Comparison of the Membrane Subproteomes during Growth of a New Pseudomonas Strain on Lysogeny Broth Medium, Glucose, and Phenol Dimitrios G. Papasotiriou,† Stavroula Markoutsa,† Bjoern Meyer,‡ Anastasia Papadioti,† Michael Karas,‡ and Georgios Tsiotis*,† Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes Heraklion, Greece, and Institut fu ¨ r Pharmazeutische Chemie, Johann Wolfgang Goethe-Universita¨t, Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany Received March 14, 2008
Study of the bacterial membrane proteome is a field of growing interest in the research of nutrient transport and processing. Pseudomonas sp. strain phDV1, a Gram-negative bacterium selected for its ability to degrade aromatic compounds, was monitored under different growth substrate conditions, using lysogeny broth medium (LB), glucose, and phenol as sole carbon source. The aim of this study was to characterize the membrane subproteomes of the Pseudomonas strain by proteomic means to assess the protein composition of this subcellular compartments, which appears fundamental for the biodegradation of aromatic compounds. A total number of 129 different proteins have been identified by MALDI-TOF/TOF, 19 of which are membrane proteins that belong to the inner membrane and 10 that belong to the outer membrane. Two membrane proteins were only expressed in the presence of the aromatic substrate. We identified a membrane protein involved in aromatic hydrocarbon degradation as well as a probable porin which may, in fact, function as an aromatic compound-specific porin. Although the presence of different transporters have been reported for different aromatic compounds such as toluene and benzoic acid, to our knowledge, these are the first phenol-inducible membrane transporters identified. Keywords: pseudomonas • mass spectrometry • membrane proteome • two-dimensional polyacrylamide gel electrophoresis
Introduction The most important phenol-degrading microorganism that showed high biodegradability is a Gram-negative bacterium of the genus Pseudomonas. A strain with high phenol-removal efficiency is Pseudomonas sp. strain phDV1, which was isolated from enriched mixed culture from samples of petroleumcontaminated soil in Denmark.1 Previous studies have shown that phenol is metabolized as a sole source of carbon and energy. We determined that this aromatic compound is degraded via the meta cleavage pathway.2,3 The applicative importance of studying the degradative pathways, by which bacteria catabolize aromatic compounds, is evident if we consider the environmental pollution of soil and water caused by fuels, oils, and solvents in agricultural and industrial activities. Gram-negative bacteria have a cell wall composed of two membranes: the outer membrane (OM) and the inner membrane (IM). The two membranes enclose a region between * To whom correspondence should be addressed. Georgios Tsiotis, Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box2208,GR-71003Voutes,Heraklion,Greece.E-mail:
[email protected]. Tel: +30 2810 545006. Fax: +30 2810 545001. † University of Crete. ‡ Johann Wolfgang Goethe-Universita¨t.
4278 Journal of Proteome Research 2008, 7, 4278–4288 Published on Web 08/16/2008
them known as the periplasm. Outer membrane proteins (OMPs), located between the outmost of the cell and its external natural environment, are important for the response to environmental changes in osmolarity, temperature, drugs, chemicals, and host defense.4,5 Inner membrane proteins (IMPs) are involved in a vast array of cellular processes such as ion channel conductance and cell signaling.6 The proteins embedded in the two membrane types differ; in the inner membrane, there is a prevalence of R-helical proteins, rich in aliphatic amino acids, whereas in the outer membrane, repeating chains of hydrophobic-hydrophilic amino acids are present, giving rise to an antiparallel beta-barrel structure.7 The requirements for a successful membrane proteome analyses are mainly two. The first is dependent upon the separation of the total proteome to subproteome fractions, thus simplifying the sample. A concern for cellular fraction purity level immediately arises, as the fraction should ideally be free of contaminating material from other cellular locations. The second challenge is to find an analytic method to separate and identify the whole set of membrane proteins, including the integral membrane proteins. Several methods have been developed to separate the outer membrane.8 Sodium lauryl sarcosinate has been found to disrupt the cytoplasmic membrane selectively under conditions 10.1021/pr800192n CCC: $40.75
2008 American Chemical Society
Comparison of the Membrane Subproteomes in which Triton X-100 and dodecyl sodium sulfate solubilize both membranes.9 Most available information has come from analysis of the membranes of Haemophilus influenzae and Escherichia coli, yielding a pure outer membrane with the inner membrane removed.8-11 However, sodium lauryl sarcosinate may lead to protein loss from the outer membrane.12 Coming to the protein separation and identification techniques available, one classical approach to proteome analyses couples 2-DE (IEF/SDS-PAGE) for protein separation with mass spectrometric identification. Membrane proteins, apart from being notoriously difficult to extract and to keep soluble in general, are notoriously under-represented due to significant solubility problems during isoelectric focusing.13-16 Shotgun approaches using LC-MS/MS have been introduced as highly efficient alternatives.17 However, LC-based strategies have no direct access to the molecular mass of the identified proteins. Additionally, by using a gel based approach, proteins are visually depicted as individual gel spots. Therefore, identified and unidentified proteins are displayed on the gel, whereas LCbased approaches restrict this feature, as only identified proteins can be taken in account.18 Furthermore, the protein mass is monitored by a marker which provides useful additional information as far as protein identification is concerned. Other strategies involve fractionation of complex protein mixtures and use of (16BAC)/SDS [benzyldimethyl n-hexadecylammonium chloride] or CTAB/SDS [Cetyl trimethyl-ammonium bromide] 2-DE gels.19,20 Another two-dimensional electrophoresis system for the isolation of prefractionated membrane protein samples is doubled SDS-PAGE (dSDSPAGE).18 Protein spots are separated after the coupling of two SDS-gels of different total acrylamide concentration. After dSDS-PAGE, using 6 M urea in the first-dimension, hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the technique. The separating effect is explained in part by protein-dependent variation of electrophoretic mobilities in the presence and absence of urea which presumably is caused by altered SDS-binding, and by anomalous migration of highly hydrophobic proteins in gels with different acrylamide concentration.21 The technique is well suited for the mass spectrometric analysis of subproteomes and identification of their constituent hydrophobic proteins. The aim of our study was to characterize the membrane subproteomes of a new Pseudomonas strain by proteomic means in order to assess the protein composition of this subcellular compartment, which appears fundamental for the biodegradation of aromatic compounds. The comparison of OM and IM fractions between different carbon sources (lysogeny broth medium, glucose and phenol) of the Pseudomonas strain using dSDS-PAGE and tandem mass spectrometry may explain the aromatic compound transport system.
Experimental Details Growth Conditions. Ten milliliters of liquid culture in LB (OD600 ) 0.4) were inoculated in 500 mL minimal medium22 with a 200 mg/L final concentration of glucose or phenol. Cultures were grown at 30 °C under vigorous shaking and the growth was monitored by measuring the optical density at 600 nm. The ring fission product from catechol was analyzed23 using an SLM AMINCO DW-2000 UV/vis spectrophotometer (SLM Instruments, Inc., Rochester, NY). Ten milliliters from the culture (OD600 ) 0.6) were centrifuged at 5000 g. The pellet was washed with 3 mL 50 mM phosphate buffer (pH 7) and centrifuged again at 5000 g for 10 min at 4 °C. After resuspend-
research articles ing the newly formed pellet in 1 mL 50 mM phosphate buffer, 100 µL of toluene/acetone (1:1) solution was added. Extraction of the cytoplasmic enzymes was achieved by incubation at room temperature for 10 min. Catechol was added as the ring fission substrate and spectra from 250 to 450 nm were acquired. Detection of 2-hydroxymuconic semialdehyde (375 nm) or cis,cis-muconic acid (260 nm) indicates catechol 2,3-dioxygenase (meta pathway) and catechol 1,2-dioxygenase (ortho pathway) activity respectively.3 Spectroscopic measurements were performed in 3 mL quartz vials with 1 cm path length. Preparation of the Total Membrane Fraction. The purification procedure was carried out as described by Tsirogianni et al.2 Cells (5 g wet weight) were suspended in 7.5 mL 50 mM Tris-H2SO4, pH 8.0. They were broken by sonication using a Broxon ultrasonic processor at the maximum power (20 times, 15 s with 45 s intervals), while the temperature was maintained below 10 °C, with a freezing bath. Unbroken cells and cell debris were removed from the resulting suspension by centrifugation at 10 000 g for 15 min at 4 °C. The supernatant was then centrifuged at 100 000 g for 1 h at 4 °C, after which a pellet was formed representing the total membrane fraction. Protein concentration of the total membrane fraction was determined using the Bradford method.24 Separation of the Two Membrane Types. The separation procedure was carried out as described by Ravaoarinoro et al. with slight modifications.8 Approximately 800 µg of total membrane proteins were incubated in 5 mL of 2% (w/v) sodium lauryl sarcosinate solution at room temperature for 1 h, followed by centrifugation at 100 000 g for 1 h at 4 °C. The resulting pellet, which represents the outer membrane fraction, was resuspended in 100 µL 50 mM Tris-H2SO4, pH 8.0. On the other hand, the sarcosinate soluble membrane fraction, containing inner membrane proteins, was precipitated using 10% TCA. One Dimensional Electrophoresis (Tris-Tricine SDS-PAGE). The procedure of Scha¨gger was used.25 Samples (80 µg) were incubated for 10 min at 40 °C before loading onto 10% acrylamide SDS-gels (23 cm separation length). Electrophoresis was started at 60 V, until the entry of the protein sample into the stacking gel, and continued for 14-16 h at 100 V. The gels were then subjected to colloidal brilliant blue staining.26 Doubled SDS-PAGE (dSDS-Tricine PAGE). This procedure was carried out according to Rais et al. with slight modifications.18 Samples (80 µg) were loaded in the first dimension which consists of a 4% polyacrylamide stacking gel and a 10% polyacrylamide separation gel containing 6 M urea. Electrophoresis was initially set up at 60 V for 1 h followed by 100 V for 14-16 h. Gel strips (1 cm width) were excised from the onedimensional SDS-Tricine PAGE and incubated for 30 min in acidic solution (100 mM Tris, 150 mM HCl, pH∼2; 50-100 times the gel volume). A 10% polyacrylamide gel with a trace amount of bromophenol blue (as a tracking dye) was used as a stacking gel in the second dimension, followed by a 16% highacrylamide gel for protein separation. Electrophoresis was carried out at 100 V for 1 h followed by 190 V for 14-16 h. Gels were fixed in 45% methanol, 1% acetic acid for at least 4 h and stained with homemade colloidal Coomassie; 34% methanol, 0.5% acetic acid, 17% (w/v) (NH4)2SO4, 0.1% (w/v) Coomassie G250 overnight.26 In-Gel Digestion. Dissected gel spots were placed in a 96 well culture-plate. The gel pieces were destained in 40% methanol, 10% acetic acid and subjected to the in-gel digestion protocol according to Shevchenko et al.27 Each gel spot was Journal of Proteome Research • Vol. 7, No. 10, 2008 4279
research articles reduced in 100 µL dithiothreitol (10 mM) at 57 °C for 1 h, alkylated in 100 µL iodoacetamide (55 mM) at room temperature in the dark for 1 h, and subsequently digested overnight using 0.12 µg bovine trypsin (proteomics grade, Sigma, Steinheim, Germany) in 50 mM ammonium bicarbonate aqueous solution. The extracts were collected initially using a 50% acetonitrile/1% TFA aqueous solution followed by 100% acetonitrile for the collection of highly hydrophobic peptides. The samples were then dried and stored at -20 °C. MALDI-TOF and MALDI-TOF/TOF. MS and MS/MS experiments were performed on a 4800 MALDI TOF/TOF Analyzer (Applied Biosystems, Darmstadt, Germany). Samples were dissolved in 5 µL 70% acetonitrile, 0.3% Trifluoroacetic acid. Cocrystallization of each peptide solution (0.5 µL) was performed using 2.5 mg/mL alpha-cyano-4-hydroxy-cinnamic acid at an equal volume. MALDI-TOF mass spectra were externally calibrated with a peptide mixture containing des-Arg-Bradykinin (904.47 Da), angiotensin I (1296.69 Da), Glu-fibrinopeptide B (1570.68 Da), ACTH (2093.0, 2465.20, 3657.93 Da), yielding 75 ppm mass accuracy. MS/MS spectra were externally calibrated from the fragment ions of angiotensin I (1296.69 Da), resulting in 75 ppm mass accuracy for the precursor and 0.8 Da for the fragment ions. A number of 1200 single scans were accumulated for MS spectra from a mass range of 700 to 4500, whereas 2000 single scans were acquired for each MS/MS spectrum. After the acquisition of MS spectra from each sample spot, result-dependent MS/MS measurements took place using GPS Explorer V3.6 (Applied Biosystems, Darmstadt, Germany). Precursor selection was performed by automatically selecting the 15 strongest in intensity precursors from each MS spectrum, although the minimum S/N filter was set up to 20. Processing, prior to the database search, was done using the data explorer V4.8 software (Applied Biosystems, Darmstadt, Germany). The MS and MS/MS spectra were smoothed, noise-filtered (correlation factor, 0.7), deisotoped and peak labeled, resulting in mass lists containing approximately 90 masses for MS and 100 for MS/MS. A combined PMF and MS/MS search was carried out for each spot using the online version of Mascot v2.2.28 The sequence database used was: NCBInr 20071214 (5742110 sequences; 1979781838 residues). The taxonomy used was other proteobacteria (1557762 sequences). The following parameters were set up for the database search; maximum number of missed cleavages, 1; peptide mass tolerance, 75 ppm; MS/MS mass tolerance, 0.8 Da; enzyme, trypsin; possible amino acid modifications, carbamidomethyl of cysteins and oxidation of methionines. The standard scoring algorithm of Mascot was used for the combined PMF and MS/MS search. Only protein hits above the significance threshold were accepted, apart from a few logical exceptions (see Results). Protein scores greater than 74, were considered significant, which is Mascot’s threshold score for identity at 95% confidence. All MS/MS spectra, used for verification, revealed prominent fragments of the corresponding peptides (see Supporting Information). Protein Prediction. The prediction of transmembrane R-helices and β-barrels for all identified inner membrane proteins was achieved using TMpred29 and PRED-TMBB30 respectively. The localization of all identified proteins was taken out of the database information or predicted using the program PSORTB v.2.0.31 Information concerning protein function of identified proteins was also acquired from the database. The grand average of hydrophobicity (GRAVY) value of the identified membrane proteins was calculated using ProtParam program.32 4280
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Results Culture Conditions of the Pseudomonas sp. Strain phDV1. Pseudomonas cells were grown in LB, sucrose and phenol. In LB and glucose, the cells display the same fast growth-rate, whereas phenol causes a longer duplication time. During the course of phenol degradation at the concentration of 200 mg/L, a bright-yellow coloration appeared in the culture medium during the later stages of growth. This is typical of the accumulation of 2-hydroxymuconic semialdehyde (HMSA) and is a common observation for aromatic compounds which are degraded via the catechol meta-cleavage pathway. The identification of this product as HMSA was confirmed by the peak absorption of the cell extract at 375 nm in the presence of catechol. Separation of the Outer from the Inner Membrane. Lauryl sarcosinate, as stated above, has been used in numerous studies because of its ability to efficiently and selectively solubilize the IM of ruptured Gram-negative bacteria.8,9 IMPs are solubilized in an detergent solution, whereas the OM peptidoglycan complex is not affected. Recovery of the OM fraction is therefore possible by ultracentrifugation after detergent exposure. In order to optically view the differences in the subcellular fractions, proteins were separated by Tricine SDS-PAGE and visualized by colloidal brilliant blue G-250 staining. Figure 1 presents a subtractive pattern for the total membrane (1, 4) versus OM (2, 5) and IM fractions (3, 6) from glucose (1-3) and phenol (4-6) cultivations. The sarcosinate-insoluble OM fraction reveals prominent protein bands at 55 kDa, 36 kDa and a cluster of protein bands between 14 kDa and 32 kDa. These protein spots can also be detected in the total membrane fraction, but they are excluded from the sarcosinate-soluble fraction (boxed in Figure 1). Analytical evaluation, of these and possibly additional protein differences, can only be achieved by protein identification of the presented spots through mass spectrometry. Comparison of Membrane Subproteomes. OM and IM fractions were subjected to dSDS-PAGE in order to identify and catalogue the OMP and IMPs. Figure 2 and Figure 3 display the OM and IM fractions from Pseudomonas phDV1 grown in LB (A), glucose (B), and phenol (C), respectively. A total amount of three replicas were created for each gel. Distinct protein spots were excised from the gels and submitted to protein assignment by MALDI-TOF/TOF. As a result, reproducibility of spot positions, spot intensities and protein identification was observed in all cases. A complete overview of all identified proteins, as well as their locations in the 2-DE gels, can be found in the Supporting Information section. In Figures 2 and 3, the OMP and IMP’s labeled, are annotated in the used database or predicted via PSORTB as such. In total, we separated and excised 247 inner membrane fraction spots from all carbon sources out of which 194 were identified. 85 outer membrane spots were separated and excised from all carbon sources, out of which 48 were identified. Out of 242 Proteins, only 8 were borderline identifications marginally below Mascot’s significant threshold (see Supporting Information). These proteins were taken in account, as they were significantly identified in other carbon sources at the same gel position. In total, 129 unique proteins could be identified by MS and MS/ MS analysis. A number of 98 unique proteins were found to be only in the inner membrane fraction and 9 only in the outer membrane fraction, whereas 22 proteins were found in both (see Supplemental data). We only accepted proteins as real OMP or IMP’s if they were annotated in the database or
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Figure 1. 1-DE SDS-PAGE from different subcellular fractions of Pseudomonas phDV1. Total membrane (1, 4), outer membrane (2, 5) and inner membrane fractions (3, 6) rom glucose (1-3) and phenol (4-6) cultivations can be depicted.
predicted via PSORTB as such and at the same time are present in the OM or IM fraction in order to guarantee unambiguous classification. Finally, a total number of 10 outer membrane proteins and 19 inner membrane proteins were identified from different carbon sources and unambiguously assigned to the outer or inner membrane (Tables 1 and 2), as displayed in Figures 2 and 3. All 10 OMP’s feature transmembrane domains (mainly β-barrels), whereas in the case of the IMP’s, 12 out of the 19 are integral membrane proteins. It is important to point
out that we observed significant differences between the subproteomes of the three different carbon sources (see Discussion). Figure 4A presents the predicted functions of the identified unique proteins, whereas Figure 4B shows the predicted localization of those proteins in the bacterial cell. Theoretical molecular weight and pI information is displayed in Figure 4C. As demonstrated, the membrane fractions are resolved and conserved over a Mr range of 10-110 kDa. However, due to Journal of Proteome Research • Vol. 7, No. 10, 2008 4281
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Figure 2. 2-DE pattern of the outer membrane subproteome of the bacterium grown in (A) LB, (B) glucose, and (C) phenol. Only identified membrane proteins are marked. A complete overview of all identified proteins can be found in the Supporting Information. (Mr, decreasing molecular weight proteins). 4282
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Figure 3. 2-DE pattern of the inner membrane subproteome of the bacterium grown in (A) LB, (B) glucose, and (C) phenol. Only identified membrane proteins are marked. A complete overview of all identified proteins can be found in the Supporting Information. (Mr, decreasing molecular weight proteins). Journal of Proteome Research • Vol. 7, No. 10, 2008 4283
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Table 1. List of the Identified Outer Membrane Proteins from dSDS-PAGE no.
protein name
1 2
probable porin membrane protein involved in aromatic hydrocarbon degradation porin, LamB type OprF OprG OmpA/MotB hypothetical protein Pmen_2977 OmpA 17 kDa surface antigen OprI
3 4 5 6 7 8 9 10 a
Swiss-Prot no.
carbon sourcea2
MW/pI
GRAVY score
A4VM31 Q479D9
46982/5.53 45938/6.66
-0.463 -0.016
Phenol Phenol
A4XRF5 Q9X4S1 A4VHR3 A4XS71 A4XWL6 A4XRS7 A4XS89 O85420
45764/5.33 33877/4.92 24997/4.73 21999/4.65 21626/6.28 17746/5.30 15534/9.50 8799/7.87
-0.540 -0.405 +0.104 -0.088 -0.391 -0.340 +0.120 -0.486
Glucose LB, Glucose, LB, Glucose, Glucose LB, Glucose, LB, Glucose, LB, Glucose, LB, Glucose,
Phenol Phenol Phenol Phenol Phenol Phenol
Carbon source in which each identified protein is found, see also Figure 2.
Table 2. List of the Identified Inner Membrane Proteins from dSDS-PAGE no.
1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 a
protein name
methyl-accepting chemotaxis sensory transducer hypothetical protein Pfl_1761 hypothetical protein Pmen_2077 ATP synthase F1, alpha subunit arginine/ornithine antiporter ATP synthase F1, beta subunit MotA/TolQ/ExbB proton channel Cytochrome b/b6-like efflux transporter, RND family, MFP subunit ATP synthase F1, gamma subunit binding-protein-dependent transport systems inner membrane component cell division protein ZipA MscS Mechanosensitive ion channel conserved TM helix repeat-containing protein ABC transporter related MotA/TolQ/ExbB proton channel ATP synthase F1, delta subunit ATP synthase B chain 6,7-dimethyl-8-ribityllumazine synthase
Swiss-Prot no.
TM helices
GRAVY score
carbon sourcea3
A4XPB3
71799/4.47
2
+0.026
Glucose
Q3KFF2 A4XU22 A4Y189 P18275 A4Y187 A4XZ52 A4XQQ6 A4XYP7
57092/9.07 56703/7.87 55349/5.40 52027/6.53 49615/4.94 48319/5.64 46011/7.68 40542/6.18
7 6 0 13 0 4 9 0
+0.328 +0.310 -0.160 +0.847 -0.150 +0.066 +0.622 -0.193
Glucose LB LB, Glucose, Phenol Glucose LB, Glucose, Phenol LB Glucose LB, Phenol
A4Y188 A4XRF2
31518/9.27 33553/9.51
0 6
-0.162 +0.634
LB Glucose
A4XVY6 A4XQU2
30737/5.42 29798/6.45
1 3
-0.445 +0.494
Glucose LB, Glucose, Phenol
A4XU35
28919/6.19
5
+0.978
LB, Glucose
A4XZ45
25564/6.53
0
-0.061
Glucose
A4XRS3
25409/5.95
3
+0.159
Glucose
A4Y190
18961/5.86
0
+0.152
LB, Glucose
A4Y191 A4XZ35
17059/5.53 16494/5.71
1 0
-0.170 +0.380
Glucose LB
Carbon source in which each identified protein is found, see also Figure 3.
technical limitations, higher molecular weight proteins do not appear to be resolved (Figure 4C).
Discussion Outer Membrane Proteins. OMPs are of particular interest in Pseudomonas strains due to their cell-surface exposure and their involvement in transport of nutrients, in export of extracellular virulence factors, and in anchoring the structures that mediate adhesion and motility. A total number of 10 outer membrane proteins were identified in the presence of all carbon sources (Table 1). These consist of proteins of the OprF, OmpA, OprG, OprI families, a 17 kDa surface antigen, and a hypothetical protein (Figure 2). OprF proteins (Q9X4S1), which have been described as nonspecific porins, form small water-filled channels. They play an important role in the maintenance of the cell structure when 4284
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cultivation is carried out in a low osmolarity environment, and in adhesion to various supports polar nutrients.33 The protein migrates at 35 kDa and is in correlation with the reported molecular weight of the monomeric form of the protein. An additional low molecular weight spot at 20 kDa should represent a proteolytic fragment.34 It is known that OprF interact with lipopolysaccharides (LPS). Two types of interactions have been described: slightly associated LPS (sLPS), released in the migration front during SDS-PAGE, and tightly associated LPS (tLPS), which cannot be separated from native porins.35 The observed horizontal streaking suggests the existence of OprF in association with different amounts of bound LPS. In the case of glucose (Figure 2B), an additional spot without streaking was observed which could present the LPS free protein. OmpA proteins (A4XRS7) occur at about 100 000 copies cell, making it one of the major outer membrane proteins of E. coli.
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Figure 4. (A)Function, (B) localization, and (C) theoretical Mw/pIs of all 129 unique proteins separated by dSDS-PAGE and identified through MALDI-TOF/TOF.
They play a structural role in the integrity of the bacterial cell surface. The physiological function of the OmpA protein is to provide a physical linkage between the outer membrane and the underlying peptidoglycan layer. This is a hypothesis that is substantiated by the observation that a double mutant in OmpA and the Lpp lipoprotein, another major outer membrane protein that interacts with the peptidoglycan, leads to spherical cells that can only survive under well-balanced osmotic conditions.36 OprG proteins (A4VHR3) have been suggested to be involved in low-affinity iron uptake due to the direct relationship between its expression and the iron concentration in the medium,37 which is in correlation to the presence of iron in the modified media of glucose and phenol.
Furthermore, a surface antigen protein (A4XS89) and a major outer membrane peptidoglycan-associated lipoprotein OprI (O85420) were identified in all carbon sources. The hypothetical protein (A4XWL6), also located in all carbon sources, was found to have 70% homology to OmpH family proteins. OmpH proteins play a structural role in the integrity of the bacterial cell wall. Overexpression of the divalent cation-regulated outer membrane protein H1 of Pseudomonas aeruginosa is associated with resistance to polymyxin B, aminoglycosides, and EDTA. Protein H1 is believed to act by replacing divalent cations at binding sites on lipopolysaccharide, thereby preventing disruption of the sites and subsequent self-promoted uptake of the antibiotics.38 Journal of Proteome Research • Vol. 7, No. 10, 2008 4285
research articles Apart from the above integral membrane proteins, there were also integral outer membrane proteins which were expressed only in the presence of one carbon source. An outer membrane LamB-type protein (A4XRF5) was identified only in the case of glucose as sole carbon energy source. The maltooligosaccharide-specific maltoporin LamB is expressed upon induction by maltose or maltodextrins.39 Maltodextrins up to maltoheptaose could be transported, as was shown in in vivo and in vitro uptake experiments but not glucose.40 Our findings support that, in Pseudomonas, glucose may indeed induce the expression of an integral membrane protein which has high homology to LamB proteins. After the cultivation of the bacterium in phenol as sole carbon energy source, two additional outer membrane proteins were expressed and identified. It is known that Pseudomonas strains encode outer membrane channels implicated in the uptake of aromatic substrates.41 The two integral membrane proteins identified are; a membrane protein involved in aromatic hydrocarbon degradation (Q479D9) and a probable porin (A4VM31). Although the presence of different transporters have been reported for different aromatic compounds such as toluene and benzoic acid,41,42 these are the first phenol inducible membrane transporters to our knowledge. It is important to point out that from Mascot results, a resemblance occurs between the probable porin and a putative aromatic compound-specific porin (Q88GR2). The aromatic compoundspecific porin turns up as the second probable protein for identification slightly below the significant threshold. Sequence alignment of both proteins results in high homology (Supporting Information). This strongly indicates that the expressed Pseudomonas phDV1 membrane protein may have similar properties to the aromatic compound-specific porin. Inner Membrane Proteins. Consistent with its environmental versatility, Pseudomonas has nearly 300 cytoplasmic membrane transport systems, about two-thirds of which appear to be involved in the import of nutrients and other molecules.43 Even though the detergent soluble fraction, isolated from the total membrane residues, can not be considered as a purified inner membrane fraction, a significant number of inner membrane proteins were identified (Table 2). Over half of the inner membrane proteins, identified after dSDS-PAGE, have a GRAVY Score higher than 0.3, indicating that the separation method is quite efficient for hydrophobic proteins. Unique cytoplasmic membrane proteins were found in one or two carbon sources but never in all three, with the exception of a mechanosensitive ion channel protein (A4XQU2) (Figure 3). Mechanosensitive channels are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eukarya. Mechanosensitive ion channels play a critical role inducing physical stress at the cell membrane to electrochemical response.44 In Gram-negative bacteria many biological processes are coupled to inner membrane ion gradients. Ions transit at the interface of helices of integral membrane proteins, generating mechanical energy to drive energetic processes.45 A functionally homologous protein, the conserved TM helix repeat-containing protein (A4XU35), was additionally found in glucose and LB. Another homologous protein, the MotA/TolQ/ ExbB proton channel system (A4XZ52, A4XRS3) has also been identified in both carbon sources. This family groups together integral membrane proteins that appear to be involved in the translocation of proteins across a membrane. Furthermore, we identified proteins belonging to the energetic metabolism, such as ATP synthase of the respiratory 4286
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Papasotiriou et al. chain. In particular, subunits alpha (A4Y189) and beta (A4Y187) were identified in all carbon sources, whereas the gamma subunit was found in LB (A4Y188), b subunit in glucose (A4Y191) and the delta subunit in LB and glucose (A4Y190). Although ATP synthase is present in all carbon sources, the additional identifications could indicate a higher abundance of ATP synthase in the glucose fraction. Glucose degradation, in general, is a main source of ATP-derived free energy. Therefore, when comparing the different carbon sources used in the experiment, glucose is found to be the carbon source which mostly increases ATP consumption.46 We were also able to identify, among other inner membrane proteins, an arginine/orthonine antiporter (P18275) and a Cytochrome b/b6-like protein (A4XQQ6). All of the above proteins are identified only in the glucose inner membrane fraction. The arginine/orthonine anitporter plays a crucial role in the arginine deiminase pathway, in which arginine is converted to NH3, CO2, and ornithine with the concomitant generation of 1 mol of ATP/mol of arginine.47 Conditions which lower ATP consumption decrease the arginine deiminase (ADI) pathway activity, whereas uncouplers and ionophores which stimulate ATP consumption increase the activity.48 This may very well be the reason why such antiporters are only found in the glucose fraction, as glucose provides higher ATP consumption. The Cytochrome b/b6, N-terminal domain protein is a component of the ubiquinol-cytochrome c reductase complex (complex III or Cytochrome b-c1 complex), which belongs to the respiratory chain that generates an electrochemical potential coupled to ATP synthesis. Therefore, Cytochrome b/b6 is only identified in glucose which provides higher ATP consumption, as indicated in Table 2. Furthermore, a cell division protein (A4XVY6) belonging to the ZipA family and a methyl-accepting chemotaxis sensory transducer (A4XPB3) that induces bacterial movement were exclusively observed in glucose. It is quite reasonable that cell division and movement of bacteria is enhanced if more ATP is provided due to glucose degradation. During cultivation, the faster cell duplication time was observed when using glucose as growth medium. The BLAST-Search against the NCBI-database of two other proteins, the ABC transporter related protein (A4XZ45) and the binding-protein-dependent transporter systems inner membrane component (A4XRF2), showed that both proteins are involved in sugar transport. Therefore, it makes sense that both proteins are only present in glucose. Poor resolution of the 2-DE (IEF/SDS) PAGE at alkaline pH makes the identification of different ABC transporters mostly difficult.49 The dSDS-PAGE system, used in the current study, allows the identification of low molecular mass proteins (MW < 35 kDa) in the pI range of 4-12 (Figure 4C). This allows the identification of basic proteins such as the binding-protein-dependent transport system of the inner membrane (A4XRF2). The 6,7-dimethyl-8-ribityllumazine synthase (A4XZ35) was only identified in LB medium, consisting of amino acids as energy source. This enzyme is involved in the riboflavin biosynthesis and riboflavin is the educt for biosynthesis of the flavin adenine dinucleotide (FAD). The higher abundance of this enzyme could therefore be rationally due to amino acid degradation via citrate circle, which should require more FAD. The respiratory chain uses the reduced FAD to generate an electrochemical potential coupled to ATP synthesis. The MFP subunit of an RND family protein (A4XYP7), which may be involved in an efflux transport system, was exclusively
research articles
Comparison of the Membrane Subproteomes identified in phenol and LB medium. Any explanation of this is beyond our intentions as it is too speculative. Furthermore, two hypothetical proteins (Q3KFF2; A4XU22) were discovered in LB medium and glucose respectively. The sequence alignment of both protein sequences resulted in a high homology. A BLAST-Search against the NCBI-database yielded no functional information. Cytoplasmic and periplasmic proteins, which serve as contaminants, have been found in IM and OM fractions (Supplementary data). Among others, heat shock proteins Hsp 90, Hsp 52 from the protein export system, were identified. The presence of a large number of ribosomal proteins was also observed. The occurrence of ribosomal proteins indicates the strong association of these proteins with the membrane during the localization of the membrane proteins, cotranslationally, into the inner membrane.13,50 Different reports also indicated that some soluble metabolic enzymes are observed in the 2-DE gels of bacterial membrane fractions, which may be peripherally associated proteins bound to OMPs/IMPs.13,51 We found that a large number of metabolic enzymes, were identified in the IMP subproteome, and a portion of these enzymes were associated with OMPs or IMPs. An interesting finding is that many enzymes which are induced by phenol were located in the outer membrane fraction of the prokaryotic cell, such as Phenol hydroxylase P1 protein (Q52171) (Supporting Information).
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Conclusion A profile of the membrane fraction from a new Pseudomonas strain, cultivated in three different carbon sources, was characterized by the analysis of soluble and insoluble sarcosyl fractions. We present a first reference map of the three carbon sources by dSDS-PAGE, and identify glucose and phenol inducible outer membrane proteins. Our results will facilitate the identification of inducible membrane protein complexes, which are involved in the transport of aromatic compounds in the cell. Abbreviations: MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; OM, outer membrane; IM, inner membrane; OMP, outer membrane protein; IMP, inner membrane protein; 2-DE, two-dimensional gel electrophoresis; dSDS-PAGE, doubled SDS polyacrylamide gel electrophoresis; LB, lysogeny broth; BLAST, Basic Local Alignment Search Tool; NCBI, National Center for Biotechnology Information.
Acknowledgment. This research program was supported by the University of Crete, the Greek Ministry of Education and the General Secretary for Research and Technology (PENED 03ED863 and 05NON-EU-426). The project is cofunded by the European Social Fund and National Resources - EPEAEK-PYTHAGORAS II. D.G.P. thanks the Erasmus program and student exchange for financial support to visit the laboratory of M. Karas. Supporting Information Available: Further details as mentioned in text. This information is available free of charge via the Internet at http://pubs.acs.org. References (1) Polymenakou, P.; Stefanou, E. Effect of temperature and additional carbon sources on phenol degradation by an indigenous soil pseudomonad. Biodegradation 2005, 16 (5), 403–413. (2) Tsirogianni, I.; Aivaliotis, M.; Karas, M.; Tsiotis, G. Mass spectrometric mapping of the enzymes involved in the phenol degrada-
(13)
(14)
(15)
(16)
(17) (18)
(19)
(20)
(21)
(22)
(23)
(24)
(25) (26)
tion of an indigenous soil pseudomonad. Biochem. Biophys. Acta 2004, 1700, 117–123. Tsirogianni, I.; Aivaliotis, M.; Karas, M.; Tsiotis, G. Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil Pseudomonad by MALDI-TOF MS using a column separation. Biodegradation 2005, 16 (2), 181–186. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Mol. Biol. Rev. 2003, 67 (4), 593–656. Walsh, N. P.; Alba, B. M.; Bose, B.; Gross, C. A.; Sauer, R. T. OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain. Cell 2003, 113, 61–71. Sheetz, M. P. Cellular plasma membrane domains. Mol. Membr. Biol. 1995, 12, 89–91. Buchanan, S. K.; Smith, B. S.; Venkatramani, L.; Xia, D.; Esser, L.; Palnitkar, M.; Chakraborty, R.; van der Helm, D.; Deisenhofer, J. Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat. Struct. Biol. 1999, 6 (1), 56–63. Ravaoarinoro, M.; Ciurli, C.; Toma, E.; Morisset, R. Rapid Method for isolating detergent-insoluble outer membrane proteins from Pseudomonas Aeruginosa. Electrophorsis 1994, 15, 594–596. Filip, C.; Fletcher, G.; Wulff, J. L.; Earhart, C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent Sodium-Lauryl Sarcosinate. J. Bacteriol. 1973, 115 (3), 717–722. Carlone, G. M.; Thomas, M.; Rumschlag, H.; Sottnek, F. O. Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J. Clin. Microbiol. 1986, 24 (3), 330–332. Schnaitman, C. A. Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100. J. Bacteriol. 1971, 108 (1), 545– 552. Stull, T. L.; Mack, K.; Haas, J. E.; Smit, J.; Smith, A. L. A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b. Anal. Biochem. 1985, 150 (2), 471– 480. Aivaliotis, M.; Corvey, C.; Tsirogianni, I.; Karas, M.; Tsiotis, G. Membrane proteome analysis of the green-sulfur bacterium Chlorobium tepidum. Electrophoresis 2004, 25 (20), 3468–3474. Aivaliotis, M.; Haase, W.; Karas, M.; Tsiotis, G. Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum. Proteomics 2006, 6 (1), 217–232. Lescuyer, P.; Strub, J. M.; Sylvie Luche, S.; Diemer, H; Martinez, P.; van Dorsselaer, A.; Lunardi, J.; Rabilloud, T. Progress in the definition of a reference human mitochondrial proteome. Proteomics 2003, 3 (2), 157–167. Santoni, V.; Molloy, M.; Rabilloud, T. Membrane proteins and proteomics: Un amour impossible. Electrophoresis 2000, 21 (6), 1054–1070. Wu, C. C.; Yates, J. R., III. The application of mass spectrometry to membrane proteomics. Nat. Biotechnol. 2003, 21 (3), 262–267. Rais, I.; Karas, M.; Scha¨gger, H. Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification. Proteomics 2004, 4 (9), 2567–2571. Hartinger, J.; Stenius, K.; Ho¨gemann, D.; Jahn, R. 16-BAC/SDSPAGE: A Two-Dimensional Gel Electrophoresis System Suitable for the Separation of Integral Membrane Proteins. Anal. Biochem. 1996, 240 (1), 126–133. Navarre, C.; Degand, H.; Bennett, K. L.; Crawford, J. S.; Mørtz, E.; Boutry, M. Subproteomics: Identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae. Proteomics 2002, 2 (12), 1706–1714. Swank, R. T.; Munkres, K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal. Biochem. 1971, 39 (2), 462–477. Kunz, D. A.; Chapman, P. J. Catabolism of pseudocumene and 3-ethyltoluene by Pseudomonas putida (arvilla) mt-2: evidence of a new function of the TOL (pWW0) plasmid. J. Bacteriol. 1981, 146 (1), 179–191. Nelson, N. J.; Montgomery, S. O.; Mahaffey, W. R.; Pritchard, P. H. Biodegradation of Trichloroethylene and Involvement of an Aromatic Biodegradative Pathway. Appl. Environ. Microbiol. 1987, 53 (5), 949–954. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. Scha¨gger, H. Tricine-SDS PAGE. Nat. Protoc. 2006, 1 (1), 16–22. Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing
Journal of Proteome Research • Vol. 7, No. 10, 2008 4287
research articles
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
(37)
(38)
4288
gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9 (6), 255–262. Shevchenko, A.; Tomas, H.; Havli, J.; Olsen, J. V.; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2007, 1 (6), 2856–2860. Perkins, D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20 (18), 3551–3567. Hofmann, K.; Stoffel, W. TMbase - A database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 1993, 374, 166. Bagos, P. G.; Liakopoulos, T. D.; Spyropoulos, I. C.; Hamodrakas, S. J. PRED-TMBB: a web server for predicting the topology of betabarrel outer membrane proteins. Nucleic Acids Res. 2004, 32, W400–404. Gardy, J. L.; Laird, M. R.; Chen, F.; Rey, S.; Walsh, C. J.; Ester, M.; Brinkman, F. S. L. PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 2005, 21 (5), 617–623. Gill, C. S.; von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182 (2), 319–326. Orange, N. Growth temperature regulates the induction of β-lactamase in Pseudomonas fluorescens through modulation of the outer membrane permeation of a beta-lactam-inducing antibiotic. Microbiology 1994, 140 (11), 3125–3130. El Hamel, C.; Freulet, M. A.; Jaquinod, M.; De´, E.; Molle, G.; Orange, N. Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties. Biochim. Biophys. Acta 2000, 1509, 237–244. Wiese, A.; Schro¨der, G.; Brandenburg, K.; Hirsch, A.; Welte, W.; Seydel, U. Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans. Biochim. Biophys. Acta 1994, 1190, 231–242. Sonntag, I.; Schwarz, H.; Hirota, Y.; Henning, U. Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins. J. Bacteriol. 1978, 136 (1), 280–285. Yates, J. M.; Morris, G.; Brown, M. R. Effect of iron concentration and growth rate on the exression of protein G in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 1989, 49, 259–262. Bell, A.; Hancock, R. E. Outer membrane protein H1 of Pseudomonas aeruginosa purification of the protein and cloning and nucleotide sequence of the gene. J. Bacteriol. 1989, 171 (6), 3211– 3217.
Journal of Proteome Research • Vol. 7, No. 10, 2008
Papasotiriou et al. (39) Benz, R.; Schmid, A.; Nakae, T; Vos-Scheperkeuter, G. H. Pore formation by LamB of Escherichia coli in lipid bilayer membranes. J. Bacteriol. 1986, 165 (3), 978–986. (40) Gehring, K.; Cheng, C. H.; Nikaido, H.; Jap, B. K. Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli. J. Bacteriol. 1991, 173 (6), 1873–1878. (41) Cowles, C. E.; Nichols, N. N.; Harwood, C. S. BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. J. Bacteriol. 2000, 182 (22), 6339–6346. (42) Ramos, J. L.; Duque, E.; Gallegos, M. T.; Godoy, P.; RamosGonzalez, M. I.; Rojas, A.; Teran, W.; Segura, A. Mechanisms of solvent tolerance in gram-negative bacteria. Annu. Rev. Microbiol. 2002, 56, 743–768. (43) Stover, C. K.; Pham, X. Q.; Erwin, A. L.; Mizoguchi, S. D.; Warrener, P.; Hickey, M. J.; Brinkman, F. S.; Hufnagle, W. O.; Kowalik, D. J.; Lagrou, M.; Garber, R. L.; Goltry, L.; Tolentino, E.; Westbrock_Wadman, S.; Yuan, Y.; Brody, L. L.; Coulter, S. N.; Folger, K. R.; Kas, A.; Larbig, K.; Lim, R.; Smith, K.; Spencer, D.; Wong, G. K.; Wu, Z.; Paulsen, I. T.; Reizer, J.; Saier, M. H.; Hancock, R. E.; Lory, S.; Olson, M. V. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 2000, 406, 959–964. (44) Pivetti, C. D.; Yen, M. R.; Miller, S.; Busch, W.; Tseng, Y. H.; Booth, I. R.; Saier, M. H. J. Two families of mechanosensitive channel proteins. Microbiol. Mol. Biol. Rev. 2003, 67 (1), 66–85. (45) Koebnik, R. The molecular interaction between components of the TonB-ExbBD-dependent and of the TolQRA-dependent bacterial uptake systems. Mol. Microbiol. 1993, 7 (3), 219. (46) Lagunas, R. Energy metabolism of Saccharomyces cerevisiae discrepancy between ATP balance and known metabolic functions. Biochim. Biophys. Acta 1976, 440 (3), 661–674. (47) Driessen, A. J.; Molenaar, D.; Konings, W. N. Kinetic Mechanism and Specificity of the Arginine-Ornithine Antiporter of Luctococcus Zuctis. J.Biol.Chem. 1989, 264 (18), 10361–10370. (48) Poolman, B.; Driessen, A. J.; Konings, W. N. Regulation of arginineornithine exchange and the arginine deiminase pathway in Streptococcus lactis. J. Bacteriol. 1987, 169 (12), 5597–5604. (49) Pennington, K.; McGregor, E.; Beasley, C. L.; Everall, I.; Cotter, D.; Dunn, M. J. Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue. Proteomics 2004, 4 (1), 27–30. (50) Lee, C.; Beckwith, J. Cotranslational and Posttranslational Protein Translocation in Prokaryotic Systems. Annu. Rev. Cell Biol. 1986, 2, 315–332. (51) Boyce, J. D.; Cullen, P. A.; Nguyen, V.; Wilkie, I.; Adler, B. Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host. Proteomics 2006, 6 (3), 870–880.
PR800192N
