PROTEOMICS ANALYSIS OF PSEUDOMONAS PUTIDA IN BIODEGRADATION OF AROMATIC COMPOUNDS CAO BIN NATIONAL UNIVERSITY OF SINGAPORE 2007 PROTEOMICS ANALYSIS OF PSEUDOMONAS PUTIDA IN BIODEGRADATION OF AROMATIC COMPOUNDS CAO BIN (B. Eng., Beijing University of Aeronautics and Astronautics, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTER OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS In the current scientific realm, no research endeavor is ever carried out in solitude. This thesis would not have been possible without the assistance and encouragements of many individuals to whom I owe my deepest gratitude. First and foremost, I would like to thank my thesis advisor, Associate Professor Loh Kai-Chee for giving me the opportunity to work in the very interesting and exciting interdisciplinary research field of Proteomics and Biodegradation. My deepest gratitude is expressed for his scientific guidance, continuous support and encouragement during my years at NUS. I would like to thank all my former and current labmates, including Dr. Geng Anli, Dr. Li Yi, Mr. Ne Lin, Ms. Wu Tingting, Ms. Karthiga Nagarajan, Mr. Satyen Gautam, Mr. Vivek Vasudevan, and Mr. Bulbul Ahmed for the helpful discussions and for their assistance during my PhD study. Ms. Chow Pek and Ms. Chew Su Mei Novel, our former and current lab officer, respectively; Mr. Han Guangjun and Ms. Li Xiang, the professional officer and lab officer for bio-research facilities, respectively, deserve separate acknowledgements. Their assistance and support made my life easier in the laboratories. I would like to thank my parents and my wife for their love and support. They are always beside me whenever I need encouragements. i Last but not least my special thanks go to Dr. Peng Zanguo for his support in both academic and non-academic areas and for being a helpful friend on whom I can always count. This work was supported by a research grant from the Ministry of Education Academic Research Fund (R-279-000-181-112). I also want to thank NUS for the research scholarship provided to me. I am also grateful for the generous NIH-AES travel grants from the American Electrophoresis Society, which provided invaluable opportunities for me to attend the AIChE Annual Meetings to present my work and learn from the international community. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS iii SUMMARY vi LIST OF TABLES ix LIST OF FIGURES xi LIST OF ABBREVIATIONS AND SYMBOLS INTRODUCTION 1.1 Research Background and Motivations 1.2 Research Objectives and Scope 1.3 Thesis Organization LITERATURE REVIEW 2.1 Aromatic Pollutants and Bioremediation 2.2 Bacterial Utilization of Aromatic Pollutants 2.2.1 2.2.2 2.2.3 2.2.4 2.3 2.4 Major Bacteria Biodegradation Mechanisms Current Status Molecular Biology Proteomics Technologies in Bioremediation 2.3.1 2.3.2 Proteomics Technologies Proteomics and Bioremediation Proteomics Studies of P. putida 2.4.1 2.4.2 2.4.3 2.4.4 xiv Introduction Proteomics Approaches Used to Study P. putida Elucidation of Catabolic Pathways Understanding physiological responses 11 11 13 19 35 48 48 60 63 63 65 72 78 MATERIALS AND METHODS 84 3.1 84 Bacterial Strains iii 3.2 Culture Media and Growth Conditions 84 3.3 Cell Growth and 2-Hydroxymuconic Semiadehyde Formation 86 3.4 Preparation of Cell Extracts 86 3.5 Catechol Dioxygenases Assays 87 3.6 Determination of Protein Concentration 87 3.7 2-DE 88 3.8 Gel Staining 89 3.8.1 3.8.2 3.8.3 3.9 Silver Stain Plus Kit (Bio-Rad) Bio-Safe Coomassie Stain Destaining Quantitative Analysis 89 90 90 91 3.10 In-gel Digestion and Peptides Extraction 92 3.11 Protein Identification by MS 93 METABOLIC PATHWAY AND CELLULAR RESPONSES OF P. PUTIDA DURING GROWTH ON BENZOATE 95 4.1 Introduction 95 4.2 Experimental Design 98 4.2.1 4.2.2 4.2.3 4.3 Results and Discussion 4.3.1 4.3.2 4.3.3 4.4 Sample Description Quantitative Analysis Bioinformatics Tools Benzoate Catabolic Pathways in P. putida P8 Stress Responses of P. putida P8 to Growth on Benzoate Adaptation of Other Metabolic Routes to Growth on Benzoate Concluding Remarks 98 98 98 99 99 122 125 130 GLOBAL PHYSIOLOGICAL UNDERSTANDING OF P. PUTIDA IN BIPHASIC GROWTH ON A MIXTURE OF PHENOL, 4-CHLOROPHENOL AND SODIUM GLUTAMATE 131 5.1 Introduction 131 5.2 Experimental Design 133 iv 5.2.1 5.2.2 5.3 Results and Discussion 5.3.1 5.3.2 5.3.3 5.3.4 5.4 Sample Description Quantitative Analysis Biphasic Growth of P. putida P8 Analysis of Proteome Profiles Differentially Expressed Catabolic Enzymes Other Metabolic Changes Concluding Remarks 133 133 133 133 135 142 142 148 COMETABOLISM OF CARBAZOLE IN PRESENCE OF SALICYLATE AND P-CRESOL: GLOBAL PHYSIOLOGICAL RESPONSES OF P. PUTIDA ATCC 117484 149 6.1 Introduction 149 6.2 Experimental Design 151 6.2.1 6.2.2 6.3 Results and Discussion 6.3.1 6.3.2 6.3.3 6.4 Sample Description Quantitative Analysis Cell Growth Proteome Analysis of P. putida ATCC 17484 Global Physiological Responses Concluding Remarks 151 151 151 151 153 153 160 CONCLUSIONS AND RECOMMENDATIONS 161 7.1 Conclusions 161 7.2 Recommendations for Future Work 163 REFERENCES 166 LIST OF PUBLICATIONS AND PRESENTATIONS 192 LIST OF AWARDS 194 v SUMMARY Although proteomics research has been, hitherto, confined mainly to areas of drug discovery, diagnostics and molecular medicine, it offers a new and important perspective for studies of microbial physiological responses in biodegradation systems. Proteomics has been applied to elucidate biodegradation pathways, to monitor physiological consequences after metabolic engineering, and to advance the understanding of microbial growth and adaptation to mixed pollutants. In this research, proteomics analysis was used to study three previously reported phenomena in biodegradation involving Pseudomonas putida. The three model systems selected were: i) biodegradation of benzoate by P. putida P8 at high substrate concentration; ii) cometabolic biodegradation of phenol, 4chlorophenol and sodium glutamate by P. putida P8; and iii) cometabolic biodegradation of carbazole, sodium salicylate and p-cresol by P. putida ATCC 17484. Two-dimensional gel electrophoresis (2-DE) was used to separate proteins extracted from P. putida cells harvested from the biodegradation systems. The 2-DE gel profiles 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/ionizationtime of flight mass spectrometry (MALDI-TOF MS). In the first system, eight catabolic enzymes involved in both the orthocleavage (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 vi 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. Furthermore, the down-regulation of the ortho-cleavage pathway in P. putida cells grown on 800 mg/L of benzoate compared to those grown on 100 mg/L of benzoate was suggested. In addition, another 28 differentially expressed proteins were also identified, including proteins involved in i) detoxification and stress response (AhpC, ATPase-like ATPbinding 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. A total of 49 protein spots were selected and identified in the 2-DE gels from P. putida P8 grown on the ternary substrate cometabolic system containing 200 mg/L of phenol, 200 mg/L of 4-chlorophenol and 1000 mg/L sodium glutamate. Among them, 16 protein spots were found differentially expressed in the two exponential growth phases during the biphasic growth, including catabolic enzymes (DmpC, DmpD, DmpE, DmpF, DmpG and AspA) for substrate utilization. The expression levels of these enzymes during growth in the two growth phases correlated well with the substrate utilization patterns observed in previous kinetics studies. The expression of other proteins involved in detoxification and stress responses (DnaK, GroEL, HtpG and AhpC etc.), carbohydrate and energy metabolism (AtpD, AtpH, Tal, Eno), and environmental information processing (several periplasmic binding proteins of ABC vii transporters) as well as a multifunctional xenobiotic reductase (XenA) was quantitatively analyzed and discussed. In the final model system, 25 protein spots were identified in P. putida ATCC 17484 during growth in the ternary substrate cometabolic biodegradation system of carbazole (0.5 mg/L), sodium salicylate (200 mg/L) and p-cresol (10 mg/L or 70 mg/L). There were significant differences in the abundances of proteins during growth at two typical p-cresol concentrations (10 mg/L and 70 mg/L). Specifically, GalU and beta-ketothiolase were involved in the biosynthesis of cell envelope and cytoplasmic membrane; GlnA and periplasmic putrescine-binding component of putrescine ABC transporter were involved in amino acid metabolism, and aldehyde dehydrogenase (ALDH) family protein was involved in the substrate utilization. Collectively, these results enhanced our understanding of the catabolic pathways and the physiological status of P. putida during biodegradation of aromatic compounds. 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Bin Cao and Kai-Chee Loh (2007) Paradigm in biodegradation using Pseudomonas putida – A review of proteomics studies, Enzyme and Microbial Technology (accepted), doi: 10.1016/j.enzmictec.2008.03.004. 4. Bin Cao and Kai-Chee Loh (2007) Physiological changes of Pseudomonas putida in biphasic growth during co-metabolism of 4-chlorophenol in the presence of phenol and glutamate: a proteomics approach, submitted to Biodegradation. 5. Bin Cao and Kai-Chee Loh (2007) Physiological impact of p-cresol on Pseudomonas putida during co-metabolic transformation of carbazole in the presence of salicylate, submitted to World Journal of Microbiology and Biotechnology. 6. Bin Cao and Kai-Chee Loh (2007) Physiological Understanding of P. putida P8 In Biphasic Growth On Mixture of Phenol, 4-Chlorophenol and Glutamate, presented at the Annual AIChE Meeting, Nov 4-9, Salt Lake City, Utah, USA. 7. Bin Cao and Kai-Chee Loh (2006) Proteomic Analysis of P. putida During Biodegradation of High Concentrations of Benzoate: Activation of meta-Pathway and Physiological Responses, presented at the 23rd Annual American Electrophoresis Society Meeting, held in conjunction with the Annual AIChE Meeting, Nov 12-17, San Francisco, California, USA. 8. Bin Cao and Kai-Chee Loh (2005) Proteomic Analysis for Biphasic Growth Pattern in a Cometabolic Mixture of Phenol, Sodium Glutamate and 4-Chlorophenol, 192 presented at the 22nd Annual American Electrophoresis Society Meeting, held in conjunction with the Annual AIChE Meeting, Oct 30- Nov 4, Cincinnati, Ohio, USA. 9. Bin Cao and Kai-Chee Loh (2004) Proteomic Analysis of Sodium Benzoate Degradation pathway in P. putida ATCC49451, presented at the 21st Annual American Electrophoresis Society Meeting, held in conjunction with the Annual AIChE Meeting, Nov 7-12, Austin, Texas, USA. 193 LIST OF AWARDS 1. Bin Cao, NIH-AES Expense Grant Award. AIChE 2006, 23rd Annual AES Meeting: Twenty awardees (18 from USA, was from UK, and Bin Cao was the only one from Asia) selected from over 150 applicants. (http://www.aesociety.org/meetings/2006/ExpenseGrantAwardeesInfo.pdf) 2. Bin Cao, 2nd Prize in AES Best Poster Presentation Award. AIChE 2006, 23rd Annual AES Meeting: A total of 18 posters were judged, and were awarded 1st, 2nd, and 3rd prizes. (http://www.aesociety.org/meetings/2006/memories.php) 3. Bin Cao, NIH-AES Expense Grant Award. AIChE 2005, 22nd Annual AES Meeting: Twenty Awardees (17 from USA, from Canada, from UK, and Bin Cao was the only one from Asia) selected from 72 oral presentations. (http://www.aesociety.org/meetings/2005/AES2005Review.pdf) 194 [...]... species P putida, in particular, are paradigms due to their extraordinary capabilities in degrading aromatic compounds It is exciting to note that the genome of Pseudomonas putida KT2440 was completely sequenced and recently published in 2002 (Nelson et al 2002), which makes physiological understanding of P putida based on proteome analysis feasible Using proteomics tools, some interesting biodegradation. .. presence of phenol and sodium glutamate; 3) Obtain molecular insights into the inhibition of p-cresol to the cometabolic transformation of carbazole in the presence of salicylate by P putida ATCC 17484 The research conducted here was an initial demonstration of the power of proteomics in environmental science applications, which investigated biodegradation systems at the protein level only Although information... tools to biodegradation in order to elucidate the physiological responses of P putida during its biodegradation of aromatic compounds Specifically, the research programme comprised the following: 1) Elucidate the degradation pathways and obtain the cellular responses of P putida P8 during growth on benzoate; 2) Understand the physiological changes of P putida P8 during the biphasic growth on 4-cp in the... Characterization of phenotypic changes within the bacteria is also an important aspect This has so far been ignored, due mainly to the lack of appropriate analytical 2 instrumentation Proteomics is a good complementary tool to kinetics analysis of biodegradation systems By applying proteomics methods to biodegradation, a more comprehensive understanding of the biodegradation systems can be obtained Proteomics. .. hydration in the anaerobic processes is thermodynamically highly unfavorable As a result, aerobic catabolism of aromatic pollutants is more prevalent in the biosphere In aerobic degradation of aromatic compounds, well-defined channels within biodegradation pathways have evolved for most commonly encountered aromatic compounds The evolution is not at all surprising in view of the vast turnover of 14 aromatic. .. roles in degrading a variety of xenobiotics Some enzymes that are associated with biodegradation of aromatic compounds are shown in Table 2-7 In the anaerobic biodegradation of aromatic compounds, the peripheral pathways converge to benzoyl-CoA (occasionally to resorcinol or phloroglucinol) (Figure 2-2a) Specific multi-component reductase catalyzes the dearomatizing reaction, where energy in the form of. .. obtained Proteomics can provide critical insights into the biodegradation kinetics of a pollutant mixture and the physiological responses of the bacteria during the biodegradation process For example, through proteomics analysis of the microorganisms, information on the mechanisms of defense, detoxification and adaptation to chemicals can be obtained Proteome profiling of the microorganisms will generate... of the state -of- the-art techniques in proteomics and the applications of proteomics in biodegradation are extensively reviewed in Chapter 2 Chapter 3 details the materials and methods used in this research Chapter 4 presents the results obtained from a proteomics study focused on the biodegradation of benzoate to elucidate the degradation pathways and the cellular responses of P putida during the pathway... of aromatic compounds from benzene to benzo(pyrene) During the past two decades, anaerobic biodegradation of aromatic pollutants has also been a subject of extensive research Major groups of anaerobic bacteria in the degradation of aromatic pollutants are listed in Table 2-5 which is modified from Table 1 of Zhang and Bennett (2005) Table 2-5 Major groups of anaerobic bacteria in aromatic biodegradation. .. Some of the major bacterial respirations during biodegradation of aromatic compounds and their electron acceptors are shown in Table 2-6 Using benzoate as a model aromatic compound, the redox potential and the energetics (free-energy changes) of the biodegradation are also indicated in the table (Diaz 2004) For the biodegradation of aromatic pollutants, the use of electron acceptors other than oxygen depends . PROTEOMICS ANALYSIS OF PSEUDOMONAS PUTIDA IN BIODEGRADATION OF AROMATIC COMPOUNDS CAO BIN NATIONAL UNIVERSITY OF SINGAPORE 2007 PROTEOMICS ANALYSIS OF PSEUDOMONAS PUTIDA. understanding of the catabolic pathways and the physiological status of P. putida during biodegradation of aromatic compounds. A comprehensive understanding of the bacterial physiology during biodegradation. 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