AphC) was induced under all aromatic compounds conditions. These results suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.
Kim
Young Hwan
YH
Proteomics Team, Korea Basic Science Institute, Yusung-Ku, Daejeon, Korea.
Cho
Kun
K
Yun
Sung-Ho
SH
Kim
Jin Young
JY
Kwon
Kyung-Hoon
KH
Yoo
Jong Shin
JS
Kim
Seung Il
SI
D000345:Affinity Labels; D001426:Bacterial Proteins / Q000378:metabolism*; D001547:Benzaldehydes / Q000494:pharmacology; D001565:Benzoates / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D007202:Indicators and Reagents; D007553:Isotope Labeling; D010226:Parabens / Q000494:pharmacology; D010627:Phenethylamines / Q000494:pharmacology; D020543:Proteome / Q000032:analysis* / Q000187:drug effects; D040901:Proteomics; D016958:Pseudomonas putida / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000345:Affinity Labels; D001426:Bacterial Proteins; D001547:Benzaldehydes; D001565:Benzoates; D007202:Indicators and Reagents; D010226:Parabens; D010627:Phenethylamines; D020543:Proteome; C100058:vanillin; C038193:4-hydroxybenzoic acid