Article pubs.acs.org/jpr
Survival During Long-Term Starvation: Global Proteomics Analysis of Geobacter sulf urreducens under Prolonged Electron-Acceptor Limitation Reema Bansal,† Ruth A. Helmus,† Bruce A. Stanley,‡ Junjia Zhu,§ Laura J. Liermann,∥ Susan L. Brantley,∥ and Ming Tien*,† †
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 305 South Frear Laboratory, University Park, Pennsylvania 16802, United States ‡ Section of Research Resources, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033, United States § Department of Public Health Sciences, The Pennsylvania University State College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033, United States ∥ Earth and Environmental Systems Institute, The Pennsylvania State University, 2217 Earth-Engineering Sciences Building, University Park, Pennsylvania 16802, United States S Supporting Information *
ABSTRACT: The bioavailability of terminal electron acceptors (TEAs) and other substrates affects the efficiency of subsurface bioremediation. While it is often argued that microorganisms exist under “feast or famine”, in the laboratory most organisms are studied under “feast” conditions, whereas they typically encounter “famine” in nature. The work described here aims to understand the survival strategies of the anaerobe Geobacter sulf urreduces under TEA-starvation conditions. Cultures were starved for TEA and at various times sampled to perform global comparative proteomic analysis using iTRAQ to obtain insight into the dynamics of change in proteins/enzymes expression associated with change in nutrient availability/ environmental stress. Proteins varying in abundance with a high level of statistical significance (p < 0.05) were identified to understand how cells change from midlog to (i) stationary phase and (ii) conditions of prolonged starvation (survival phase). The most highly represented and significantly up-regulated proteins in the survival phase cells are involved in energy metabolism, cell envelope, and transport and binding functional categories. The majority of the proteins were predicted to be localized in the cell membranes. These results document that changes in the outer and cytoplasmic membranes are needed for survival of Geobacter under starvation conditions. The cell shuts down anabolic processes and becomes poised, through changes in its membrane proteins, to sense nutrients in the environment, to transport nutrients into the cell, and to detect or utilize TEAs that are encountered. Under TEA-limiting conditions, the cells turned from translucent white to red in color, indicating higher heme content. The increase in heme content supported proteomics results showing an increase in the number of cytochromes involved in membrane electron transport during the survival phase. The cell is also highly reduced with minimal change in energy charge (ATP to total adenine nucleotide ratio). Nonetheless, these proteomic and biochemical results indicate that even under TEA starvation cells remain poised for bioremediation. KEYWORDS: Geobacter sulfurreducens, starvation phase, survival strategy, terminal electron acceptor (tea), iTRAQ, LC-MS/MS, proteomics, heme content, energy charge, uranium bioremediation
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INTRODUCTION
anaerobes can utilize a wide range of both solid and soluble metals as TEAs.2 This respiratory process is referred to as dissimilatory metal reduction (DMR). Interest in this process intensified when researchers discovered that microorganisms catalyzing DMR are also able to reduce U(VI) and Tc(VII) to
Microorganisms play a central role in the redox cycling of inorganic compounds on Earth. This biological redox cycling is thought to be involved in a large range of transformations that can result in both dissolution and precipitation reactions.1 One example of a geochemically relevant biological redox process involves the utilization of metals as terminal electron acceptors (TEAs). In the absence of oxygen, facultative and obligate © 2013 American Chemical Society
Received: March 24, 2013 Published: August 27, 2013 4316
dx.doi.org/10.1021/pr400266m | J. Proteome Res. 2013, 12, 4316−4326
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Article
changes in protein expression between midlog, stationary, and survival phases.12 Our present study is not the first to characterize differential protein expression under different growth conditions for Geobacter. Ding et al.13 compared Geobacter protein profiles from midlog batch, late-log batch, and chemostat (steady state) cultures grown with fumarate and iron citrate as electron acceptors. Khare et al.14 and Ding et al.15 also characterized the proteomes of metabolically active and growing Geobacter cultures with fumarate and ferric citrate and ferric oxide and ferric citrate as TEAs, respectively. Nunez et al.,16 used proteomics approach to characterize the RpoS regulated genes by comparing gene expression and protein profiles of the wild-type strain and rpoS mutant. None of these previous studied characterized the proteome of cells undergoing prolonged TEA starvation, which is the focus of the present study. The goal of the present study was to examine the survival strategy through global proteome analysis using iTRAQ, of prolonged starving cultures of Geobacter, a condition relevant to oligotrophic environments. In addition to this global analysis, we also measured the change in the energy charge between the exponentially growing and the starved cells. We also determined the total cellular heme content of the midlog, stationary, and survival phase cells.
U(IV) and Tc(IV), respectively, resulting in their precipitation and thus immobilization.2 To stimulate the reductive bioremediation of these radionuclides in the oligotrophic environments where they are found, researchers often inject electron donors directly into wells.3 A Gram-negative DMR bacterium commonly found in such environments is Geobacter sulf urreducens. Geobacter has rapidly become a model organism for the study of DMR due to its ubiquity in oligotrophic environments that harbor these radioactive contaminants.4 In addition, the organism has a sequenced genome5 and genetic systems for manipulation.4 Throughout the region of contamination, G. sulf urreducens, like other environmental microbes living in natural oligotrophic environments, exists under a “feast or famine” model of survival.6 Famine may be the norm under typical conditions. TEA limitation is thought to be an environmentally relevant condition, as field studies have shown that increased microbial activity or dilution from rainfall can result in transient and regional depletion of suitable TEA concentrations.7 Furthermore, famine may even persist after time elapses after carbon addition through injection wells. Under normal conditions, microorganisms may typically have access to TEA but be limited in electron donors (carbon source). However, in the presence of excess electron donors such as that found near an injection well, the area may become highly reduced and thus be limited in availability of TEA. In fact, this is often a model remediation strategy, that is, to maintain a highly reduced environment such that the micro-organisms are primed to reduce any influx of U(VI) or Tc(VII). Understanding how a target organism exists in contaminated waste sites can help in planning efficient bioremediation strategies. Previously, we demonstrated that G. sulfurreducens displays five typical stages of growth when grown under electron-acceptor (fumarate)-limiting conditions: lag, log, stationary, decline, and survival phases.8 In the laboratory, our results showed that the organism can sustain a stable population of ∼106 cells/mL for over 2 years without replenishment of growth medium and acquires a growth advantage adaptation that begins to emerge during prolonged starvation.8 This adaptation is termed “growth advantage in stationary phase,” or GASP.8 The older GASP strains can outcompete younger cultures when cocultured under similar conditions.8 The work described here is aimed at understanding the survival strategies of Geobacter under TEA-limiting conditions, conditions that we suggest would evolve at injection wells after prolonged carbon addition. The present study probes the changes in the total proteome. We present the first look at the physiological state of long-term stationary phase cells starved for TEA and the differences in protein expression between growing cells and starved cells of G. sulf urreducens. As documented in the literature, changes in nutrient availability or in environmental stress are always associated with changes in expression of new or different suites of proteins/enzymes. For example, cells can synthesize storage compounds like glycogen9 and polyphosphates,10 which help them through periods of energy-poor conditions. Stationary-phase cells can also produce protective substances such as trehalose for thermotolerance.11 Therefore, studying the differential protein expression between different growth phases grown under TEA limitation will elucidate how G. sulf urreducens persists under stress conditions. We employed iTRAQ to obtain information about relative
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MATERIALS AND METHODS
Reagents and Chemicals
All reagents and chemicals were acquired from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Bacterial Growth and Fumarate Reduction
Liquid batch cultures of G. sulf urreducens strain PCA were in anaerobic serum bottles at 30 °C in ATCC medium (1957) containing 30 mM acetate and 50 mM fumarate as the TEA, as recommended by ATCC. The culture bottles were kept like that thereafter with no further nutrient supplementation. All culture manipulations were performed under strict anaerobic conditions. Growth of cultures was monitored by measuring the OD600 of 1 mL samples from each culture. Simultaneously, consumption of fumarate and acetate and accumulation of succinate was also monitored using HPLC. Samples were collected at regular intervals and were analyzed using an Agilent HPLC 1260 Infinity system equipped with a Supelcogel C610H column, using 0.1% phosphoric acid as eluent, and detection at 210 nm. The flow rate was 0.5 mL/min. Cells were harvested for differential proteomics analysis from three replicate cultures in the midlog phase (41 h), two replicate cultures in the early stationary phase (72 h), and three replicate cultures in the survival phase (395 days). To minimize protein degradation during preparation, all samples were maintained under nitrogen during work up and kept at 4 °C. Protein Extraction and Preparation for LC-MS/MS
Proteins were isolated from whole-cell pellets harvested by centrifugation from 30 mL (midlog and early stationary phases) or 100 mL (survival phase) cultures. Cell pellets were disrupted by sonication on ice with pipetting in 3 mL (5 mL for survival phase) of 0.5 M triethyl ammonium bicarbonate (TEAB) with 0.05% SDS, 1 mM phenylmethanesulfonylfluoride (Bio-Rad; Hercules, CA), and DNase. The cell suspension was then boiled for 10 min and centrifuged in a microfuge for 10 min at full speed, after which the supernatant was collected and stored at −20 °C until analysis. Protein concentration was determined 4317
dx.doi.org/10.1021/pr400266m | J. Proteome Res. 2013, 12, 4316−4326
Journal of Proteome Research
Article
using a Bio-Rad Protein Assay Kit (Bio-Rad; Hercules, CA) following the manufacturer’s instructions. Each sample (150 μg of protein) was dried using a Savant SpeedVac (Thermo Scientific; Waltham, MA) and then resuspended in 20 μL of 0.5 M TEAB, pH 8.5. Two μL of 2% SDS was added, the sample was vortexed, and then 1 μL of concentrated tris-(2carboxyethyl) phosphine (TCEP) was added for a final concentration of 5 mM TCEP. Each sample was vortexed and incubated for 1 h at 60 °C. Then, 1 uL of freshly prepared 84 mM solution of iodoacetamide was added to each sample, and samples were incubated in the dark at 23 °C for 30 min. Ten μL of trypsin (1 ug/ul) was added to each sample, and samples were incubated at 48 °C overnight.17 All eight samples were then labeled with separate iTRAQ reagents from an iTRAQ Multiplex (8-plex) kit (Applied Biosystems; Carlsbad, CA) following the manufacturer’s postalkylation instructions. Labeled samples were combined and then washed four times by sequential drying using a SpeedVac, followed by resuspension in water. The washed combined protein preparation was resuspended in 12 mM ammonium formate in 25% acetonitrile at pH 2.5 to 3.0. The pH was then adjusted to 2.5 to 3.3 using formic acid.
into the instrument. A total of 15 MALDI target plates were analyzed on ABI 4800 MALDI TOF-TOFs in a data-dependent manner (up to 15 MS/MS spectra per MALDI spot, with the spot containing the most abundant MS peak representing each unique peptide mass across the plate chosen for MS/MS fragmentation of that unique peptide mass). 5500 MALDI spots were analyzed, averaging 500 laser shots per spot at Laser Power 3400. The total number of MS/MS spectra taken was 21 260, with up to 2500 laser shots per spectrum at Laser Power 3700, with CID gas Air at 1.2 to 1.3 × 10−6 Torr. As a MALDI experiment, the charge state of all peptides analyzed was +1. Mass Spectrometric Data Analysis
Protein identification and quantification was performed using the Paragon algorithm, as implemented in Protein Pilot 3.0 software. A database containing 3465 protein sequences of G. sulf urreducens from the J. Craig Venter Institute (December 2009) along with a concatenated reverse decoy data set of these proteins and 389 common laboratory contaminants was used as the reference database. Proteins identified with (1) an unused ProtScore of
