Cao Bin, Loh Kai-Chee
Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore.
Biotechnol Bioeng. 2008 Dec 15;101(6):1297-312. doi: 10.1002/bit.21997.
The catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate were studied through proteomics approach. Two-dimensional gel electrophoresis (2-DE) gel profiles of P. putida cells grown on 100 and 800 mg/L benzoate were quantitatively compared using threshold criteria and statistical tools. Protein spots of interest were identified through database searching based on peptide mass fingerprints (PMFs) obtained using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight catabolic enzymes involved in both the ortho-cleavage (CatB, PcaI, and PcaF) and the meta-cleavage (DmpC, DmpD, DmpE, DmpF, and DmpG) pathways for benzoate biodegradation were identified in P. putida grown on 800 mg/L of benzoate while no meta-cleavage pathway enzymes were observed in the 2-DE gel profiles of P. putida grown on 100 mg/L of benzoate. The activation of both the ortho- and the meta-cleavage pathways in P. putida P8 grown on high benzoate concentration was confirmed directly at the protein level. In addition, another 28 differentially expressed proteins were also identified, including proteins involved in (i) detoxification and stress response (AhpC, ATPase-like ATP-binding region, putative DNA-binding stress protein, SodB and catalase/peroxidase HPI); (ii) carbohydrate, amino acid/protein and energy metabolism (isocitrate dehydrogenase, SucC, SucD, AcnB, GabD, ArcA, ArgI, Efp and periplasmic binding proteins of several ABC-transporters); and (iii) cell envelope and cell division (bacterial surface antigen family protein and MinD). Based on the data obtained, physiological changes of P. putida in response to growth on benzoate at different concentrations were discussed.
通过蛋白质组学方法研究了恶臭假单胞菌P8在以苯甲酸盐为碳源生长过程中的分解代谢途径和细胞应答。使用阈值标准和统计工具对在100 mg/L和800 mg/L苯甲酸盐上生长的恶臭假单胞菌细胞的二维凝胶电泳（2-DE）凝胶图谱进行了定量比较。基于使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）获得的肽质量指纹（PMF），通过数据库搜索鉴定了感兴趣的蛋白质斑点。在以800 mg/L苯甲酸盐生长的恶臭假单胞菌中鉴定出了参与苯甲酸盐生物降解的邻位裂解（CatB、PcaI和PcaF）和间位裂解（DmpC、DmpD、DmpE、DmpF和DmpG）途径的8种分解代谢酶，而在以100 mg/L苯甲酸盐生长的恶臭假单胞菌的2-DE凝胶图谱中未观察到间位裂解途径的酶。直接在蛋白质水平上证实了在高苯甲酸盐浓度下生长的恶臭假单胞菌P8中邻位和间位裂解途径均被激活。此外，还鉴定出另外28种差异表达蛋白，包括参与（i）解毒和应激反应（AhpC、ATP酶样ATP结合区域、假定的DNA结合应激蛋白、SodB和过氧化氢酶/过氧化物酶HPI）；（ii）碳水化合物、氨基酸/蛋白质和能量代谢（异柠檬酸脱氢酶、SucC、SucD、AcnB、GabD、ArcA、ArgI、Efp和几种ABC转运蛋白的周质结合蛋白）；以及（iii）细胞膜和细胞分裂（细菌表面抗原家族蛋白和MinD）的蛋白。基于所获得的数据，讨论了恶臭假单胞菌在不同浓度苯甲酸盐上生长时的生理变化。
