Proteomic Analysis of Cell Envelope from - American Chemical Society

Proteomic Analysis of Cell Envelope from Staphylococcus xylosus C2a, a Coagulase-Negative Staphylococcus Stella Planchon,† Christophe Chambon,‡ Mickae1 l Desvaux,† Ingrid Chafsey,† Sabine Leroy,† Re´ gine Talon,† and Michel He´ braud*,†,‡ INRA Centre de Clermont-Ferrand/Theix, UR454 Microbiologie, Equipe Qualite´ et Sećurite´ des Aliments, and Plate-Forme Proteómique, 63122 Saint-Gene`s Champanelle, France Received March 14, 2007

Staphylococcus xylosus is a saprophytic bacterium commonly found on skin of mammals but also used for its organoleptic properties in manufacturing of fermented meat products. This bacterium is able to form biofilms and to colonize biotic or abiotic surfaces, processes which are mediated, to a certain extent, by cell-envelope proteins. Thus, the present investigation aimed at evaluating and adapting different existing methods for cell-envelope subproteome analyses of the strain S. xylosus C2a. The protocol selected consisted initially of a lysostaphin treatment producing protoplasts and giving a fraction I enriched in cell wall proteins. A second fraction enriched in membrane proteins was then efficiently recovered by a procedure involving delipidation with a mixture of tributyl phosphate, methanol, and acetone and solubilization with a buffer containing ASB14. Proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 168 protein spots was identified corresponding to 90 distinct proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach was carried out on proteins identified within cell envelope of S. xylosus C2a. Thirty-four proteins were predicted as membrane-associated with 91% present, as expected, within fraction II enriched in membrane proteins: 24 proteins were predicted as membranal, 3 as lipoproteins, and 7 as components of membrane protein complex. Eighteen out of 25 (72%) proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins: 6 proteins were predicted as secreted via Sec translocon, and the remaining 19 proteins were predicted as secreted via unknown secretion system. Eighty-one percent (25/31) of proteins predicted as cytoplasmic were found in fraction II: 8 were clearly predicted as interacting temporarily with membrane components. By coupling conventional 2-DE and bioinformatic analysis, the approach developed allows fractionating, resolving, and analyzing a significant and important set of cell envelope proteins from a coagulase-negative staphylococcus, that is, S. xylosus C2a. Keywords: Cell envelope proteins • Staphylococcus xylosus • two-dimensional gel electrophoresis • MALDI-TOF MS

Introduction Staphylococcus xylosus is a non-motile Gram-positive coccus, which belongs to the Staphylococcus saprophyticus group. Unlike Staphylococcus aureus, S. xylosus is a coagulase-negative staphylococci (CNS). Within CNS, some of them like Staphylococcus epidermidis are involved in nosocomial infections, while others are commensals strains. S. xylosus is commonly found on the skin of a variety of mammals and occasionally on human skin.1 Consequently, it has been isolated from dairy and meat products. Some strains are used as a starter culture * To whom correspondence should be addressed. INRA, Centre de Clermont-Ferrand/Theix, UR454 Microbiologie, Equipe Qualite´ et Sećurite´ des Aliments, F-63122 Saint-Gene`s Champanelle, France. Tel: +33-(0)4-7362-46-70. Fax: +33-(0)4-73-62-45-81. E-mail: [email protected]. † UR454 Microbiologie, Equipe Qualite´ et Sećurite´ des Aliments. ‡ Plate-Forme Proteómique.

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Published on Web 07/17/2007

in sausage manufacturing, in which they contribute to the flavor and the color of the final products.2 This species is also isolated from environmental sources like soil and surfaces of processing lines.2,3 Its capacity to colonise the skin and various surfaces is probably due to its ability to form biofilm.4,5 The process of biofilm formation is mediated by cell surface associated macromolecules. Surface proteins play key roles in bacterial interaction and adaptation to the environment; in adhesion to abiotic or biotic surfaces, to host cells, or to other bacterial cells; and in invasion process.6,7 In Gram-positive bacteria, or more exactly Monodermata, surface proteins are associated either with the cell wall or with the cytoplasmic membrane.8 The study of these proteins is a real challenge, considering they are difficult to extract, to solubilize, and to analyze due to their intrinsic hydrophobic nature, alkaline pI, and the number of transmembrane span10.1021/pr070139+ CCC: $37.00

 2007 American Chemical Society

Proteomic Analysis of Cell Envelope from S. xylosus C2a

ning regions.6,9 Extraction of the cell wall proteins has been previously described in Gram-positive bacteria. One method used high salt concentrations, such as lithium chloride for S. aureus9 and Bacillus subtilis,10 and Tris and potassium isothiocyanate for Listeria monocytogenes.11 Another method used sodium dodecyl sulfate (SDS) to release surface proteins in S. aureus9 and L. monocytogenes.12 Finally, a method used enzymes that destroyed the cell wall integrity and released covalently anchored cell wall proteins.9,13 Authors have used the lysostaphin alone,14 or combined with mutanolysin,15 to recover surface proteins in S. aureus. Extraction of membrane proteins was also performed by lysostaphin treatment followed by ultracentrifugation in S. aureus.9,16 Lipids were further solubilized and removed following a delipidation step, which allows to release membrane proteins entrapped in lipid bilayer.17,18 Several delipidation solvents were described in the literature: (i) a mixture of methanol and chloroform;19 (ii) a mixture of tri-n-butylphosphate (TBP), acetone, and methanol;17 (iii) the Triton X100;20 and (iv) a mixture of ammonium bicarbonate, chloroform, and trifluoroethanol (TFE).21 Finally, many chemical agents have been used to solubilize membrane proteins such as (i) 3-[3-chloamidopropyl dimethylammonio]1-propanesulfonate (CHAPS), a conventional zwitterionic surfactant;22 (ii) an organic solvent trifluoroethanol (TFE);16 and (iii) the amidosulfobetaine (ASB14).23 Subsequent analyses of surface proteins in Gram-positive bacteria, using various proteomic techniques, have been reported. In staphylococci, studies of surface proteins concerned mainly the opportunist pathogen S. aureus. Authors have used 2-DE conventional techniques,9 1-DE combined with liquid chromatography (LC)-MS/MS,16 and two-dimensional (2-D) nanoliquid chromatography coupled with ion-trap mass spectrometry.15 Recently, Gatlin et al.15 have successfully isolated 48 cell envelope proteins by combining anion chromatography with either iso-electrofocusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or LC-MS/MS. To date, this study represents the most extensive description of cell surface proteins in S. aureus and in staphylococci. However, no data is as yet available on surface proteins of coagulase-negative staphylococci such as S. xylosus. The aim of the present study was to develop a reproducible and efficient method to fractionate and separate by 2-DE a significant set of the cell envelope proteome of S. xylosus.

Materials and Methods Bacterial Strain and Culture Conditions. The strain S. xylosus C2a isolated from human skin (University of Tu¨bingen) was subcultured three times under shaking (150 rpm) at 30 °C, in tryptic soya broth medium supplemented with 0.6 g/L yeast extract (TSB, Difco, France). The third subculture was used in stationary phase to inoculate culture in TSB supplemented with 20 g/L sodium chloride at initial absorbance at 600 nm (OD600) of 0.6-0.7. The culture was incubated under shaking (150 rpm) at 30 °C during 48 h. Cells in stationary phase (40 mL at OD600 of 10) were centrifuged at 7500g for 15 min at 20 °C. The cell pellet was washed twice with the buffer 1, pH 7.5 (20 mM Tris, 5 mM MgCl2, and 5 mM ethylenediaminetetraacetic acid [EDTA]) then resuspended in this buffer and stored frozen at -20 °C until required. Cell Envelope Protein Extraction. Frozen cells were thawed, centrifuged at 7500g for 15 min at 20 °C, and resuspended in digestion buffer (pH 8) containing 50 mM Tris-HCl, 20 mM MgCl2, 30% sucrose, cocktail of protease inhibitor according

research articles to the manufacturer’s instructions (Roche, Germany) and 120 µg of lysostaphin (Sigma, Saint Louis, MO) for 1 mL of sample at OD600 of 40 according to Vytvytska et al.14 The suspension was incubated at 37 °C for 35 min under shaking at 150 rpm. Then, protoplasts were sedimented by centrifugation at 6000g for 30 min at 4 °C and frozen at -20 °C in 2 mL of buffer 1. The supernatant containing proteins was filtered and treated with DNAse and RNase (20 µg/mL of each) at room temperature during 15 min.14 Proteins were precipitated with trichloroacetic acid 20% (v/v) at 4 °C and harvested by a 13 000g centrifugation for 45 min at 4 °C. The precipitate was rinsed three times with glacial absolute ethanol and centrifuged at 13 000g for 15 min at 4 °C. It was air-dried and resuspended in buffer 2 containing 7 M urea, 2 M thiourea, 2 mM tributyl phosphine, 10 mM Tris, and 1% ASB14. Proteins of this fraction I enriched in cell wall proteins were stored at -80 °C until use. Then, frozen protoplasts in buffer 1 were thawed and vortexed three times for 1 min with glass beads (145 µm diameter, Sigma, Saint Louis, MO). Unbroken protoplasts were sedimented by centrifugation at 3500g for 15 min at room temperature, and the supernatant was collected. This step was repeated three times, and all supernatants were pooled and treated with DNase and RNase (20 µg/mL of each) during 15 min at room temperature. Membrane pellet was harvested by ultracentrifugation at 200 000g for 30 min at 4 °C and then washed twice with Tris base buffer (40 mM, pH 8.5). It was resuspended in 500 µL of Tris base buffer and submitted to a delipidation by using either (i) the method of Santoni et al.20 with 0.2% Triton X100, or (ii) a mixture containing 150 µL ammonium bicarbonate in 1 mL of 2:1 TFE/chloroform as described by Deshusses et al.,21 or (iii) a solution composed of TBP, acetone, and methanol (1:12:1) according to Mastro and Hall.17 After treatment, the delipidated protein fraction was dissolved in buffer 2 or in this buffer where the ASB14 detergent was replaced either by TFE16 or by CHAPS.22 Proteins of this fraction II enriched in membrane proteins were stored frozen at -80 °C. Two to three 2-DE analytical gels were performed from each preparation. The amount of proteins was measured by the Bradford method using BSA as a standard. Samples in buffer 2 were diluted by 1/10 for assay compatibility. 2-DE and Image Analysis. The first dimension was performed with the IEF Cell (Bio-Rad, Hercules, CA) system and pH 4-7 or nonlinear pH 3-10, 17 cm Immobiline pH Gradient (IPG) strips (Bio-Rad). The samples were mixed with 2 mM TBP, 0.3% ampholytes 4-6 and 5-7 or 3-10 for proteins from the fractions I and II, respectively, and completed to 400 µL with rehydration buffer 2. After passive rehydration (9 h), IEF was carried out at 19 °C for a total of 48 000 Vh. Second dimension was performed overnight in 10% acrylamide gels. Analytical gels were carried out with 60 µg of proteins and silver stained according to Rabilloud.24 Six 2-DE gels with samples from two different cultures were performed to evaluate the reproducibility of the methods. The gels were compared using Image Master 2-D Platinum software v.5.0 (GE Healthcare, Uppsala, Sweden). Proteins were identified from semipreparative gels loaded with either 200 µg of proteins of fraction I and silver stained according to Yan et al.,25 or 600 µg of proteins of fraction II and stained with colloidal Coomassie Blue.26 Protein Identification by MALDI-TOF MS. The protein spots of interest were excised from the semipreparative gels using pipet cones cut to the diameter of each spot.27 The in-gel protein destaining and trypsin digestion and then the MALDIJournal of Proteome Research • Vol. 6, No. 9, 2007 3567

research articles TOF MS analyses were performed according to previously described methods.28 As the genome sequencing of S. xylosus C2a is in progress in our laboratory, some spots were identified from the sequences currently available. From this genomic data, S. xylosus C2a proteins were identified by homology using AGMIAL, a genome annotation system developed at INRA.29 Accession numbers for those sequences were recorded in GenBank. Mascot searches were further carried out using the Firmicutes database and deposited sequences of S. xylosus C2a. Database searches were conducted using a mass accuracy of 25 ppm, and carbamidomethyl modification of cysteine residues and oxidation of methionine residues were allowed. Bioinformatic Analyses. Bioinformatic analyses were performed from Web-based servers or under Unix environment from Topaze server homed at MIG (Mathe´matiques Informatique et Geńomes) Research Unit (INRA, Jouy-en-Josas, France). Cleavable signal peptide predictions were performed from (i) SignalP v2.0 and v3.0 trained on Gram-positive bacteria using both neural networks and hidden Markov model with truncation disabled,30,31 (ii) PrediSi trained on Gram-positive bacteria with truncation disabled,32 and (iii) Phobius.33 Tat signal peptide prediction was performed from TatP v1.0.34 Prediction of non-classical secreted protein, that is, lacking a signal peptide, was performed from SecretomeP v2.0 trained on bacteria.35 For the identification of lipoproteins, sequences were scanned by ScanProsite.36 Moreover, these results were combined with those from servers dedicated to the recognition of lipoproteins, that is, DOLOP37 and LipoP v1.0.38 Transmembrane R-helices were predicted combining (i) TMpred,39 (ii) MEMSAT v3.0,40 (iii) HMMTOP v2.0,41 (iv) SOSUI v1.1,42 (v) TopPred v2.0,43 (vi) THUMBUP,44 (vii) UMDHMMTMHP v1.0,44 and (vii) HMMTM.45 To verify annotation of protein sequences and identify domains involved in cell wall attachment,8 Pfam searches were performed with E-value cutoff set at 0.001.46 This analysis was completed by combining results from PSORTb v2.0 trained on Gram-positive bacteria which predicts cellular localization of a protein.47 COG searches were also performed with E-value cutoff set at 0.001.48 COG and KEGG were interrogated to assist protein assignement into functional categories.49

Results and Discussion Cell Envelope Protein Extraction. In Gram-positive bacteria, cell envelope proteins include proteins associated with the cytoplasmic membrane and with the cell wall (Figure 1). To analyze this subproteome, the cell envelope of S. xylosus was fractionated (Figure 2). The first fraction enriched in cell wall proteins was obtained by a classical lysostaphin treatment; this enzyme cleaves specifically the cross bridges of the staphylococcal peptidoglycan and, thus, allows the release of proteins attached to the cell wall.9,15 To recover a second fraction enriched in membrane proteins, a combination of basic techniques described in the literature was first used, namely, separation following ultracentrifugation22,50 and washing with Tris base buffer.51 Subsequently, different delipidation procedures were tested: (i) Triton X100 usually applied to plant cells,20 (ii) the method of Deshusses et al.21 generally used with Gram-negative bacteria, and (iii) the method of Mastro and Hall,17 habitually applied to eukaryotic cells and using TBP lipophilic solvent, which is thought to extract lipids by forming micelles. While it is well-known that 3568

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Triton X100 does not solubilize membrane proteins but breaks the lipids-proteins and lipids-lipids interactions, this method applied to S. xylosus resulted sometimes in smearing on 2-DE gels and no reproducible efficiency of protein extraction (Supplementary Figure 1SA in Supporting Information). Similarly, the method of Deshusses et al. applied with some modifications to S. xylosus failed to yield reproducible gels (Supplementary Figure 1SB in Supporting Information). The method of Mastro et al. was finally chosen for its reproducibility, efficiency, and feasibility for delipidation of membrane proteins in S. xylosus (Supplementary Figure 2S in Supporting Information). It is worth mentioning that, when combining trin-butylphosphate lipophilic solvent with acetone/methanol solution, this method allows the precipitation and desalting of membrane proteins.17 At this stage, three different buffers containing TFE, CHAPS, or ASB14 were tested to solubilize membrane proteins (Supplementary Figure 2S in Supporting Information). It appeared that the latter, that is, the ASB14based buffer, greatly improved resolution and separation of proteins of the fraction II enriched in membrane proteins of S. xylosus as well as solubilization of basic proteins when compared to the two other buffers. Cell Envelope Protein Identification. The fraction I enriched in cell wall proteins and the fraction II enriched in membrane proteins were separated in pH 4-7 (Figure 3A) and nonlinear pH 3-10 (Figure 3B) IPG strips, respectively. The analysis of 2-DE patterns with the Image Master 2-D platinum v5.0 software showed a good reproducibility between the gels of each fraction and allowed to resolve 421 ( 72 and 535 ( 43 spots in fractions I and II, respectively. A total of 168 protein spots, numbered on the 2-DE patterns, was identified in the two fractions by MALDI-TOF MS (Figure 3, Tables 1-3). Numerous proteins were resolved as two or more spots on the 2-DE, which may be caused by post-translational processing. The 168 proteins spots corresponded to 98 proteins, but 8 were common to the two fractions, so finally 90 distinct proteins were identified. As summarized in Figure 1, a large variety of proteins can be found within the cell envelope of Gram-positive bacteria.8 Fraction I enriched in cell wall proteins should contain proteins exhibiting covalent or noncovalent cell wall binding domains and proteins part of macromolecular structure, as well as extracytoplasmic proteins interacting with cell envelope proteins.8 From fraction II enriched in membrane proteins, recovery of membrane proteins is expected as well as peripheral membrane proteins, which include lipoproteins, subunits of membrane-associated complex, and proteins interacting with membrane components, that is, other membrane-associated proteins or proteins associated with lipid bilayer due to electrostatic and/or hydrophobic/steric properties.50 To categorize the 90 proteins identified within the cell envelope of S. xylosus C2a, bioinformatic analyses were carried out. The rationale behind this approach is presented in Figure 4 and summary of predictions are given in Supporting Information (Supplementary Table 1S in Supporting Information). This approach leads to classify proteins of S. xylosus C2a cell envelope in three categories presented in Tables 1, 2, and 3. First, proteins were scanned for the presence of N-terminal signal peptides with SignalP v3.0 and v2.0, using both neural networks and hidden Markov model, as well as PrediSi and Phobius, giving rise to 6 individual predictions. A protein was predicted as secreted if at least 3 tools out of 6 gave a significant positive result. Further in silico analyses failed to identify Tat


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Figure 1. Schematic representation of the different types of cell envelope proteins found in Gram-positive bacteria. Six protein secretion systems allowing protein translocation across the cytoplasmic membrane are currently recognized in these bacteria: (i) the Sec (Secretion) pathway, (ii) the Tat (Twin arginine translocation), (iii) the FPE (Fimbrilin-protein exporter, (iv) the Wss [WXG100 (proteins with WXG motif of ∼100 residues) secretion system), (v) the holins (hole-formers), and (vi) the FEA (Flagella export apparatus).52,55 Proteins secreted via the three first systems usually bear N-terminal signal peptide. Protein secretion can also occur via unknown secretion system.35 Once translocated, a cell envelope protein is either a membrane-associated protein, a cell-wall associated protein, or part of macromolecular protein structure.8 Membrane associated proteins include IMP (integral membrane protein), lipoprotein, and peripheral proteins (not indicated on the schema). Cell-wall associated proteins include (i) proteins covalently linked and bearing LPXTG motif, and (ii) protein bearing cell wall binding domain allowing noncovalent binding, i.e., CWD1 (Cell-wall binding domain of Type 1), CWBD2 (Cell-wall binding domain of Type 2), LysM (Lysin motif), GW module or SLHD (S-layer homology domain). Proteins can also remain anchored via uncharacterized domains or interactions. Macromolecular protein surface structure include S-layer, pilus (involving sortases), flagellum (involving the FEA), and cellulosome (not indicated on the schema).8 In addition, trans-cell-wall structure forms by fimbrilin assembly via the FPE can be found within the cell wall.

(Twin-arginine translocation) signal peptide or prepilin using TatP and Pfam, respectively. Eleven proteins displaying an N-terminal signal peptide are most certainly secreted via Sec translocon (Table 2). Remaining proteins were screened with SecretomeP and from literature survey in order to identify those lacking a signal peptide and secreted via FEA (Flagellin Export Apparatus), holins, Wss [WXG100 (proteins with WXG motif of ∼100 residues) secretion system], or unknown secretion system (Figure 1). Twenty proteins were predicted as secreted by unknown secretion system (Table 2). Proteins predicted as Secdependent were further scanned for the presence of lipobox using DOLOP, LipoP, and ScanProsite with both PS51257 profile and G+LPP pattern46 giving rise to 4 individual predictions. A protein was predicted as lipoprotein if at least 2 out of 4 gave a significant positive result, that is, 3 proteins (Table 1). Additional proteins could not be identified following Pfam searches for lipoprotein-associated domains. Except for predicted lipoproteins, all proteins were further analyzed with Pfam in order to identify cell-wall binding domain. Interestingly, a fibronectin binding protein A present

in fraction I enriched in cell wall proteins, exhibited a Secdependent signal peptide with a YSIRK motif (PF04650; E-value ) 1.8 × 10-8) and a C-terminal LPXTG motif (PF00746; E-value ) 8.0 × 10-9). In S. aureus, YSIRK motif is required for efficient signal peptide processing.52 This motif is systematically associated with a LPXTG motif; even so, the opposite is not true. Furthermore, while LPXTG motif allows covalent anchoring of a protein to peptidoglycan via sortase,53,54 YSIRK motif is not essential for cell wall protein anchoring.52 All proteins were also analyzed for the presence of transmembrane R-helices using TMpred, MEMSAT, HMMTOP, SOSUI, TopPred, THUMBUP, UMDHMMTMHP, and HMMTM. A protein was predicted as possessing transmembrane domain (TMD) if at least 4 tools out of 8 gave a significant positive result. Twenty-four proteins displayed between 1 and 6 R-helical TMDs and were thus predicted as integral membrane proteins (Table 1). It is worth stressing that the presence of an N-terminal signal peptide and position of TMD were carefully taken into consideration for prediction of membrane proteins and individually checked.45 In fact, signal peptides always Journal of Proteome Research • Vol. 6, No. 9, 2007 3569

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Figure 2. Diagram of subproteome fractionation procedure applied to S. xylosus C2a. TFE/CHCl3, trifluoroethanol/chloroform; CHAPS, 3-[3-chloamidopropyl dimethylammonio]-1-propanesulfonate; ASB14, amidosulfobetaine.

possess a hydrophobic H-domain; nevertheless, those proteins are not systematically membrane proteins. For example, in the fibronectin binding protein A predicted as translocated via Sec and covalently bound to cell-wall, between 1 and 2 TMDs are predicted (Table 2). However, the first TMD corresponds to the H-domain of the cleavable N-terminal signal peptide, and the second TMD to the hydrophobic domain of the C-terminal LPXTG motif; this protein was, thus, considered as secreted. In contrast, Type I signal peptidase found in fraction II enriched in membrane proteins is predicted with a cleavable signal peptide but is most certainly uncleaved and remains anchored to the membrane via its hydrophobic signal peptide Hdomain.51 Another case is the elastin binding protein identified in fraction II enriched in membrane proteins and exhibiting a LysM domain (PF01476; E-value ) 1.9 × 10-6); however, a signal peptide could not be identified, and between 1 and 4 transmembrane domains were found away from the N-terminal region leading to the final prediction of this protein as membranal (Table 1). All remaining proteins were further submitted to PSORTb. Thirty-three proteins were only predicted as cytoplasmic (Supplementary Table 1S in Supporting Information). Nineteen primarily predicted as cytoplasmic were also predicted as extracytoplasmic, since they were secreted via unknown secretion system (Table 2). Together with proteins predicted as secreted via Sec, 25 proteins were predicted as extracytoplasmic (Table 2). Five proteins were predicted both in the cytoplasm and membrane (Supplementary Table 1S in Supporting Information). A closer look revealed those proteins were systematically ATPase component of ABC (ATP Binding Cassette) transporter and were, thus, part of membrane protein complex (Table 1). Considering this investigation concerns the cell envelope proteome of a Gram-positive bacterium, that is, S. xylosus C2a, three main categories of proteins finally emerge from this bioinformatic analysis: membrane associated proteins (Table 1), secreted proteins (Table 2), and cytoplasmic proteins (Table 3). The plausibility of all assignment was finally checked by a comparison with the functional annotations available and the results of Pfam and COG searches. Ninety-one percent (31/ 3570


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34) of proteins predicted as membrane associated were, as expected, present within fraction II enriched in membrane proteins (Table 1). Seventy-two percent (18/25) of proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins (Table 2). Eighty-one percent (25/ 31) of proteins predicted as cytoplasmic were found in fraction II (Table 3). (i) Membrane Associated Proteins. This category gathered proteins predicted as membrane integrated proteins, lipoproteins, and components of membrane protein complex (Table 1). As expected, a large majority of these 34 proteins were present in the fraction II enriched in membrane proteins, that is, 31 proteins including 3 present in both fractions I and II. A majority of membrane integrated proteins had no putative signal peptides but were all found in fraction II enriched in membrane proteins (Table 1.1 and Supplementary Table 1S in Supporting Information). In bacteria, all cytoplasmic membrane proteins are inserted in Sec-facultative but YidC-dependent mechanism;8 in Gram-positive bacteria, two orthologues of YidC are found, that is, SpoIIIJ and its paralogue YqjG.55 Among the 24 membrane integrated proteins here identified in S. xylosus, about half of them are involved in cell envelope biogenesis (Table 1.1). In fact, acetyl-CoA carboxylase R-subunit (spot 72) is responsible for fatty acid biosynthesis.56 Four enzymes are involved in glycero-phospholipid metabolism (spots 8-10, 82, 83, 96, and 122) such as the glycerol-3phosphate dehydrogenase which is also membrane-associated in S. aureus.57,58 Three proteins are directly involved in cell wall biogenesis and in peptidoglycan biosynthesis (spots 17, 18, 33, 34, and 95) for example, UDP-N acetylglucosamine transferases.59 Interestingly, some membrane proteins involved in carbohydrate metabolism could also be involved in cell wall biogenesis such as glucose-6-phosphate isomerase (spots 16 and 17) as shown in Mycobacterium smegmatis,60 or lactate dehydrogenase (spots 59, 60-76) considering L-lactate could be a cell wall component as observed in Lactobacillus plantarum.61 Among membrane proteins identified as involved in carbohydrate metabolism, several were related to (i) glycolysis with pyruvate dehydrogenase E1 component complex (spots 109 and 110) also isolated from membrane fraction in S. aureus,62,63 (ii) tricarboxylic acid (TCA) cycle and respiratory chain with succinate dehydrogenase (spot 90) also described as a membrane-bound dehydrogenase in S. aureus and B. subtilis,64,65 or lactate dehydrogenase as shown in Corynebacterium glutamicum.66 Three membrane associated proteins were predicted as lipoproteins and were all identified within membrane fraction II (Table 1.2). Lipoproteins are characterized by a specific signal peptide of class 2 exhibiting a conserved lipobox sequence;8 following translocation across the cytoplasmic membrane, such proteins are subjected to several post-translational modifications leading to protein anchoring to membrane long chain fatty acid. The present investigation allowed the identification of a peptidyl-prolyl isomerase (spot 27) which would play a major role in protein secretion by helping the post-translocational extracellular folding of several secreted proteins.67 The two other lipoproteins are periplasmic substrate-binding subunits of ABC transporters; one is predicted as involved in metal transport (spots 46, 48, and 73) and the second in amino acid transport (spots 141 and 142). Seven proteins were predicted as integral components of membrane protein complex and 6 were identified within fraction II (Table 1.3). These proteins included 5 ATPase


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Figure 3. (A) 2-DE pattern of S. xylosus C2a fraction I enriched in cell wall proteins. The extracts were harvested after lysostaphin treatment of cells grown under shaking until late stationary phase. Protein extracts (200 µg) were separated by IEF over a linear pH gradient of 4-7 followed by a 10% SDS-PAGE. (B) 2-DE patterns of S. xylosus C2a fraction II enriched in membrane proteins after delipidation with a mixture of tributylphosphate, acetone, and methanol and solubilization in buffer containing ASB14. Protein extracts (600 µg) were separated by IEF over a nonlinear pH gradient of 3-10 followed by a 10% SDS-PAGE. For the two fractions, proteins were revealed with silver staining, and the spot numbers are listed in Tables 1-3. Journal of Proteome Research • Vol. 6, No. 9, 2007 3571

3572

Aerobic glycerol-3-phosphate dehydrogenase Glycerol-3-phosphate dehydrogenase

Inositol monophosphatase family protein

Phosphoglycerol transferase and related proteins, alkaline phosphatase superfamily Transketolase

8 9 10 122

82 83

96


Quinone/NADPdependent oxidoreductase

Type I signal peptidase

ATP-dependent Clp

105 106 107-108 113 115-116

65

91-92

90

94958238

94958268

94958282

94958280

57866658

EF456680*

70726860

Pyruvate dehydrogenase E1 component

Dihydrolipoamide transferase E2 component of 2-oxoglutarate dehydrogenase complex Succinate dehydrogenase flavoprotein subunit Malate quinone oxidoreductase

94958234

dehydrogenase

L-lactate

59 60 76 135-138 137-136 109-110

86-87 88

73663122

Glucose-6-phosphate isomerase A

73662725

94958260

gb

gb

gb

gb

ref

gb

ref

gb

ref

ref

gb

gb

gb

Ef456679*

94958276

ref

gb

73662768

EF456673*

gb

ref

73662630

EF456678*

gb

DBc

94958258

GIb

16-17

5-6

72

95

Penicillin binding protein 4 UDP-N-acetylglucosamine N-acetylmuramylpentapeptide pyrophosphorylundecaprenol transferase UDP-N-acetylglucosamine 1-carboxyvinyl transferase Acetyl-CoA carboxylase a-subunit

protein annotation

17 18 33-34

spot id

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. epidermidis RP62A S. xylosus C2a

S. xylosus C2a

S. hemeolyticus JCSC1435

S. xylosus C2a

S. saprophyticus ATCC 15305 S. saprophyticus ATCC 15305

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. saprophyticus ATCC 15305

S. xylosus C2a

S. xylosus C2a


S. xylosus C2a

Staphylococcus strain pI

MW (kDa)

62.260

35.129

6.05

5.94

4.98

6.32

5.59

36.759

55.881

65.631

45.428

41.528

4.98 4.78

34.781

49.796

4.84 5.00

72.183

74.827

4.95

6.50

30.146

35.345

5.05

5.46

44.996

5.62

(1) Membrane Integrated Proteins Folding, Sorting and 5.40 22.373 Degradation Folding, Sorting and 4.98 21.225

Respiratory chain

Carbohydrate metabolism, TCA cycle, Respiratory chain Carbohydrate metabolism, CA cycle, Respiratory chain

Carbohydrate metabolism, Glycolysis Amino acid metabolism Carbohydrate metabolism, TCA cycle

Carbohydrate metabolism, Glycolysis, Cell wall biogenesis Carbohydrate metabolism, Glycolysis Amino acid metabolism, Respiratory chain, Cell wall biogenesis

Cell wall biogenesis, Peptidoglycan biosynthesis Lipid metabolism, Fatty acid metabolism, Carbohydrate metabolism Membrane biogenesis, Lipid metabolism, Glycero-phospholipid metabolism Membrane biogenesis, Lipid metabolism, Glycero-phospholipid metabolism Membrane biogenesis, Lipid metabolism, Glycero-phospholipid metabolism Membrane biogenesis, Lipid metabolism, Glycero-phospholipid metabolism Carbohydrate metabolism

(1) Membrane Integrated Proteins Cell wall biogenesis, 7.76 36.636 Peptidoglycan biosynthesis Cell wall biogenesis, 6.63 40.239 Peptidoglycan biosynthesis

functional categoryd

theoriticale

Table 1. Proteins Identified by MALDI TOF Mass Spectrometry and Predicted as Membrane Associateda

5.30-5.10

5.10

6.05 6.10 6.20-6.3 4.50 4.65-4.8

5.20

3.45-3.50 3.55

4.25-4.45

5.00 5.20

4.80-4.90

4.90-4.95

5.20

5.30 5.45

5.50

5.05 6.15-6.20

4.50

5.15

7.45 7.85 7.60-8.10

pI

20.7-20.7

21

55.4 55.2 55.4-55.3 40.2 38-4-38.4

60.1

55.3-55.3 64.8

45.4-45.3

34.0 33.8

48.4-48.4

70.9-70.9

49.4

29.7 29.6

34.9

64.2 63.8-64.4

35.9

52.4

46.8 46.7 40.3-40.0

MW (kDa)

experimental

21-21

52

54 59 54-54 28 62-62

34

35-34 34

17 35 54 39-37 44-44 27

42

29

34

45 55

31 28 26 39

45

53

21 42 31

sequence coveragef

35*-32*

109*

206* 280* 265*-252* 55* 148*-175*

161

126*-81* 81*

36* 65* 152* 93-140* 139-154* 74 70

96 106 100-107

116*

70* 111*

114 132 117 94*

130*

207*

51* 101* 93-94

Mascot scoreg

5/56-4/37

12/47

22/50 34/62 27/44-27/44 9/68 17/60-17/43

24/64

16/45-12/57 12/57

5/69 9/58 16/36 13/54-13/24 15/33-14/26 9/32 9/35

16/64 16/64 15/77-15/69

22/92

8/46 10/33

22/76 19/45 17/46 13/51

17/48

22/39

5/43 12/41 12/44-12/43

peptides match

1-2

1

1-2

1-2

1-4

1-2

1-2

1-2

1-3

2-4

4-6

1

2-3

1

1-2

1-4

1-2

1

TMDh

I

II

II

II

II

II

II

II

I

I

I

II

II

II

II

II

II

II

II

CEFi

research articles Planchon et al.

protease proteolytic subunit Phosphate starvationinducible protein, PhoH Elastin binding protein Methyl-accepting chemotaxis-like domain protein Pantothenate metabolism flavoprotein

Oxidoreductases related to aryl-alcohol dehydrogenases Conserved staphylococcal protein

Foldase protein PrsA precursor ABC-type metal ion

transporter system, extracellular binding protein ABC-type amino acid

61 62-63 26 127 1 59-60 151

119

27

48 73

ABC-type transport system involved in Fe-S cluster assembly, ATPase component ABC-type metal ion transport system, ATP binding component ABC-type multidrug transport system, ATPase component ABC-type antimicrobial peptide transport system, ATPase component ABC transport system, ATPase component F0F1-type ATP synthase b-subunit

153

94958278

94958266

94958264

EF456685*

94958236

73661551

EF456684*

EF456683*

94958288

94958270

gb

gb

gb

gb

gb

ref

gb

gb

gb

gb

gb

gb gb

94958284 94958274

EF456682*

gb

DBc

EF456681*

GIb

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a


S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a S. xylosus C2a

S. xylosus C2a

Staphylococcus strain

6.25

4.94

6.24

4.55 6.41

6.33

pI

9.14

29.519

34.929

37.427

33.258

35.332

44.440

57.724 18.132

34.819

MW (kDa)

Membrane transport, Defense mechanism

Membrane transport, Defense mechanism

Membrane transport, Metal transport

6.39

6.19

6.30

28.002

33.993

27.239

(3) Membrane Complex Components Metabolism of cofactors, Fe-S 4.73 28.242 cluster biogenesis

Membrane transport, Amino acid transport

(2) Lipoproteins Folding, Sorting and 8.58 Degradation Membrane transport, Metal 5.41 transport

Unknown no COG

Biosynthesis of polyketides and nonribosomal peptides, Metabolism of cofactors, Pantothenate and CoA biosynthesis Xenobiotics biodegradation and Metabolism

Signal transduction Phospholipids metabolism Cellular adhesion Bacterial chemotaxis

Degradation


theoriticale

6.45

6.25

6.90

3.90

8.65

8.40

5.55 5.10

5.55

6.20

6.05

4.55

6.00 6.10

4.50 4.70-4.95 6.15 6.35 3.35 5.85-5.85 6.10

pI

28.3

36.7

28.1

28.3

29.2

29.4

33.8 36

35.1

37.3

30.8

35.5

46.1 46.2

21.5 21.52.6 34.7 34.8 97.7 18.8-19 18.9

MW (kDa)

experimental

25

55

34

83

28

28

65 46

22

38

41

59

53 56

38 21-21 41 30 29 48-48 48

sequence coveragef

38*

115*

78*

153*

69

68

179* 54*

38*

98*

77*

144*

163* 188*

66* 53*-51* 92* 57* 101* 76*-68* 48*

Mascot scoreg

5/119

17/69

8/35

19/53

11/45

10/47

18/49 11/103

5/30

12/35

9/37

16/65

21/76 20/58

7/31 6/25-6/27 10/32 8/40 13/46 8/26-6/32 5/42

peptides match

0

0

0

0

1

1

1

1-2

1

1-2

1-4 1-2

1

TMDh

II

II

II

II

II

II

I

II

II

II

II

II II

II

II

CEFi

Membrane transport, Defense 7.91 24.831 7.85 24 37 70* 7/29 0 II mechanism EF456686* gb S. xylosus C2a Energy metabolism, Oxidative 4.65 51.220 4.60 48.2 32 79* 10/40 0 I 15 phosphorylation, ATP synthesis 25 F0F1 ATP EF456687* gb S. xylosus C2a Energy metabolism, Oxidative 6.20 31.878 6.15 31.9 44 133* 13/29 0 II 130 synthaseg-subunit phosphorylation, ATP 6.00 31.9 27 47* 8/41 synthesis a Subcellular localization was predicted using various bioinformatic tools as described in the text. b GenInfo Identifier (GI). Asterisk indicates GenBank temporary assignement number for S. xylosus C2a proteins. c Database (DB), i.e., gb (GenBank), ref (RefSeq), or emb (EMBL). d According to COG and KEGG. e Theoritical molecular weight (MW) is calculated using average mass values of amino acids and theoritical pI using pK values of amino acids from Bjellqvist reference table. f Expressed as percentage. g Mascot scores were obtained against Firmicutes database where scores greater than or equal to 68 are significant (p < 0.05). Asterisk indicates protein identification against S. xylosus C2a partial genomic database where scores greater than or equal to 32 are significant (p < 0.05). h Number of transmembrane domain (TMD) from predictions involving TMpred, MEMSAT, HMMTOP, SOSUI, TopPred, THUMBUP, UMDHMMTMHP, and HMMTM. i Cell envelope fraction (CEF) obtained as described in the Material and Methods, where I is enriched in cell wall proteins and II is enriched in membrane proteins.

139

43

20

45

transport system, extracellular componen

142

141

46

134

13 14

protein annotation

spot id

Table 1. (Continued)


research articles

Journal of Proteome Research • Vol. 6, No. 9, 2007 3573

3574


73663225

94958242

EF456692*

DegV family protein

R-D-1.4-glucosidase

Glyceraldehyde-3phosphate dehydrogenase

Fructose-bisphosphate aldolase class I Enolase

132

12

34-35 36-37 38-39 117 52 118 19

Transcription antitermination factor Metallo-β-lactamase family protein

94958250

gb

gb

emb

410516

EF456669*

gb emb

gb

ref

emb

gb

gb

ref

93463978 410515

EF456699*

94958246

27468547

gb

gb

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a S. xylosus pUra41 S. xylosus pUra41

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a


S. xylosus C2a

S. xylosus C2a

S. epidermidis ATCC 12228 S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus DSM 20266

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

Staphylococcus strain pI

MW (kDa)

5.58

5.76

5.33

5.94

34.765

31.011

40.535

25.379

Defense mechanism

Transcription

Carbohydrate metabolism, Glycolysis Carbohydrate metabolism, Glycolysis, Amino acid metabolism Carbohydrate metabolism, Amino acid metabolism Amino acid metabolism Nucleotide metabolism, Amino acid metabolism Nucleotide metabolism, Amino acid metabolism

Stress response, Alkaline stress Protein folding, sorting and degradation Protein folding, sorting and degradation Protein folding, sorting and degradation Protein folding, sorting and degradation Lipid metabolism, Fatty acid metabolism Carbohydrate metabolism Carbohydrate metabolism, Glycolysis

4.96

5.18

5.33

4.82 5.29

4.69

4.55

4.74

4.89

4.63

5.75

5.23

4.59

4.83

4.50

4.82

23.112

20.623

61.813

32.860 15.432

35.187

47.135

33.145

36.203

63957

30.703

54.171

57.749

10.332

66.507

18.520

(2) Secreted via Unknown Secretion System Stress response, 4.51 21.043 Oxidative stress Stress response, 4.88 22.535 Oxidative stress Stress response, 5.43 57.127 Oxidative stress

Transcription, Cell wall biogenesis

Cell wall biogenesis

Surface antigen, Virulence factor Membrane transport, Defense mechanism

(1) Secreted via Sec Translocon Cellular adhesion, 4.58 75.890 Virulence factor Surface antigen, 4.65 21.641 Virulence factor


theoriticale

5.15

4.80 5.55 5.30 5.05-5.15 5.25 5.35 5.40

4.70

4.70 3.95 4.60

4.90-5.05 4.95-4.90 4.90-4.85

3.55

5.95

5.50

3.45

4.60

4.85-4.90 4.85-4.95 5.30 5.40 5.40 4.80 4.85 3.45

4.60

4.90

5.60 5.85-6.15

4.40-4.50 4.40 4.45 5.55 5.30 4.95-5.05

4.35

pI

25.7

34.9 18.9 20.5 62.1-62.1 61.9 61.9 21.5

33.8

35.6 35.3 43.3

38.9-38.9 38.9-38.0 36.4-6.3

56.2

31.6

48.9

58

7.6

24.3-24.2 22.9-22.5 56.8 56.7 55.1 8.8 18.9 66.2

21.1

30.1

29.7 29.7-29.6

19.6-9.6 18.7 18.6 30.9 30.9 41.4-41.3

75.6

MW (kDa)

experimental

30

39 63 70 39-35 51 49 65

39

34-43 38-32 34-36 26 26 50 22

42

34

17

25

75

22-21 35-76 43 53 52 34 59 20

47

45 41 51 15

37-37 37 23 21 26 16-21

33

sequence coveragef

47*

91* 101 113 167-163 208 179 63*

45*

82-101 81-90 79-91 74 52* 120* 40*

134*

70*

54*

90

71*

37*-32* 55*-139* 188 234 202 51* 98* 68*

46*

67* 58* 66* 44*

51*-55* 63* 43* 53* 52* 42*-38*

112*

Mascot scoreg

5/32

11/54 9/48 10/37 18/38-19/44 27/57 21/48 7/51

9/90

9/37-11/44 10/51-12/58 10/41-10/58 10/44 7/44 15/53 7/51

17/50

8/40

5/31

13/51

7/46

3/38-8/87 6/41-1/35 19/42 25/51 22/46 5/30 10/46 9/33

6/65

10/82 8/60 12/103 6/36

6/50-6/42 6/31 5/38 6/25 9/38 5/23-6/48

17/64

peptides match

0

0

0

0 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

1-2

TMDh

I

II

I I II II

I

II II I II I

I

II

II

I

II

I

II

I

II

I

I

I

I

I II I

I

I

CEFi

a Subcellular localization was predicted using various bioinformatic tools as described in the text. b GenInfo Identifier (GI). Asterisk indicates GenBank temporary assignement number for S. xylosus C2a proteins. c Database (DB), i.e., gb (GenBank), ref (RefSeq) or emb (EMBL). d According to COG and KEGG. e Theoritical molecular weight (MW) is calculated using average mass values of amino acids and theoritical pI using pK values of amino acids from Bjellqvist reference table. f Expressed as percentage. g Mascot scores were obtained against Firmicutes database where scores greater than or equal to 68 are significant (p < 0.05). Asterisk indicates protein identification against S. xylosus C2a partial genomic database where scores g32 are significant (p < 0.05). h Number of transmembrane domain (TMD) from predictions involving TMpred, MEMSAT, HMMTOP, SOSUI, TopPred, THUMBUP, UMDHMMTMHP, and HMMTM. i Cell envelope fraction (CEF) obtained as described in the Material and Methods, where I is enriched in cell wall proteins and II is enriched in membrane proteins.

81

Arginase Urease β-subunit urea amidohydrolase Urease R-subunit

474177

Cytosol aminopeptidase

13

54 100 66 4-5 6 7 67

gb

94958286

Chaperonin GroEL

11

Phospho-transcetylase

gb

94958272

Co-chaperonin GroES

106

57

gb

EF456689*

Alkaline shock protein 23 Chaperone protein DnaK

EF456690*

gb

68137816

Catalase B

emb

gb

gb

8977980

94958240

94958248

gb

gb

94958230

EF456688*

gb

gb

gb

DBc

94958254

94958256

94958252

GIb

84-85 86-87 91 92 93 98 99 89

Immunodominant antigen A RND family efflux transporter, MFP component Cell-shape determining protein MreC Cell envelope-related transcriptional attenuator LytR

Fibronectin-binding protein A Immunodominant antigen B

protein annotation

Alkyl hydroperoxide reductase subunit C Superoxide dismutase

93

68

70 72-73

94-95 96 97 51 23 23-24

4

spot id

Table 2. Proteins Identified by MALDI TOF Mass Spectrometry and Predicted as Secreteda


100

89 90 50 29 30 31 32 8-9

75 76 77 81

79-80

103

2

150

22 29 121 123 51 145 146 143 144 47-48

78

77

spot id

DAHP synthetasechorismate mutase

Alanine dehydrogenase UDP-N-acetylmuramyl tripeptide synthase Dihydrolipoamide dehydrogenase

Nucleosidediphosphate-sugar epimerase Glucosamine-6phosphate isomerase

Glucose-1dehydrogenase Transaldolase

Xylose isomerase

ref

73661973

gb

EF456696*

gb

gb

EF456677*

EF456697*

gb

gb

gb

gb

emb

emb

gb

EF456693*

94958244

94958262

EF456695*

2226002

48835

EF456691*

ref

ref

73663526

ref

50S ribosomal protein L1 50S ribosomal protein L4 50S ribosomal protein L5 Trigger factor 73661984

DBc

ref ATCC 15305

ref

ref

73662353

73662821

70725018

73662569

GIb

30S ribosomal protein S4

Signal receiver domain SrrA of SrrAB system (staphylococcal respiratory response protein) Response regulator VicR of VicRK system, homologue to YycFG 30S ribosomal protein S2

protein annotation

functional categoryd pI

MW (kDa)

theoriticale

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. saprophyticus ATCC 15305 S. saprophyticus ATCC 15305 S. saprophyticus ATCC 15305 S. xylosus C2a


S. saprophyticus

S. hemeolyticus JCSC1435

Carbohydrate metabolism,cell envelope biogenesis Carbohydrate metabolism, Cell wall biogenesis Amino acid metabolism, Cell wall biogenesis Cell wall biogenesis, Peptidoglycan biosynthesis Carbohydrate metabolism, TCA cycle, Amino acid metabolism Amino acid metabolism

Protein folding, sorting and degradation (2) Other Proteins Carbohydrate metabolism Carbohydrate metabolism Carbohydrate metabolism

Translation

Translation

Translation

Translation

Translation

Signal transduction, Two-component system

5.53

4.95

7.64

5.02

5.22

5.21

4.51

4.92

4.99

4.23

8.73

10.16

9.14

9.69

5.52

5.05

40.611

49.451

49.148

39.903

22.398

23.694

25.548

28.599

50140

48.879

20215

22427

24965

23144

29729

27149

(1) Proteins Interacting with Membrane Components S. saprophyticus Signal transduction, 5.14 28057 ATCC 15305 Two-component system


Table 3. Proteins Identified by MALDI TOF Mass Spectrometry and Predicted as Cytoplasmica

5.70

5.20 5.35 5.10 5.35 5.45 6.30 6.50 4.85-4.75

4.55 4.60 4.65 5.15

4.95-5.05

6.07

3.3

8.45

5.90 6.35 5.40 5.60 5.40 5.60 8.80 8.85 9.00 5.15-5.40

4.80

5.15

pI

45.6

22.6 22.2 22.8 39.1 39 50 49.7 50.6-50.6

28.8 26.2 26.3 29.8

28.4-28.3

48.4

66.4

21.9

29.1 29.3 24.7-24.4

35.8 41.8 36.1 36 25.2 27.2

30.5

32.6

MW (kDa)

experimental

33

30 46 35 16 33 37 38 27 28

22 30 35 74 43 38

18

43

27 35 27 33 45 46 51 36 32 42 40 56

54

55

sequence coveragef

90*

51* 98* 53* 40* 88* 127* 80* 46* 77*

46* 32* 60* 154* 121* 57*

13/52

6/43 9/36 6/42 4/36 9/30 13/31 15/76 9/73 9/34

5/27 6/84 7/45 17/61 11/36 6/38

5/27

18/64

111*

49*

9/22 12/50 9/23 12/26 8/54 9/53 11/66 9/23 8/18 8/32 7/28 16/29

13/69

13/36

peptides match

77 76 75 111 69 80 82 103 99 88 81 136

85

151

Mascot scoreg

II

I

II

II I

I

II

I

I

II

II

II

II

II

II

II

II

II

CEFh


research articles


3576 EF456698* EF456668*

RecA protein

ATPase AAA family and related to the helicase subunit of the Holliday junction resolvase Transcription termination factor Transcriptional regulator Transcription pleiotropic repressor CodY ATP-dependent RNA helicase General stress protein 20U Pyridoxine biosynthesis protein Allophanate hydrolase subunit 2 SpoU rRNA methylase family protein HD superfamily hydrolase GTP-binding protein Era

Journal of Proteome Research • Vol. 6, No. 9, 2007 ref

73663269 gb

gb

EF456675*

EF456676*

gb

ref

gb

ref

ref

gb

ref

EF456674*

73663546

EF456671*

70725957

73662822

EF456670*

27468637

gb

gb

gb

gb

DBc

S. saprophyticus ATCC 15305 S. xylosus C2a

S. xylosus C2a

S. saprophyticus ATCC 15305 S. xylosus C2a

S. hemeolyticus JCSC1435 S. xylosus C2a


S. epidermidis ATCC 12228 S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a

S. xylosus C2a


Unknown (COG1159)

Stress response, Oxidative stress Stress response, Oxidative stress Xenobiotics biodegradation Defense mechanism, Translation Unknown (COG1896)

Transcription

Transcription

Transcription

Transcription

(2) Other Proteins Lipid metabolism, Fatty acid metabolism Nucleotide metabolism, Metabolism of cofactors and vitamins DNA replication, recombination, and repair DNA replication, recombination, and repair


6,78

5,03

6.31

6,44

5.13

4.60

9.45

5.44

5.77

7.77

7.21

5.44

4.75

5.70

pI

34.506

25.054

27.064

37.913

31.907

16.981

57.029

28.525

27.765

49.793

47.399

37.807

26.140

28.076

MW (kDa)

theoriticale

6.75-6.95

7.80 8.15 6.65 7.15 4.91

5.00

4.85

5.35

5.95-6.05

6.15

8.75

6.40 6.65

5.40-5.55

4.85

5.80

pI

34-33.7

38.9 38.8 28.2 28 25.1

34.9

16.1

52.2

28.6-28.7

28.2

51.5

46.4 46.3

45.2-45.8

27.4

31.6

MW (kDa)

experimental

38 54

16 43 55 32 48

30

61

26

42 29

47

30

50 26 48 72

27

29

sequence coveragef

75* 129*

35* 108* 100* 43* 95

69

67*

71

102 77

112*

104

170* 38* 148* 302*

41*

80*

Mascot scoreg

12/53 19/56

4/20 11/32 13/43 8/52 10/39

10/43

8/75

14/70

11/41 9/32

10/33

15/51

20/43 8/67 15/36 26/41

5/36

9/31

peptides match

II

II

II

II

II

I

II

II

II

II

II

II

I

II

CEFh

c

a Subcellular localization was predicted using various bioinformatic tools as described in the text. b GenInfo Identifier (GI). Asterisk indicates GenBank temporary assignement number for S. xylosus C2a proteins. Database (DB), i.e., gb (GenBank), ref (RefSeq) or emb (EMBL). d According to COG and KEGG. e Theoritical molecular weight (MW) is calculated using average mass values of amino acids and theoritical pI using pK values of amino acids from Bjellqvist reference table. f Expressed as percentage. g Mascot scores were obtained against Firmicutes database where scores greater than or equal to 68 are significant (p < 0.05). Asterisk indicates protein identification against S. xylosus C2a partial genomic database where scores greater than or equal to 32 are significant (p < 0.05). h Cell envelope fraction (CEF) obtained as described in the Material and Methods, where I is enriched in cell wall proteins and II is enriched in membrane proteins.

37 38

35 36 44 46 155

74

104

94

84-85

57

19

EF456694*

98 99 15 16

78

EF456672*

GIb

Trans-2-enoyl-ACP reductase Purine nucleoside phosphorylase

protein annotation

131

spot id

Table 3. (Continued)



research articles

Figure 4. Schematic representation of the rationale behind the bioinformatic analyses carried out in order to categorize the 90 proteins identified within the cell envelope of S. xylosus C2a. Shaded boxes, membranes associated proteins; horizontally striped box, secreted proteins; vertically striped box, cytoplasmic proteins.

components which are predicted as associated to transmembrane permease of ABC (ATP Binding Cassette) transporter68,69 involved either in metal (spots 45 and 153), multidrug (spot 20), or antimicrobial peptide transport responsible for bacterial resistance (spot 43). β- and γ-F0-F1 ATP synthase subunits were also identified (spots 15 and 25-130, respectively). These subunits are integral parts of F0-F1 ATP synthase and are essential for normal assembly and function of this large membrane-bound protein complex involved in oxidative phosphorylation by chemioosmotism.70 (ii) Secreted Proteins. In Gram-positive bacteria, secreted proteins correspond to extracytoplasmic proteins that are entirely outside the outermost lipid bilayer, that is, the cytoplasmic membrane and, thus, include released and surfaceassociated proteins.71 Secreted proteins here regroup (i) proteins predicted with N-terminal cleavable signal peptides and secreted via the Sec pathway30,31 (Table 2.1), and (ii) proteins translocated via unknown secretion systems35 (Table 2.2). Altogether, 25 proteins were predicted as secreted and, thus, expected in the cell-envelope of S. xylosus C2a; the large majority was found in fraction I enriched in cell wall proteins, that is, 18 including 4 present in both fractions I and II. Six proteins were predicted as secreted in a Sec-dependent manner and were systematically present in fraction I enriched in cell wall proteins (Table 2.1). Together with membrane bound elastin exhibiting a LysM domain (Table 1.1 and Supplementary Table 1S in Supporting Information), fibronectin binding protein A would be involved in bacterial adhesion to extracellular matrix of mammalian cells.72 Despite the absence of cell wall binding motif,8 some proteins have been described as associated to the bacterial cell surface like the immunodominant antigens A and B (IsaA, IsaB) in S. aureus73,74 or the cell shape determining protein (MreC) involved in murein synthesis, that is, in peptidoglycan synthesis75 (Table 2.1). It is worth stressing that 3 secretion systems are currently known to permit translocation of proteins lacking a signal peptide in Gram-positive bacteria, namely, the FEA (Flagellin

Export Apparatus), the holins, and the Wss [WXG100 (proteins with WXG motif of ∼100 residues) secretion system],52 (Figure 1); still, no proteins secreted via these systems could be identified here. Even though 19 proteins primarily predicted as cytoplasmic were also predicted as secreted via unknown secretion system as revealed by SecretomeP and literature surveys35 (Table 2.2), 12 were found in fraction I enriched in cell wall proteins, including 3 found in both fractions I and II. For example, superoxide dismutase here found in fraction I is primarily described as a cytoplasmic protein involved in oxidative stress,76 but it has been reported to be also cell-surface displayed in S. aureus77 and secreted in a SecA2-dependent manner in Mycobacterium tuberculosis.78 While SecA2 is a paralogue of SecA, involvement of the Sec translocon in SecA2dependent secretion has not been as yet addressed in the literature. While cytoplasmic chaperones such as DnaK, GroEL, or GroES, here found in fractions I or II (Table 2.2), are known to participate in bacterial protein secretion and consequently interact with Sec system,79 they have also been reported to be on cell surface of several bacteria.80 Interestingly, some cytoplasmic proteins primarily involved in carbohydrate metabolism such as glyceraldehyde-3phosphate dehydrogenase or enolase, both found in fraction I enriched in cell wall protein (Table 2.2), are actually moonlighting outside the cell in Streptococcus pneumoniae as they exhibit plasmin(ogen)-binding activity.81,82 Furthermore, despite the absence of a signal sequence and typical motifs required for cell-surface display, the attachment of these glycolytic enzymes to bacterial cell surfaces was observed in streptococci,83 listeria11 or staphylococci.15 For most other proteins found in fraction I, such as arginase,85 their potential extracellular roles remain unknown. As already pointed out for Type 1 signal peptidase, it cannot be ruled out that some of these proteins remained anchored within the membrane via mispredicted and/or uncleaved signal peptide. Similarly, uncharacterised domains might be involved in cell-wall attachment for some of these proteins in S. xylosus.8 Journal of Proteome Research • Vol. 6, No. 9, 2007 3577

research articles (iii) Proteins Predicted as Cytoplasmic. Besides the 19 cytoplasmic proteins also predicted as secreted (Table 2.2), this category gathered proteins interacting temporarily with membrane components and other cytoplasmic proteins (Table 3 and Supplementary Table 1S in Supporting Information). Thirtyone proteins were only predicted as cytoplasmic. Most of them were present in fraction II enriched in membrane proteins, that is, 25 including 1 present in both fractions I and II. Eight proteins were predicted as interacting with membranebound components and were systematically present in fraction II enriched in membrane proteins (Table 3.1). Among them, 2 were response regulators interacting with a membrane-bound sensor in two-component systems.86 Five proteins were ribosomal proteins. In the SRP (Signal Recognition Particule)dependent pathway in particular, ribosomal proteins interact with the Sec translocon in the course of co-translational translocation.87 Trigger factor is a major ribosome-associated chaperone involved in protein folding process. It is worth noting that ribosomal proteins constitute a large proportion of the total proteome, for example, approximately 20% in S. aureus,16 and consequently could mask low-abundance proteins in pI basic regions. Among the 23 remaining cytoplasmic proteins, 17 were found in fraction II enriched in membrane proteins, including one present in both fractions I and II. These proteins were neither predicted nor previously reported as secreted, as associated to the bacterial cell-surface, nor as interacting with membrane components (Table 3.2). Nevertheless, some of them might be suspected to be present in the cell envelope such as glucose-1-dehydrogenase, which takes part in an alternative mechanism for glucose uptake through the membrane of S. xylosus,88 or glucosamine-6-phosphate isomerase, which is a precursor of cell wall and polysaccharide synthesis in S. aureus.89 As already pointed out for proteins secreted via unknown secretion system, some of these proteins may have multiple subcellular localizations and moonlight in S. xylosus.90 Furthermore, signal peptide, lipobox, cell-wall binding domain, or transmembrane domain might be present, but an unpredicted, uncharacterised secretion system or unravelled physiological reason may also explain their localization within the cell envelope. Alternatively, since cytoplasmic proteins could also be associated with the lipid bilayer by electrostatic forces and/or hydrophobic or steric properties, these proteins may be, by no means, functionally associated with the membrane; membrane interaction may be artefactual and occurs only under the conditions used during isolation and thus be considered as contaminants.

Conclusions With 90 distinct proteins identified, this study represents the first proteomic analysis of cell envelope proteins of S. xylosus, a coagulase-negative staphylococci. Besides envelope-associated proteins, it appeared some of them had multiple subcellular localization. In fact, some proteins reported as located in the cytoplasmic compartment were also associated with the bacterial cell surface. These proteins supposedly transit between the cytosol and the surface compartments, and consequently, their localization, rather than to be strictly compartmentalized, could also depend on physiological and/or environmental conditions. Moreover, their moonlighting role at different subcellular localizations remains to be elucidated in S. xylosus. Captivatingly, from a recent study in S. pneumo3578


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niae, it appears that release of the cytoplasmic proteins originates from lysis of noncompetent cells and is triggered by competent cells.91 This tightly controlled phenomenon, which involves several cell wall hydrolases, was named allolysis. Occurrence of allolysis and its involvement in cytoplasmic protein release/cell-surface display has not been questioned in other bacteria, including staphylococci, but would undoubtedly require further investigations. Fractionation appeared efficient and specific with limited contamination considering that only 8 out of 90 proteins were found in common between fractions I and II and that the proportion of proteins experimentally found and expected in a given fraction was high in each category, that is, for proteins predicted as membrane associated (31/34), secreted (18/25), and cytoplasmic (25/31). Nevertheless, only one protein with a LPXTG motif was found in fraction I enriched in cell wall proteins, whereas the remaining proteins were devoid of predictable cell wall-binding motifs. Membrane proteins are notoriously difficult to analyze by proteomic approach.92,93 Indeed, proteins extracted in the fraction II enriched in membrane-associated proteins were mainly predicted as integral membrane proteins, but most of them had only one predicted transmembrane domain. It should also be stressed, however, that approximately 30% of proteins encoded in the genome of S. aureus are supposedly membrane proteins and 25% of predicted membrane proteins exhibit only a single transmembrane domain.16 Whereas lysostaphin and tributyl phosphate/acetone/butanol treatments seem appropriate for extracting proteins of cell wall and membrane fractions in S. xylosus, investigations of these subproteomes might be improved by applying techniques other than conventional 2-DE. In fact, other techniques are now used to investigate membrane proteins with several transmembrane domains and cell wall attached proteins, that is, 1-DE combined with LC-MS/MS, or 2-D nanoliquid chromatography coupled with ion-trap mass spectrometry.15,94 Such alternative and complementary approaches are currently underway in our laboratory.

Acknowledgment. Stella Planchon received a Ph.D. grant from the French Ministry of National Education and Research. We thank Nicole Garrel and Brigitte Duclos for their technical assistance, Sibille Farrer for her assistance with MALDI-TOF MS analyses, Thierry Sayd for his advice regarding membrane protein extraction, and Martine Morzel for helpful discussions about this manuscript. The authors thank anonymous reviewers for constructive comments. Supporting Information Available: Figures showing examples of patterns of fraction II enriched in membrane proteins delipidated using Triton X100 or the method using a mixture of chloroform and trifluoroethanol; and comparative patterns of fraction II enriched in membrane proteins delipidated using a mixture of tributylphosphate, acetone, and methanol; table listing a summary of predictions. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kloos, W. E.; Schleifer, K. H. In Bergey’s Manual of Systematic Bacteriology; Sneath, P. H. A., Mair, N. S., Sharpe, M. E., Holt, J. G., Eds.; The Williams Wilkins Co.: Baltimore, MD, 1986; pp 1013-1035. (2) Talon, R.; Leroy-Se´trin, S.; Fadda, S. In Research Advances in Quality of Meat and Meat Products; Fidel Toldra´, Ed.; Research Signpost: India, 2002; pp 175-191.

Proteomic Analysis of Cell Envelope from S. xylosus C2a (3) Corbiere Morot-Bizot, S.; Leroy, S.; Talon, R. Int. J. Food Microbiol. 2006, 108, 210-217. (4) Norwood, D. E.; Gilmour, A. Lett. Appl. Microbiol. 2001, 33, 320324. (5) Planchon, S.; Gaillard-Martinie, B.; Dordet-Frisoni, E.; BellonFontaine, M. N.; Leroy, S.; Labadie, J.; Hebraud, M.; Talon, R. Int. J. Food Microbiol. 2006, 109, 88-96. (6) Nouwens, A. S.; Cordwell, S. J.; Larsen, M. R.; Molloy, M. P.; Gillings, M.; Willcox, M. D.; Walsh, B. J. Electrophoresis 2000, 21, 3797-3809. (7) Silveira, M. G.; Baumgartner, M.; Rombouts, F. M.; Abee, T. Appl. Environ. Microbiol. 2004, 70, 2748-2755. (8) Desvaux, M.; Dumas, E.; Chafsey, I.; Hebraud, M. FEMS Microbiol. Lett. 2006, 256, 1-15. (9) Nandakumar, R.; Nandakumar, M. P.; Marten, M. R.; Ross, J. M. J. Proteome Res. 2005, 4, 250-257. (10) Antelmann, H.; Yamamoto, H.; Sekiguchi, J.; Hecker, M. Proteomics 2002, 2, 591-602. (11) Schaumburg, J.; Diekmann, O.; Hagendorff, P.; Bergmann, S.; Rohde, M.; Hammerschmidt, S.; Jansch, L.; Wehland, J.; Karst, U. Proteomics 2004, 4, 2991-3006. (12) Muller, S.; Hain, T.; Pashalidis, P.; Lingnau, A.; Domann, E.; Chakraborty, T.; Wehland, J. Infect. Immun. 1998, 66, 3128-3133. (13) Jonquieres, R.; Bierne, H.; Fiedler, F.; Gounon, P.; Cossart, P. Mol. Microbiol. 1999, 34, 902-914. (14) Vytvytska, O.; Nagy, E.; Bluggel, M.; Meyer, H. E.; Kurzbauer, R.; Huber, L. A.; Klade, C. S. Proteomics 2002, 2, 580-590. (15) Gatlin, C. L.; Pieper, R.; Huang, S. T.; Mongodin, E.; Gebregeorgis, E.; Parmar, P. P.; Clark, D. J.; Alami, H.; Papazisi, L.; Fleischmann, R. D.; Gill, S. R.; Peterson, S. N. Proteomics 2006, 6, 1530-1549. (16) Scherl, A.; Francois, P.; Bento, M.; Deshusses, J. M.; Charbonnier, Y.; Converset, V.; Huyghe, A.; Walter, N.; Hoogland, C.; Appel, R. D.; Sanchez, J. C.; Zimmermann-Ivol, C. G.; Corthals, G. L.; Hochstrasser, D. F.; Schrenzel, J. J. Microbiol. Methods 2005, 60, 247-257. (17) Mastro, R.; Hall, M. Anal. Biochem. 1999, 273, 313-315. (18) Candiano, G.; Musante, L.; Bruschi, M.; Ghiggeri, G. M.; Herbert, B.; Antonucci, F.; Righetti, P. G. Electrophoresis 2002, 23, 292297. (19) Molloy, M. P.; Herbert, B. R.; Williams, K. L.; Gooley, A. A. Electrophoresis 1999, 20, 701-704. (20) Santoni, V.; Kieffer, S.; Desclaux, D.; Masson, F.; Rabilloud, T. Electrophoresis 2000, 21, 3329-3344. (21) Deshusses, J. M.; Burgess, J. A.; Scherl, A.; Wenger, Y.; Walter, N.; Converset, V.; Paesano, S.; Corthals, G. L.; Hochstrasser, D. F.; Sanchez, J. C. Proteomics 2003, 3, 1418-1424. (22) Hermann, T.; Finkemeier, M.; Pfefferle, W.; Wersch, G.; Kramer, R.; Burkovski, A. Electrophoresis 2000, 21, 654-659. (23) Schluesener, D.; Fischer, F.; Kruip, J.; Rogner, M.; Poetsch, A. Proteomics 2005, 5, 1317-1330. (24) Rabilloud, T. Methods Mol. Biol. 1999, 112, 297-305. (25) Yan, J. X.; Wait, R.; Berkelman, T.; Harry, R. A.; Westbrook, J. A.; Wheeler, C. H.; Dunn, M. J. Electrophoresis 2000, 21, 3666-3672. (26) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Electrophoresis 1988, 9, 255-262. (27) Folio, P.; Chavant, P.; Chafsey, I.; Belkorchia, A.; Chambon, C.; He´braud, M. Proteomics 2004, 4, 3187-3201. (28) Neyraud, E.; Sayd, T.; Morzel, M.; Dransfield, E. J. Proteome Res. 2006, 5, 2474-2480. (29) Bryson, K.; Loux, V.; Bossy, R.; Nicolas, P.; Chaillou, S.; van de Guchte, M.; Penaud, S.; Maguin, E.; Hoebeke, M.; Bessieres, P.; Gibrat, J. F. Nucleic Acids Res. 2006, 34, 3533-3545. (30) Bendtsen, J. D.; Nielsen, H.; von Heijne, G.; Brunak, S. J. Mol. Biol. 2004, 340, 783-795. (31) Nielsen, H.; Engelbrecht, J.; Brunak, S.; von Heijne, G. Int. J. Neural Syst. 1997, 8, 581-599. (32) Hiller, K.; Grote, A.; Scheer, M.; Munch, R.; Jahn, D. Nucleic Acids Res. 2004, 32, W375-W379. (33) Kall, L.; Krogh, A.; Sonnhammer, E. L. J. Mol. Biol. 2004, 338, 1027-1036. (34) Bendtsen, J. D.; Nielsen, H.; Widdick, D.; Palmer, T.; Brunak, S. BMC Bioinf. 2005, 6, 167. (35) Bendtsen, J. D.; Kiemer, L.; Fausboll, A.; Brunak, S. BMC Microbiol. 2005, 5, 58. (36) de Castro, E.; Sigrist, C. J.; Gattiker, A.; Bulliard, V.; LangendijkGenevaux, P. S.; Gasteiger, E.; Bairoch, A.; Hulo, N. Nucleic Acids Res. 2006, 34, W362-W365. (37) Sutcliffe, I. C.; Harrington, D. J. Microbiology 2002, 148, 20652077. (38) Madan Babu, M.; Sankaran, K. Bioinformatics 2002, 18, 641-643. (39) Hofmann, K.; Stoffel, W. 1993, 374, 166.

research articles (40) Jones, D. T.; Taylor, W. R.; Thornton, J. M. Biochemistry 1994, 33, 3038-3049. (41) Tusnady, G. E.; Simon, I. Bioinformatics 2001, 17, 849-850. (42) Hirokawa, T.; Boon-Chieng, S.; Mitaku, S. Bioinformatics 1998, 14, 378-379. (43) Claros, M. G.; von Heijne, G. Comput. Appl. Biosci. 1994, 10, 685686. (44) Zhou, H.; Zhou, Y. Protein Sci. 2003, 12, 1547-1555. (45) Bagos, P. G.; Liakopoulos, T. D.; Hamodrakas, S. J. BMC Bioinf. 2006, 7, 189. (46) Sonnhammer, E. L.; Eddy, S. R.; Durbin, R. Proteins 1997, 28, 405420. (47) Gardy, J. L.; Laird, M. R.; Chen, F.; Rey, S.; Walsh, C. J.; Ester, M.; Brinkman, F. S. Bioinformatics 2005, 21, 617-623. (48) Tatusov, R. L.; Fedorova, N. D.; Jackson, J. D.; Jacobs, A. R.; Kiryutin, B.; Koonin, E. V.; Krylov, D. M.; Mazumder, R.; Mekhedov, S. L.; Nikolskaya, A. N.; Rao, B. S.; Smirnov, S.; Sverdlov, A. V.; Vasudevan, S.; Wolf, Y. I.; Yin, J. J.; Natale, D. A. BMC Bioinf. 2003, 4, 41. (49) Ogata, H.; Goto, S.; Sato, K.; Fujibuchi, W.; Bono, H.; Kanehisa, M. Nucleic Acids Res. 1999, 27, 29-34. (50) Klein, C.; Garcia-Rizo, C.; Bisle, B.; Scheffer, B.; Zischka, H.; Pfeiffer, F.; Siedler, F.; Oesterhelt, D. Proteomics 2005, 5, 180197. (51) Molloy, M. P.; Herbert, B. R.; Walsh, B. J.; Tyler, M. I.; Traini, M.; Sanchez, J. C.; Hochstrasser, D. F.; Williams, K. L.; Gooley, A. A. Electrophoresis 1998, 19, 837-844. (52) Desvaux, M.; He´braud, M. FEMS Microbiol. Rev. 2006, 30, 774805. (53) Bae, T.; Schneewind, O. J. Bacteriol. 2003, 185, 2910-2919. (54) Foster, T. J.; Hook, M. Trends Microbiol. 1998, 6, 484-488. (55) Tjalsma, H.; Antelmann, H.; Jongbloed, J. D.; Braun, P. G.; Darmon, E.; Dorenbos, R.; Dubois, J. Y.; Westers, H.; Zanen, G.; Quax, W. J.; Kuipers, O. P.; Bron, S.; Hecker, M.; van Dijl, J. M. Microbiol. Mol. Biol. Rev. 2004, 68, 207-233. (56) White, S. W.; Zheng, J.; Zhang, Y. M.; Rock, C. O. Annu. Rev. Biochem. 2005, 74, 791-831. (57) Lascelles, J. J. Bacteriol. 1978, 133, 621-625. (58) Nishihara, M.; Koga, Y. J. UOEH 2000, 22, 13-18. (59) Scheffers, D. J.; Pinho, M. G. Microbiol. Mol. Biol. Rev. 2005, 69, 585-607. (60) Tuckman, D.; Donnelly, R. J.; Zhao, F. X.; Jacobs, W. R., Jr.; Connell, N. D. J. Bacteriol. 1997, 179, 2724-2730. (61) Goffin, P.; Deghorain, M.; Mainardi, J. L.; Tytgat, I.; ChampomierVerges, M. C.; Kleerebezem, M.; Hols, P. J. Bacteriol. 2005, 187, 6750-6761. (62) Adler, L. A.; Arvidson, S. J. Bacteriol. 1988, 170, 5337-5343. (63) Vilhelmsson, O.; Miller, K. J. Appl. Environ. Microbiol. 2002, 68, 2353-2358. (64) Kubak, B. M.; Yotis, W. W. Biochim. Biophys. Acta 1981, 649, 642650. (65) Johnson, A. S.; van Horck, S.; Lewis, P. J. Microbiology 2004, 150, 2815-2824. (66) Bott, M.; Niebisch, A. J. Biotechnol. 2003, 104, 129-153. (67) Maruyama, T.; Furutani, M. Front. Biosci. 2000, 5, D821-D836. (68) Otto, M.; Gotz, F. Res. Microbiol. 2001, 152, 351-356. (69) Higgins, C. F. Res. Microbiol. 2001, 152, 205-210. (70) Muller, V.; Gruber, G. Cell. Mol. Life Sci. 2003, 60, 474-494. (71) Economou, A.; Christie, P. J.; Fernandez, R. C.; Palmer, T.; Plano, G. V.; Pugsley, A. P. Mol. Microbiol. 2006, 62, 308-319. (72) Westerlund, B.; Korhonen, T. K. Mol. Microbiol. 1993, 9, 68794. (73) Cassat, J. E.; Dunman, P. M.; McAleese, F.; Murphy, E.; Projan, S. J.; Smeltzer, M. S. J. Bacteriol. 2005, 187, 576-592. (74) Resch, A.; Leicht, S.; Saric, M.; Pasztor, L.; Jakob, A.; Gotz, F.; Nordheim, A. Proteomics 2006, 6, 1867-1877. (75) Stewart, G. C. Mol. Microbiol. 2005, 57, 1177-1181. (76) Barrie`re, C.; Leroy-Se´trin, S.; Talon, R. J. Appl. Microbiol. 2001, 91, 514-519. (77) Hampton, M. B.; Kettle, A. J.; Winterbourn, C. C. Infect. Immun. 1996, 64, 3512-3517. (78) Braunstein, M.; Espinosa, B. J.; Chan, J.; Belisle, J. T.; Jacobs, W. R., Jr. Mol. Microbiol. 2003, 48, 453-464. (79) Desvaux, M.; Parham, N. J.; Scott-Tucker, A.; Henderson, I. R. Trends Microbiol. 2004, 12, 306-309. (80) Bergonzelli, G. E.; Granato, D.; Pridmore, R. D.; Marvin-Guy, L. F.; Donnicola, D.; Corthesy-Theulaz, I. E. Infect. Immun. 2006, 74, 425-434. (81) Bergmann, S.; Rohde, M.; Chhatwal, G. S.; Hammerschmidt, S. Mol. Microbiol. 2001, 40, 1273-1287.


research articles (82) Bergmann, S.; Rohde, M.; Hammerschmidt, S. Infect. Immun. 2004, 72, 2416-2419. (83) Hughes, M. J.; Moore, J. C.; Lane, J. D.; Wilson, R.; Pribul, P. K.; Younes, Z. N.; Dobson, R. J.; Everest, P.; Reason, A. J.; Redfern, J. M.; Greer, F. M.; Paxton, T.; Panico, M.; Morris, H. R.; Feldman, R. G.; Santangelo, J. D. Infect. Immun. 2002, 70, 1254-1259. (84) Page, M. G.; Burton, K. Biochem. J. 1978, 174, 717-725. (85) McGee, D. J.; Radcliff, F. J.; Mendz, G. L.; Ferrero, R. L.; Mobley, H. L. J. Bacteriol. 1999, 181, 7314-7322. (86) Foussard, M.; Cabantous, S.; Pedelacq, J.; Guillet, V.; Tranier, S.; Mourey, L.; Birck, C.; Samama, J. Microbes Infect. 2001, 3, 417424. (87) Valent, Q. A.; Scotti, P. A.; High, S.; de Gier, J. W.; von Heijne, G.; Lentzen, G.; Wintermeyer, W.; Oudega, B.; Luirink, J. EMBO J. 1998, 17, 2504-2512.

3580


Planchon et al. (88) Fiegler, H.; Bassias, J.; Jankovic, I.; Bruckner, R. J. Bacteriol. 1999, 181, 4929-4936. (89) Komatsuzawa, H.; Fujiwara, T.; Nishi, H.; Yamada, S.; Ohara, M.; McCallum, N.; Berger-Bachi, B.; Sugai, M. Mol. Microbiol. 2004, 53, 1221-1231. (90) Jeffery, C. J. Trends Biochem. Sci. 1999, 24, 8-11. (91) Guiral, S.; Mitchell, T. J.; Martin, B.; Claverys, J. P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8710-8715. (92) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054-1070. (93) Cordwell, S. J. Curr. Opin. Microbiol. 2006, 9, 320-329. (94) Calvo, E.; Pucciarelli, M. G.; Bierne, H.; Cossart, P.; Albar, J. P.; Garcia-Del Portillo, F. Proteomics 2005, 5, 433-443.

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