Proteomic Profiling of Escherichia Coli in Response to Heavy Metals ...

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Heavy Metals Stress. Patcharee Isarankura-Na-Ayudhya. Department of Medical Technology, Faculty of Allied Health Science. Thammasat University, Pathum ...

European Journal of Scientific Research ISSN 1450-216X Vol.25 No.4 (2009), pp.679-688 © EuroJournals Publishing, Inc. 2009

Proteomic Profiling of Escherichia Coli in Response to Heavy Metals Stress Patcharee Isarankura-Na-Ayudhya Department of Medical Technology, Faculty of Allied Health Science Thammasat University, Pathum Thani 12120, Thailand Chartchalerm Isarankura-Na-Ayudhya Department of Clinical Microbiology, Faculty of Medical Technology Mahidol University, Bangkok 10700, Thailand Lertyot Treeratanapaiboon Department of Parasitology, Faculty of Medical Technology Mahidol University, Bangkok 10700, Thailand Kulanan Kasikun Department of Medical Technology, Faculty of Allied Health Science Thammasat University, Pathum Thani 12120, Thailand Kreangkrai Thipkeaw Department of Medical Technology, Faculty of Allied Health Science Thammasat University, Pathum Thani 12120, Thailand Virapong Prachayasittikul Department of Clinical Microbiology, Faculty of Medical Technology Mahidol University, Bangkok 10700, Thailand E-mail: [email protected] Tel: (662) 418-0227; Fax: (662) 412-4110 Abstract Over utilization of natural resources and growing trend of industrialization raise a global concern on environmental toxic metals to living organisms. Using two-dimensional gel electrophoresis (2-DE) in combination with peptide mass fingerprinting, significant changes of differentially expressed proteins in response to toxic doses of cadmium (0.2 mM), zinc (0.6 mM), copper (1.2 mM) and mercuric ions (0.05 mM) were detected and implied multi-involvement of cellular processes. Cadmium ions exerted the toxicity by down-regulation of enzymes involved in the energy metabolism such as aconitase, malic enzyme and enolase. These suppressing effects also took place for components involved in the active transport (dipeptide binding protein and oligopeptide transport periplasmic binding protein) and protein synthesis machinery (elongation factor-Tu and 30S ribosomal protein S1). More importantly, the presence of Cd2+ rendered a markedly increase (> 5 folds) of 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase, an enzyme implicated in riboflavin biosynthesis. Effects of zinc ions resembled those of cadmium ions on the E.

Proteomic Profiling of Escherichia Coli in Response to Heavy Metals Stress


coli metabolism and energy production. Cysteine synthase A and zinc-binding transport protein (ZnuA) were up-regulated upon exposure to copper and mercury, respectively. Taken together, it can be proposed that once the growth arrestment by toxic metals occurred, various mechanisms, i) up-regulating of some enzymes involved in catabolic regulation to maintain cellular energy production; ii) initiation of using the low-energy requiring transport system for importing of amino acid and other carbon sources; iii) facilitating synthesis of metal-binding amino acids and iv) enhancing metal-transporting system for intracellular metal regulation, are accounted for the adaptation and cellular compensation. Keywords: Escherichia coli, proteomic, electrophoresis, heavy metals





1. Introduction Environmental pollution by toxic metal ions, particularly heavy metals, is widespread receiving more attention as a global ‘health-political-economical’ concern due to their involvements in human health problem, their accumulations in the food supply chain, and their distributions in water resources (Sharma and Agrawal, 2005; Schell et al., 2006; Houston, 2007; Islam et al., 2007). Toxicity to the human-being comprises of acute- and chronic-diseases, hormonal imbalances, nutritional deficiencies, autoimmune and neurological disorders etc (Lynes et al., 2007). Plausible explanations on the occurrence of such deleterious effects of heavy metals can be drawn on i) disruptions on a vast assortment of metabolic processes; ii) alterations on a balance of pro-oxidant and antioxidant systems; and iii) competitions with nutrient trace elements for binding sites on essential metalloenzymes, receptors, metal-binding transporter and storage systems (Bertin and Averbeck, 2006; Houston, 2007). To prevent these health deteriorations, contaminations of toxic metals in foods and drinking water are, therefore, stringently monitored and regulated by national and international policies (Reiley, 2007). Investigations of detailed mechanisms on ‘how metals exerted their toxic effects in biological systems’ and ‘how organisms acclimatized themselves in response to metal stress’ are continuously carried out in several models e.g. plants, microbes, animals and mammalian cells (Balshaw et al., 2007; Haferburg and Kothe, 2007; Milner and Kochian, 2008; Thompson and Bannigan, 2008; Yoon et al., 2008). Escherichia coli (E. coli), a Gram-negative bacillus, becomes one of the utmost popular models used for studying roles of metal stress owing to its duplication time and rapid response to toxicants. Regulations of cellular processes following exposure to metal ions at both transcriptional and translational levels have been reported (Ferianc et al., 1998; Brocklehurst and Morby, 2000; Wang and Crowley, 2005; Easton et al., 2006). However, the molecular mechanisms and underlying responses of cells against various metal ions are not yet completely understood. Therefore, in the present study, proteomic analyses of E. coli strain TG1 upon exposure to effective dose (at 50% growth inhibition) of metal ions (cadmium, zinc, copper and mercury) have been carried out. Up- and down-regulation of differentially expressed proteins have been investigated. Mechanisms of cellular adaptation and compensation against different kinds of toxic metals have then been proposed.

2. Materials and Methods 2.1. Bacterial Strain E. coli strain TG1 (supE, hsdΔ5, thiΔ(lac-proAB), F’[traD36 proAB+ lacIq lacZΔM15] was used throughout the study.


Patcharee Isarankura-Na-Ayudhya, Chartchalerm Isarankura-Na-Ayudhya, Lertyot Treeratanapaiboon, Kulanan Kasikun, Kreangkrai Thipkeaw and Virapong Prachayasittikul

2.2. Effect of Metal Ions on Growth Inhibition Cells were grown in 5 ml Luria-Bertani (LB) broth (10 g/L tryptone, 5 g/L NaCl and 5 g/L yeast extract, pH 7.2) supplemented with 100 mg/L ampicillin at 37ºC, 150 rpm for overnight. Then 50 μl of overnight cultures were transferred into 5 ml broth and grown until OD reached 0.5. Cells were adjusted to equal OD = 0.05 in LB broth supplemented with 200 mg/L ampicillin. Aliquot of cell (100 μl) was seeded onto a 96 well sterile microplate and 100 μl of LB broth containing various concentrations of CdCl2, ZnCl2, CuCl2 or HgCl2 were then added. The metal chloride was used in order to prefer biosorption of metal on the cell surface to its intracellular uptake (Kotrba et al., 1999). Plate was further incubated at 37ºC with moisture for 30 hours. The growth rate was determined by monitoring the absorbance at 540 nm by microplate reader. The concentrations of metal ions those gave rise approximately 50% growth inhibition were selected for further experiments. 2.3. Preparation of Protein Samples for Proteomic Analysis Initially, cells were grown at 37ºC for overnight in 5 ml LB/Amp. Cells were subcultured and further incubated at 37ºC for 6 hours to mid-exponential phase. Cells were subsequently inoculated in 50 ml LB/Amp and incubated at 37ºC for 4 hours. Metal solutions were then added to the cultures to yield the final concentrations of 0.2 mM cadmium, 0.6 mM zinc, 1.2 mM copper and 0.05 mM mercury and the cells were incubated for additional 12 hours. Preparation of crude protein extracts was performed as previously described (Isarankura-Na-Ayudhya et al., 2008) with minor modification. Briefly, cells were collected, washed, and resuspended in Tris buffer. The suspension was then mixed with 250 μl of lysis solution (7 M urea, 2 M thiourea, 4% CHAPS; freshly prepared by supplementation with 10 mg/ml dithiothreitol (DTT) and 10 μl/ml protease inhibitor cocktail). Cells were disrupted on ice by sonic disintegration using Branson sonifier (model 450) equipped with a microtip. Collection of whole cell lysates was performed by centrifugation at 15,000 rpm for 60 min at room temperature. Bradford’s method was used for quantification of protein amounts using bovine serum albumin as a standard. The protein solution was finally mixed with 1 M acrylamide (at 1:10 of total volume) and stored at room temperature for 10 min. 2.4. Two-Dimensional Gel Electrophoresis (2-DE) Two-dimensional gel electrophoresis was carried out using 2-D Electrophoresis System (GE Health care, USA) as described previously (Panpumthong and Vattanaviboon, 2006) with some modification. Two hundred micrograms of bacterial protein extract was mixed with 350 μl of rehydration buffer (8 M urea, 4% CHAPS, 2 mM TBP, 0.001% bromphenol blue and 65 mM dithiothreitol) containing 1% 3-10 IPG buffer. The mixture was stand for 15 min at room temperature and removal of insoluble material was done by centrifugation at 15,000 rpm for 10 min at 20°C. The supernatant was further loaded on to 18-cm IPG strips with pH range of 3-10 of an isoelectric focusing system (IPGphoreTM). Samples were run through steps of strip rehydration (20°C, 12 h) and isoelectric focusing (500 volts for 1 h, 1,000 volts for 1 h, and 8,000 volts to reach 33,000 volt·h). The maximum current was maintained at 50 mA per one strip. After the complete process was accomplished, the strip was equilibrated twice times (15 min each) in equilibration buffer (50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.03% bromphenol blue) supplemented with 65 mM DTT and 135 mM iodoacetamide to allow the cysteine residues to be reduced and then carbamidomethylated. The strip was subjected to the second dimensional separation (HoeferTM DALT) using a SDS-polyacrylamide gel (12.5%). Separation of protein was executed under the applied voltage of 20 volt per gel at 15°C until the bromphenol blue dye front reached 0.5 cm from the bottom of the gel.

Proteomic Profiling of Escherichia Coli in Response to Heavy Metals Stress


2.5. Gel Staining and Differential Analysis The gels were stained with silver stain according to the standard recommendation. After staining, gel images were acquired using Image scanner II (GE Health Care, USA). Differential analysis was performed by ImageMaster 2D Platinum version 6.2.1 (GE Health Care, USA) software tool. These included spot intensity calibration, spot detection and background subtraction. Quantification of intensity of each spot was performed in term of spot volume (area intensity). The total spot volume normalization method was applied in which the percentage of each spot volume on a gel image is calculated relative to the total volume of all spots on that image. Then, determination of differentially expressed proteins was achieved by comparing the ratio of % volume values between control and treated sample gel. 2.6. MALDI-TOF Mass Spectrometry and Peptide Mass Fingerprinting (PMF) Analysis Mass spectrometry and peptide mass fingerprinting (PMF) analysis were carried out as described (Isarankura-Na-Ayudhya et al., 2008). Initially, protein spots were manually excised from the gels, transferred to microtitre plate, and then soaked in 50% methanol and 5% acetic acid for overnight. Tryptic digestion using sequencing grade of modified trypsin (Promega, UK) and protein extraction were performed on a Spot Handling Workstation (GE Health Care, USA) using the preset protocols from the manufacture. MALDI-TOF mass spectrometer (Model ReflexIV, Bruker Daltonics, Germany) was used for protein identification based on peptide fingerprint map. Briefly, the extracted peptides were mixed with solution of 10 mg/ml α-cyano-4-hydroxycinnamic acid (LaserBio Labs, France) in 66% acetonitrile and 0.1% trifluoroacetic acid (TFA) and then spotted onto a 96-well target plate. The mass spectra were acquired in the positive ion reflector delayed extraction mode using approximately 200 laser shots. Peak lists were generated using the XMASS software (Bruker Daltonics). The BioTool 2.0 software (Bruker Daltonics) integrated with the MASCOT 2.2 search engine (MatrixScience, was used for spot proteins identification by querying the trypsindigested peptide fragment data using the reference database NCBInr with 4626804 sequences and 1596079197 residues. The searching criteria were set up as follows: complete carbamidomethylation of cysteine and partial methionine oxidation were exploited; an initial mass tolerance of ± 200 ppm was used in all searches; the number of missed cleavage sites was allowed up to 1. Search result scores which is greater than 59 was considered to be of significant difference (p

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