Article pubs.acs.org/jpr
Pseudomonas putida Response to Cadmium: Changes in Membrane and Cytosolic Proteomes Anna Manara,† Giovanni DalCorso,*,† Cecilia Baliardini, Silvia Farinati, Daniela Cecconi, and Antonella Furini Dipartimento di Biotecnologie, Università degli Studi di Verona, Strada Le Grazie 15, 37134 Verona, Italy S Supporting Information *
ABSTRACT: Pseudomonas putida is a saprophytic bacterium with remarkable environmental adaptability and the capacity to tolerate high concentrations of heavy metals. The strain P. putida-Cd001 was isolated from soil contaminated with Cd, Zn and Pb. Membrane-associated and cytosolic proteomes were analyzed to identify proteins whose expression was modulated in response to 250 μM CdSO4. We identified 44 protein spots in the membrane and 21 in the cytosolic fraction differentially expressed in Cd-treated samples compared to untreated controls. Outer membrane porins from the OprD and OprI families were less abundant in bacteria exposed to Cd, whereas those from the OprF and OprL, OprH and OprB families were more abundant, reflecting the increased need to acquire energy sources, the need to maintain membrane integrity and the process of adaptation. Components of the efflux system, such as the CzcB subunit of the CBA system, were also induced by Cd. Analysis of the cytosolic proteome revealed that proteins involved in protein synthesis, degradation and folding were induced along with enzymes that combat oxidative stress, showing that the entire bacterial proteome is modulated by heavy metal exposure. This analysis provides new insights into the adaptation mechanisms used by P. putida-Cd001 to survive in Cd-polluted environments. KEYWORDS: cadmium, heavy metals, proteomics, Pseudomonas putida
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detoxification of toxic metals such as Cd and Hg.11 The presence of efflux pumps removes heavy metals from the cell, and certain highly specialized efflux pumps are found only in a few species of bacteria, rendering them heavy metal tolerant.6 There is a large and diverse literature discussing bacterial responses to chemical and physical stresses.12 In particular, whole-genome expression analysis, proteomics and metabolomics have provided a global overview of bacterial stress responses, helping to elucidate the adaptive mechanisms underlying heavy metal tolerance and therefore providing nonempirical biotechnology-based solutions for toxic metal remediation in contaminated environments. Pseudomonas is a model genus helpful in the study of responses to xenobiotic compounds.13 One of its properties is the wide variety of organic compounds used as carbon source. P. putida, for instance, is a ubiquitous saprophytic bacterium with remarkable environmental adaptability and the ability to degrade aromatic xenobiotic compounds such as toluene, benzene and ethylbenzene, making it a pivotal instrument for the analysis of biodegradation.14 This species has also the capacity to tolerate high concentrations of heavy metals.15−17 P. putida genome contains 61 open reading frames related to metal tolerance or homeostasis, and seven potentially involved in metal resistance.18 In addition, the presence of gene duplications
INTRODUCTION The heavy metal cadmium is a major pollutant,1 which is often released into the environment by anthropogenic activities such as industrial processing and agricultural practices, including the use of fertilizers containing heavy metals.2,3 Cd is released into the air, water and soil, and there is substantial transfer between these environmental compartments (http://www.cadmium. org/). Cd is extremely toxic to living organisms, and even at low concentrations it may affect multiple cellular processes, e.g. by inhibiting DNA repair,4 inducing oxidative stress5 and binding to respiratory enzymes.6 Microorganisms must survive in dynamic environments, and this requires ability to adapt rapidly to environmental stresses.7 Several metal homeostasis mechanisms have evolved, allowing microbes to immobilize toxic metals and/or export them from the cell.8 One such mechanism is the tight regulation of metal import by expressing transporter proteins with high affinity for essential metals. Although this should limit the uptake of toxic metal ions, Cd is taken into cells via the transport system for essential divalent ions such as Mn2+ and Zn2+.9 Furthermore, the abundance of membrane-localized transporters increases in metal-limiting environments, allowing the import of toxic elements when the levels of essential metal ions are restored to normal.10 Another metal homeostasis mechanism involves metal-sequestering proteins and metal ion efflux pumps.10 For example, metallothioneins are low-molecular-mass cysteine-rich cation-binding proteins that play an important role in the © 2012 American Chemical Society
Received: March 22, 2012 Published: July 16, 2012 4169
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suggests redundant functions or tolerance to different metals.17,18 The unexpectedly large number of P. putida genes implicated in metal(loid) homeostasis, tolerance and resistance suggests that the species can adapt to environments polluted with heavy metals and could be used for the transcriptomic and proteomic analysis of adaptation. A proteomic approach on P. putida KT2440 treated with Cu and Cd highlighted that these metals induced different genes; in particular, Cd positively modulated transcription regulators, outer membrane proteins and channels involved in Cd efflux from the cells.19 The attention has also focused on other Pseudomonas species, such as P. aeruginosa, whose proteome in response to Cr(VI) has been recently investigated. This analysis evidenced that proteins involved in protein synthesis, energy production and detoxification are up-regulated by Cr, as well as proteins involved in the synthesis of exopolysaccharides, functional against Cr(VI) stress and bounding Cr(VI) to the outside of the cell.20 To further investigate the Cd effect on Pseudomonas and in an attempt to characterize more Cd-responsive genes potentially involved in metal homeostasis and/or tolerance, a P. putida strain was chosen, among several bacteria identified in the rhizosphere of the Cd and Zn hyperaccumulator Arabidopsis halleri collected from a site contaminated with the heavy metals Zn, Pb and Cd in Auby, Nord Pas-du-Calais, France.21 This site has been characterized by recent industrial metallurgical activity, and it is therefore considered as highly polluted.21 The soil samples, characterized by a Cd concentration ranging between ca. 0.09 g/kg (0−5 cm deep) and ca. 0.4 g/kg (10−15 cm deep), yielded several bacterial strains that were isolated and selected in vitro for their ability to tolerate high Cd levels in the culture medium.22 A P. putida strain was chosen for proteomic analysis because of the usefulness of the experimental approach and the availability of a database for protein identification.23 We identified P. putida proteins that were modulated in response to Cd treatment, aiming eventually to use the corresponding genes in future Cd phytoremediation programs or for the reduction of Cd levels in food. We focused on the components of cytosolic and membrane-associated proteomes, in the latter case using an optimized 2-DE protocol allowing the separation and quantification of membraneassociated proteins potentially involved in heavy metal import and export. The impact of our proteomic analysis on current understanding of adaptive mechanisms in Cd-polluted environments is discussed.
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was amplified by PCR with primers F8 and R11.25 Amplification products were sequenced directly and analyzed using BLASTN.26 One of the bacterial strains was identified as Pseudomonas putida, hereafter P. putida-Cd001. Effect of Cd on P. putida-Cd001 Growth and Determination of the 50% Lethal Dose (LD50)
To measure the growth of P. putida-Cd001 and the effect of Cd, cells were grown in nutrient broth, which was supplemented with increasing concentrations of CdSO4 (0, 150, 250, 500 μM and 1.0 mM) once the OD600 reached 0.1. Over the next 48 h, the OD600 was recorded at intervals and plotted against time. To determine the LD50, P. putida-Cd001 cells were inoculated in 100 mL of nutrient broth supplemented with CdSO4 (0, 250, 500, 700 μM and 1.0, 2.0, and 3.0 mM). After 48 h at 28 °C with agitation, the cultures were diluted and plated on agarized nutrient broth at 28 °C for 24 h. Colony-forming units (CFU) were counted and plotted against increasing the Cd concentration, and the LD50 was extrapolated from the 50% CFU value. Each experiment was performed in triplicate. To test the chelating properties of nutrient broth, P. putida-Cd001 cells were treated with Cd in both nutrient broth and 0.9% aqueous solution for 15, 30, and 60 min, and the effect of Cd was measured as changes in the number of CFU/mL. The detailed procedure and results are described in Figure S1 (Supporting Information). Cd Bioaccumulation by P. putida-Cd001
For bioaccumulation analysis, anexic P. putida-Cd001 cultures in 100 mL of nutrient broth containing 100 μM CdSO4 (c. 11.3 ppm Cd) were incubated on a orbital shaker (180 rpm) for 48 h at 28 °C. The cells were harvested by centrifugation (6000g, 15 min, 4 °C), and the pellet was washed twice in 50 mL of 0.9% w/v NaCl, followed by drying at 60 °C. The Cd content was determined in triplicate after sample digestion with nitric acid, by inductively coupled plasma mass spectrometry (ICP-MS) (EPA 3051A 2007+EPA6010C 2007, http://www. epa.gov/wastes/hazard/testmethods/sw846/online/3_series. htm, http://www.epa.gov/wastes/hazard/testmethods/sw846/ online/6_series.htm). Cultivation Conditions in the Presence or Absence of Cd for Proteomic Analysis
P. putida-Cd001 cultures were grown in 250-mL Erlenmeyer flasks containing 100 mL of medium on a rotary shaker (200 rpm) at 28 °C, until the OD600 reached 0.7−0.8. These cultures were used to inoculate two 1-L Erlenmeyer flasks containing 250 mL of fresh medium. CdSO4 was added to one flask to a final concentration of 250 μM, with the untreated flask acting as the control. Cells were harvested from both cultures after incubation overnight at 28 °C shaking at 200 rpm. Cell suspensions were centrifuged for 20 min at 10000g and 4 °C, washed with 0.9% NaCl, and the pellets were stored at 80 °C until use. Each treatment was carried out in triplicate.
EXPERIMENTAL METHODS
Isolation and Identification of Bacterial Strains
Bacterial strains were isolated from the rhizosphere of Arabidopsis halleri plants growing in contaminated soil in the town of Auby, Northern France.21,24 Two grams of soil were suspended in 18 mL of 0.9% w/v NaCl in a 150-mL Erlenmeyer flask, and the suspension was incubated at 28 °C in the dark for 2 h on a rotary shaker (250 rpm). Serial dilutions of the suspensions were then prepared. Petri dishes containing agarized nutrient broth (Oxoid, Garbagnate Milanese, Italy) were streaked with 0.1 mL/plate of the diluted suspensions and were incubated aerobically for 5 d at 28 °C. Visually distinct bacterial colonies were picked and streaked on plates containing solidified nutrient broth supplemented with up to 2 mM CdSO4 to select Cd-resistant bacterial strains. Genomic DNA was extracted from surviving bacteria using the NucleoSpin Tissue Kit (Clontech, Palo Alto, CA), and the 16S rDNA gene
Preparation of Protein Fractions
Bacterial pellets were resuspended in cold 0.9% NaCl, washed and centrifuged for 10 min at 6000g. The cells were resuspended in lysis buffer (30 mM Tris-Cl pH 8; 20% sucrose, 1 mM EDTA), incubated for 10 min at room temperature with gentle shaking and centrifuged for 20 min at 12000g. The pellet was resuspended in 30 mM Tris-Cl pH 7.5, sonicated for 40 s on ice (seven cycles), and centrifuged for 1 h at 150000g and 4 °C. After this step, the supernatants represented the cytosolic fraction, and the pellets represented the membranes. The supernatant was concentrated, 4170
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5 μm particle size) analytical column and a Zorbax 300SB-C18 (40 nL, 5 μm) enrichment column. The complete system was fully controlled by the ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) software packages. The scan window ranged from 300 to 1800 m/z. For MS/MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides of each m/z value were selected to be further isolated and fragmented. The MS/MS scanning was performed in the normal resolution mode at a scan rate of 13 000 m/z per second. Five scans were averaged to obtain each MS/MS spectrum. To generate fragment ions, collision-induced dissociation (CID) was performed on isolated peptide ions with fragmentation amplitude of 1.15 V. The precursor isolation width was set to 4 m/z, moreover exclusion limits were automatically placed on previously selected mass-to-charge ratios for 0.2 min. Databases were searched using the MS/MS ion search using the online Mascot search engine (www.matrixscience.com, version 2.3) against all entries of the nonredundant NCBInr database (version 20110521 grouping 14 141 183 sequences and setting taxonomy on Bacteria - Eubacteria, corresponding to 8 113 516 sequences) considering the following parameters: specific trypsin digestion, up to one missed cleavage; fixed and variable modifications: propionamide (Cys) and oxidation (Met), respectively; peptide and fragment tolerances: ±0.9 Da and ±0.9 Da, respectively; and peptide charges: +1, +2 and +3. For positive identification, the score of the result of [−10 log(P)] needed to exceed the significance threshold (p < 0.01) and at least two different peptides (p < 0.05) had to be assigned. Spots that contained a mixture of several proteins were excluded and not further considered in the analysis, since densitometry cannot estimate the relative abundance of different proteins and their individual contribution to the total fold variation.
and nucleic acids were removed by 1% streptomycin sulfate and centrifuging for 10 min at 9000g. Cytosolic proteins were precipitated overnight by adding four volumes of cold acetone, and the pellet was resuspended in the buffer used for firstdimension electrophoresis (7 M Urea, 2 M Thiourea, 20 mM Tris, 3% Chaps). The membrane-fraction pellet was resuspended in 100 mM Na2CO3 (pH 11) and incubated on ice for 1 h with gentle shaking. Samples were centrifuged for 1 h at 150000g, 4 °C, and pellets were resuspended in the buffer used for first-dimension electrophoresis (7 M Urea, 2 M thiourea, 20 mM Tris, 1% ASB14 and 1% TritonX100. Protein concentrations were determined using the Bradford assay (Sigma-Aldrich, St. Louis, MO) for cytosolic fraction and DC-Protein-assay (Biorad) for membrane fraction. Two-Dimensional Gel Electrophoresis
Cysteine reduction and alkylation were achieved by adding 5 mM fresh tributyl-phosphine and 10 mM free acrylamide to 1.0 mg of the membrane and cytosol protein extracts. We then added 0.5% carrier ampholytes (pH 3−10) and traces of bromophenol blue to the samples. Each sample (2.2 mg/mL) was loaded by rehydration onto large-size 17 cm pH 4−7 NL IPG strips (Bio-Rad, Hercules, CA), and five replicates were made for the treated and control samples. Isoelectric focusing was carried out until 65 000 V × h (10 000 V maximum), and orthogonal separation by SDS polyacrylamide gel electrophoresis was carried out using polyacrylamide gradient gels (10−20% T) that were stained with Sypro Ruby (Bio-Rad). Protein Pattern and Statistical Analysis
Gels were scanned using the BioRad VersaDoc 1000 imaging system, with image analysis using PDQuest software (version 7.3, BioRad). Each gel was analyzed for spot detection, background subtraction, and protein spot OD intensity quantification. Gel images with the largest numbers of spots and the best protein patterns were chosen as reference templates, and spots in a gel were then matched across all gels. Spot quantity values were normalized in each gel, dividing the raw quantity of each spot by the total quantity of all the spots included in the gel. Gels were divided into two groups (treated and untreated control), and for each protein spot, the average spot quantity value and its variance coefficient in each group were determined. Statistical analysis (Student’s t-test) was performed to identify proteins that were significantly (p < 0.05) modulated across two sets of samples.
P. putida-Cd001 RNA Isolation and Northern Dot-Blot Analysis
P. putida-Cd001 cells were grown in nutrient broth containing 250 μM CdSO4, and an untreated culture was used as control. After incubation for 16 h at 28 °C shaking at 180 rpm, total RNA was isolated from both cultures using TRIzol reagent (Invitrogen, Karlsruhe, Germany). DNA probes were prepared using the nonisotopic labeling kit BrightStar Psoralen-Biotin (Ambion, Austin, TX) according to the manufacturer’s instructions. Total RNA was denatured and used for dot-blot on a positively charged nylon membrane and cross-linked by ultraviolet light. Dot-blots were prehybridized in ULTRAhyb hybridization solution (Ambion, Austin, TX) at 42 °C for 1 h and then hybridized with a specific probe (10 pM in hybridization solution) overnight at 42 °C. DNA probes were amplified by PCR using sequence specific primers (Supporting Information Table S2) purified and denatured at 100 °C for 10 min. Following hybridization, membranes were washed, and signals were detected with the nonisotopic detection kit BrightStar BioDetect (Ambion, Austin, TX). The analysis of the spot intensity was performed by scanning membranes and processing the images with the QuantityOne software (Version 4.4.1, Bio-Rad, Hercules, CA, USA).
In-Gel Digestion
Spots showing statistically significant differential expression were carefully cut from the stained gels and digested with trypsin according to Shevchenko et al.27 with minor modifications. The gel pieces were swollen in digestion buffer (50 mM NH4HCO3, 12.5 ng/μL of modified porcine trypsin, sequencing grade, Promega, Madison, WI, USA). After 30 min, 20 μL of 50 mM NH4HCO3 were added to the gel pieces, and digestion continued at 37 °C overnight. The supernatant containing tryptic peptides was dried by vacuum centrifugation. Prior to mass spectrometric analysis, the peptide mixtures were dissolved in 5 μL of 2% acetonitrile. Peptide Sequencing by nanoHPLC-Chip-MS/MS
Motility Test
Peptides from each sample were separated using reversed phase nano-HPLC-Chip technology (Agilent Technologies, Palo Alto, CA, USA) coupled online with a 3D ion trap mass spectrometer (model Esquire 6000, Bruker Daltonics, Bremen, Germany). The chip comprised a Zorbax 300SB-C18 (150 mm × 75 μm, with a
P. putida-Cd001 was inoculated in semisolid plates containing 0.3% (w/v) agarose, as described by Fonseca et al.28 The plates were incubated at 28 °C overnight (16 h) with high humidity to prevent dehydration. 4171
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observed.29 At 1 mM CdSO4, the highest concentration that was not lethal, growth was not measurable. Conversely, treatment with 500 μM CdSO4 did not completely inhibit growth but resulted in a lag phase of ∼20 h. This prolonged lag time is probably needed for P. putida cells to adapt themselves to the high CdSO4 concentration in order to restore growth. Lower concentrations allowed growth to continue, albeit more slowly than controls (Figure 1A). Plating serial culture dilutions on agarized nutrient broth allowed the number of CFUs to be established after Cd treatment, providing a measure of cell viability. As shown in Figure 1B, the LD50 after 48 h treatment at 28 °C was approximately 1.10 mM CdSO4. It is worth noting that the actual Cd concentrations effective on P. putida-Cd001 cells are probably lower than the nominal values indicated, because of a feeble chelating attitude of the nutrient broth utilized for the experiments (Supporting Information Figure S1), and therefore, the determined LD50 is referred to P. putidaCd001 grown in this culture medium. Cd tolerance of this strain was also compared with tolerance of Escherichia coli DH5α strain. As shown in Figure S3 (Supporting Information), E. coli cells are not able to grow on plates containing Cd concentrations higher than 0.5 mM, while P. putida-Cd001 cells keep growth ability also in 1.5 mM Cd containing plates.
RESULTS AND DISCUSSION
Effect of Cd on Bacterial Growth
We recovered eight candidate bacterial species with high-level of Cd tolerance from samples of A. halleri rhizospheric soil contaminated predominantly with Cd, Zn and Pb,21 following in vitro selection on Cd-supplemented medium.22 The strains were identified by 16S rDNAs sequencing, including one showing 100% sequence identity to P. putida (FN600411). We named this strain P. putida-Cd001 because of its ability to tolerate high Cd concentrations (1 mM) on agarized medium, and selected it for proteomic analysis. P. putida-Cd001 cells grown for 2 d in nutrient broth containing 11.3 ppm Cd (100 μM CdSO4) were able to adsorb or absorb up to 6 ppm (±0.3) Cd.22 Cell growth in nutrient broth was monitored for 48 h, measuring changes in OD600 following treatment with increasing concentrations of CdSO4 (0, 150, 250, 500 μM and 1 mM). As shown in Figure 1A, the addition of Cd to an
Effect of Cd on the P. putida-Cd001 Proteome
Samples for proteomic analysis were collected after 16 h exposure of exponentially growing bacterial cells to 250 μM CdSO4. These conditions were chosen to maximize the amount of proteomic data without subjecting cells to excessive stress. Cell samples from treated and control flasks were fractionated, and the cytosolic proteins were separated from membraneassociated proteins. Proteins from both fractions were then separated by charge (isoelectric focusing) and orthogonally by mass (SDS-PAGE). Master 2D maps of the membrane and cytosolic proteomes are shown in Figure 2, while representative 2D gels showing the differential protein expression upon treatment with Cd compared with untreated controls are reported in Figure S2 (Supporting Information). Protein spots showing statistically significant differential expression (considering a fold variation threshold >2) in five replicate gels were catalogued, resulting in a total of 44 spots in the membrane fraction (25 up-regulated and 19 down-regulated) and 21 in the cytosolic fraction (14 up-regulated and 7 down-regulated). These were processed for MS analysis, annotated against the NCBI database and clustered according to known or hypothetical function (Table 1). As shown, the majority of the identified proteins are homologous to known P. putida sequences. The experimental approach used (i.e., IEF followed by SDS-PAGE) led to the identification of cytosolic, periplasmic and membrane-associated proteins, indicating that integral transmembrane proteins are rather underrepresented. This is in agreement with the in silico analysis, aimed at detecting transmembrane domains, performed on the identified protein sequences (as reported in Supporting Information Table S1). In fact, many proteins examined do not show multiple transmembrane spans, typical of integral membrane proteins. Despite these inherent difficulties, reported data show that many proteins are synthesized and degraded following the addition of Cd, allowing the regulation of metal import and detoxification by the bacterial cell. MS analysis identified few proteins in the cytosolic fraction that are predicted to be periplasmic or/and to contain single transmembrane spans, as reported in Supporting Information
Figure 1. Growth curve showing the Cd sensitivity of P. putida-Cd001 monitored by recording changes in cell density over 48 h in the presence of increasing Cd concentrations (A). Growth was monitored by recording the optical density of the culture at 600 nm (OD600). Dose response curve (B), showing the Cd effect Cd on P. putidaCd001 vitality, expressed as colony forming units (CFU)/mL. The LD50 was calculated by interpolation of the dose response curve.
early, exponentially growing culture caused a longer lag time compared to untreated cells, and the extent of growth retardation correlated with Cd concentration as previously 4172
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for instance, was also demonstrated in P. f luorescens ATCC 948 treated with 0.1 mM cobalt,30 while P. aeruginosa cells undergoing Cr(VI) stress show higher abundance of ribosomal subunits such as 50S-L1, 30S−S1,20 highlighting the role of these proteins in adaptation of bacteria toward the stress condition. Proteins Involved in Membrane Structure and Channels
The exposure of P. putida-Cd001 cells to 250 μM CdSO4 also altered the abundance of several membrane-associated proteins, in particular outer membrane porins. Some of these became less abundant, e.g. members of the OprD family (spots 5−13, 45 and 46), or even disappeared altogether (OprI, spot 14). OprD expression is strongly influenced by environmental conditions.31 In P. aeruginosa, OprD is the primary route for the uptake of carbapenems, a class of broad spectrum antibiotic, and the levels of this protein are reduced in response to Zn.32 The repression of P. putida-Cd001 OprD by Cd suggests that antibiotic-resistant bacteria may be selected in environments polluted by heavy metals. Similarly, the major outer membrane lipoprotein OprI is responsible for susceptibility to cationic α-helical antimicrobial peptides/proteins in P. aeruginosa.33 OprI expression in P. putida is also inhibited by Cd, which may explain its resistance to antimicrobial peptides. Several other porins were induced by Cd treatment, including OprF (spot 15), OprL (spot 16), OprH (spot 47) and OprB (spots 17 and 18). OprF is a major outer membrane porin of the Pseudomonads. It is a multifunctional protein that binds peptidoglycans and plays a role in antimicrobial drug resistance, maintenance of cell shape and membrane integrity,34 and in the promotion of plant growth and root adhesion.35 The Cd-specific induction of OprF may explain the need to maintain membrane integrity and promote cell adaptation in the presence of heavy metals. Similarly, the peptidoglycanassociated lipoprotein OprL is also induced, and this might explain the need to maintain outer membrane integrity as previously reported.36,37 Interestingly, in P. aeruginosa other metal stress, such as that induced by Cr(VI), has been shown to induce outer-membrane lipoproteins associated to peptidoglycan, with high similarity to OprL,20 which, together with an enhanced exolipopolysaccharide biosynthesis, could produce more outer membrane, offering a kind of passive resistance mechanism, preventing metal ion entrance into the cell.20 Notably, this phenomenon could in part be responsible for a lower metal availability for plant up-take, explaining the lower shoot-accumulation of Cd observed in plants inoculated with P. putida-Cd001 in comparison with not-inoculated plants.22 Another outer membrane protein (OprH) was identified in the cytosolic fraction (spot 47), and this is strongly induced in Pseudomonas by Mg starvation and phenol treatment.36 The strain P. putida KT2440, the Outer membrane protein H1, which shares 96% positive residues with OprH (spot 47), shows great induction upon Cu addition, and even a higher accumulation following Cd treatment,19 and its possible involvement in ion binding to the lipopolysaccharide layer was suggested.19 Its accumulation in response to Cd also in P. putida-Cd001 points, therefore, to a putative role of OprH in metal tolerance. The expression of OprB (spots 17 and 18) was also induced, and this porin specifically facilitates the diffusion of glucose and other carbohydrates across the outer membrane. This suggests that one response to Cd is a requirement for more energy, resulting in the import of energy sources. This coincides with
Figure 2. Master maps of 2D-gels representing P. putida-Cd001 membrane and cytosolic fractions. Protein spots with statistically significant differences in density when comparing cultures exposed to Cd and controls between treated and untreated cells are highlighted (A). Protein spots showing clear differential expression when comparing treated and untreated samples (highlighted with arrows) (B). Spot numbers correspond to those listed in Table 1.
Table S1 (e.g., spots 45, 46, 54, 62), which may represent contamination, preparation artifacts such as protein detaching during fraction preparation (sonication), or genuine events such as protein−protein interactions resulting in the coselection of membrane-peripheral proteins as part of cytosolic complexes. For several proteins, proteomics data presented were validated by Northern dot-blot analysis. Total RNA was extracted from bacterial cells, treated with DNase and loaded onto positively charged nylon membranes. The transcription level upon Cd treatment was analyzed for ten genes, and the expression pattern observed by 2D-analysis was confirmed at least in nine cases (Figure 3), even though the extent of the differential expression differs between protein and mRNA. The tight control of protein synthesis during metal uptake and detoxification is demonstrated by the general Cdinduced increase in the abundance of spots corresponding to the Tu elongation factor, which facilitates elongation during translation (spots 1−4), and ribosomal subunits (spots 56 to 58). The up-regulation of proteins involved in protein biosynthesis is a common response to heavy metals, indeed also in P. putida-KT2440 in response to Cu and Cd, the accumulation of these factors reflects increased protein synthesis.19 The new induction of the Tu elongation factor, 4173
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Table 1. Identification of Membrane and Cytosolic Proteins That Are Differentially Expressed in Response to Treatment with 250 μM CdSO4a protein name
b
SSP
NCBI accession no.
NCBI accession organism
MW (Da)/pI theoretical
Mascot scorec
identified peptides number
fold of variationd Cd vs control
43451/5.22
102
2
N. i.e
554 434 200
11 9 4
↑ 3.8 >10 >10
47758/5.80
105
2
↓ 3.4
220 319 308
7 7 7 6 7 3 10 2
Membrane-Associated Fraction 1
Protein expression elongation factor Tu
2 3 4 5
Porins and transporters outer membrane porin OprE3 (OprD-family)
outer membrane protein (OprD-family)
14
outer membrane lipoprotein OprI
15
OprF
16 17 18 19
21 22 23 24 25 26 27 28
gi|26987193
P. putida KT2440
5401 5403 6301
6 7 8 9 10 11 12 13
20
4403
5501
gi|24981628
5502 6501 7501 7503 7601 8501 8601 1401 3
gi|26989046
211
gi|37704612
peptidoglycan-associated lipoprotein OprL
3101
gi|26987958
carbohydrate-selective porin OprB
3401 4507 7301
gi|148546300 gi|26988120
3601
gi|26986789
6901
gi|170723379
6902 3701 3703 1903 1904 2901 4901
outer membrane porin OprE (OprD-family) outer membrane porin (OprD-family)
efflux transporter, RND family, MFP subunit CzcB family cobalt/zinc/cadmium efflux membrane transporter Receptors TonB-dependent hemoglobin/transferrin/ lactoferrin family receptor hypothetical protein PputGB1_4051, similar to an Outer membrane receptor for siderophore TonB-dependent receptor TonB-dependent siderophore receptor TonB-dependent siderophore receptor
P. putida KT2440
P. putida F1
48336/5.42
gi|170723456
P. putida W619
50183/5.77
gi|26987941
P. putida KT2440 P. putida KT2440 Pseudomonas sp. MFY59 P. putida KT2440 P. putida F1
46092/4.84
327 148 471 102
8795/7.88
210
4
N. d.f
33857/4.77
63
2
N. i.
17822/5.16
138
3
↑ 2.8
49558/5.77
P. putida KT2440 P. putida KT2440
41225/5.97
358 350 155
7 8 4
↑ 4.2 ↑ 2.4 N. i.
43952/5.19
542
10
N. i.
P. putida W619
94107/5.34
113
2
↑ 6.1
gi|167035046
P. putida GB-1
45201/5.14
137
3
↑ 3.8
gi|148549481
P. putida F1
74670/5.07
gi|148546135 gi|167031919
P. putida F1 P. putida GB-1
81939/4.96 82731/5.09
gi|170724009
P. putida W619
88364/5.02
513 503 384 616 893 315
12 4 7 12 19 7
N. i. N. i. >10 ↑ 8.9 >10 >10
6202
gi|26988643
25454/5.94
84
2
↑ 2.1
68484/5.13 26786/5.03 18755/5.82
336 342 74
9 5 2
↑ 3.1 ↓ 5.0 ↑ 2.8
44734/5.37
145
2
↑ 5.7
46966/4.65 105758/5.01 47504/4.47 24289/5.52 46169/5.61
351 177 314 133 167
7 5 5 4 3
↑ 2.3 ↑ 2.2 ↓ 2.0 ↓ 2.1 >10
507 314 474 122 137
10 8 8 3 3
