Profiling the Outer Membrane Proteome during Growth and

Profiling the Outer Membrane Proteome during Growth and Development of the Social Bacterium Myxococcus xanthus by Selective Biotinylation and Analyses of Outer Membrane Vesicles Jo ¨ rg Kahnt,† Kryssia Aguiluz,† Ju ¨ rgen Koch,† Anke Treuner-Lange,† Anna Konovalova,† † Stuart Huntley, Michael Hoppert,‡ Lotte Søgaard-Andersen,*,† and Reiner Hedderich† Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Str., 35043 Marburg, Germany, and Institute of Microbiology & Genetics, Georg-August-Universita¨t Go¨ttingen, 37077 Go¨ttingen, Germany Received May 19, 2010

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO12 (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells. Keywords: Myxococcus xanthus • outer membrane proteome • outer membrane vesicles • native membrane vesicles • integral outer membrane proteins • TonB-dependent transporters • lipoproteins • biotinylation • outer membrane

Introduction The outer membrane of Gram-negative bacteria is an important physical interface between the cell and its environment.1 Proteins in the outer membrane play crucial roles in the uptake of various compounds across the outer membrane using both energized and nonenergized transport. Outer membrane proteins are also essential in the export of proteins and low molecular mass compounds, and they have important functions in cell-cell communication and cell-cell interactions. Finally, outer membrane proteins are essential for the integrity and stability of the cell envelope in Gram-negative bacteria. In this study, we focused on the analysis of the outer membrane proteome of Myxococcus xanthus. The delta-proteobacterium M. xanthus has a highly complex lifecycle that involves social behaviors.2 In their soil habitat, M. xanthus cells are predators growing as saprophytes or by lysing prey bacteria and metabolizing the macromolecules * To whom correspondence should be addressed. Tel. +49-6421-178201. Fax: +49-6421-178209. E-mail: [email protected]. † Max Planck Institute for Terrestrial Microbiology. ‡ Georg-August-Universita¨t Go¨ttingen. 10.1021/pr1004983

 2010 American Chemical Society

released.3,4 To achieve this, M. xanthus cells form spreading, cooperatively feeding colonies5 in which cells secrete numerous secondary metabolites6 and hydrolytic enzymes.7 In response to nutrient deprivation, M. xanthus cells initiate a developmental program that culminates in the formation of spore-filled fruiting bodies that each contain 100.000 spores.2 This developmental program involves two morphogenetic events, aggregation of cells into fruiting bodies and sporulation of cells that have accumulated inside fruiting bodies. The social behaviors of M. xanthus cells rely on several contact-dependent activities,8 which either involve direct cell-cell or cell-substratum interactions. M. xanthus cells move by gliding motility and possess two gliding motility systems.9 The formation of spreading colonies as well as fruiting body formation depend on the functionality of both systems.9 Both motility systems rely on contact-dependent activities. The force for the S-motility system is generated by retraction of type IV pili (T4P)10,11 and is generally only functional when cells are within contact-distance of each other.9 The cell-cell contact-dependency of T4P function is thought to depend on exopolysaccharides of the extracellular matrix stimulating retraction.12 The extracellular matrix of M. xanthus is composed Journal of Proteome Research 2010, 9, 5197–5208 5197 Published on Web 08/05/2010

research articles of exopolysaccharides and proteins in a ratio of approximately 1:1.13,14 Two models have been proposed for how the A-motility system generates mechanical force. In one model,15 force is generated by secretion of a polyelectrolyte gel from nozzlelike structures embedded in the cell envelope. The components of the nozzles remain to be identified. In the alternative model,16 A-motility depends on focal adhesion complexes distributed along the cell envelope. In this model, force is generated by a protein complex that spans the cell envelope, adheres to the substratum and pulls on a cytoskeletal element in the cytoplasm.16 Except for the cytoplasmic protein AglZ16 no other components of focal adhesion complexes have been identified. Moreover, the functionality of the two motility systems may involve contact-dependent transfer of lipoproteins between cells.17 Specifically, the outer membrane lipoprotein Tgl,18 which is required for assembly of the outer membrane secretin PilQ in the T4P system,19 and the lipoprotein CglB,20 which by an unknown mechanism is required for A-motility, have been shown to be transferred between cells. A different type of cell-cell contact-dependent activity was observed in the case of the FrzCD protein,21 which is a cytoplasmic methylaccepting chemotaxis protein ortholog and part of the Frz chemosensory system, which regulates the cellular reversal frequency.22 FrzCD localizes to clusters that continuously change size, number, and position. When cells make side-toside contacts, FrzCD clusters in adjacent cells show transient alignment. This phenomenon suggests that M. xanthus harbor a trans-envelope signaling system that allow the specific interaction between M. xanthus cells.21 Fruiting body formation depends on cell-cell communication and two intercellular signals, the A- and C-signal, have been characterized.23 The C-signal is a 17 kDa protein (p17),24-26 which is generated by proteolytic cleavage of the 25 kDa csgA protein (p25).26,27 p25 as well as p17 are anchored in the outer membrane by an unknown mechanism.26 C-signal transmission has been proposed to be cell-cell contact-dependent and involve contacts between signaling cells28,29 suggesting that the receptor for the C-signal, which remains to be identified, is an outer membrane protein. Two general experimental strategies have been used to comprehensively characterize the outer membrane proteome in Gram-negative bacteria. In one method, outer membrane proteins accessible from the cell surface as well as proteins associated with the cell surface are enriched by selective labeling of intact cells with reactive reagents that covalently attach a biotin group to primary amines. Labeling is followed by membrane isolation and affinity purification using the biotin-tag (e.g., see refs 30, 31). This method has previously been reported to also result in labeling of periplasmic, inner membrane and cytoplasmic proteins.30,31 In a different approach advantage is taken of the fact that a wide variety of Gram-negative bacteria shed outer membrane vesicles (OMV) (for review, see refs 32, 33). The physiological function of OMV is not known but they are currently viewed as an alternative protein secretion system.32,34 Only a limited number of studies have been performed to characterize the proteome of native OMV. These studies have shown that native OMVs are enriched for outer membrane proteins and entrapped periplasmic proteins thought to be cargo carried by the OMV whereas inner membrane proteins and cytoplasmic proteins are significantly underrepresented.35,36 However, it is not currently know if OMVs contain the entire outer membrane proteome or are enriched for a subset of outer membrane proteins. 5198

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Kahnt et al. Currently, little is known about the composition of the outer membrane proteome in M. xanthus and only a few proteins of the outer membrane have been characterized.17,18,26,37-39 To increase the understanding of the outer membrane of M. xanthus cells and the molecular mechanisms underlying contact-dependent activities, we performed an analysis of the outer membrane proteome of M. xanthus using both of the two experimental approaches outlined above. In both procedures proteins were identified using 1D-SDS-PAGE followed by nano-LC-MALDI-MS/MS of gel-sections digested with trypsin. Moreover, our studies were performed on vegetative cells as well as on cells that had been starved for 18 h to monitor changes in the outer membrane proteome during fruiting body formation. A comparison of proteins identified showed that the two methods are highly specific for cell envelope proteins; however, the subset of cell envelope proteins identified in general and outer membrane proteins in particular are significantly different.

Materials and Methods Growth and Development of Myxococcus xanthus. M. xanthus DK1622 wild-type cells40 were grown in liquid CTT medium (10 g bacto casitone/L, 10 mM Tris-HCl [pH 8.0], 1 mM potassium phosphate [pH 7.6], 8 mM MgSO4)9 at 32 °C to a density of 5 × 108 cells per ml. After harvesting, one-half of the culture was used for in vivo labeling of intact cells and the second half was used for development. For development, cells were resuspended in prewarmed (32 °C) MC7 starvation buffer (10 mM MOPS-KOH [pH 7.0], 1 mM CaCl2) to a calculated density of 5 × 109 cells per mL. The cell suspension was diluted 1:8 in MC7 buffer and 120 mL of the resulting suspension were transferred to a sterile 245 mm × 245 mm polystyrene dish and incubated at 32 °C. After 18 h of development, cells were harvested, and immediately used for in vivo labeling. In vivo Biotinylation of Intact Cells and Isolation of Biotinylated Proteins. Cells from vegetative cultures or after development were harvested and resuspended to a calculated density of 2.5 × 109 cells per mL in ice-cold phosphate buffered saline (PBS) (0.1 M phosphate, 0.15 M NaCl, [pH 8.0]) supplemented with 1 mM CaCl2, 0.5 mM MgCl2 and 1.6 mM D-biotin. Cells were labeled by incubation with 360 µM (final concentration) of NHS (N-hydroxysuccinimide)-PEO12 (polyethylene oxide)-biotin (Thermo Scientific, Bonn, Germany) for 30 min at 20 °C. The reaction was stopped by adding 3 volumes of stop solution (50 mM glycine [pH 7.4], 100 mM NaCl, 27 mM KCl, 1.0 mM CaCl2, 0.5 mM MgCl2) to one volume of the biotinylation mixture. After 10 min incubation at 20 °C cells were harvested by centrifugation at 6000× g. Cells were washed 3 times in 50 mM glycine (pH 7.4). Treated cells were either stored at -20 °C for further analyses as described below or exposed to light microscopy using a Leica DM6000B microscope equipped with a Leica Plan Apo 100×/NA1.40 phasecontrast oil objective to test for morphological changes and viable counts to test for viability. To isolate biotinylated proteins, labeled bacteria (∼0.5 g wet mass) were resuspended in 10 mL MOPS buffer (10 mM MOPS-KOH [pH 7.2], 1 mM CaCl2, 4 mM MgCl2) containing protease inhibitors (0.5 mM phenylmethylsulfonylfluorid, 2.9 mM benzamidine, 1 µM pepstatin, 1 µM leupeptine) and lysed on ice by sonication using a Branson-Sonifier (energy out-put: 60 W, 3 × 5 min). Cell debris and intact bacteria were removed by centrifugation at 6000× g for 15 min, 4 °C. Membranes were sedimented at 160 000× g for 2 h, 4 °C, washed once, resuspended in MOPS

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Outer Membrane Proteome of Myxococcus xanthus buffer containing protease inhibitors. Subsequently, the detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate) was added to a final concentration of 1.2% at a protein concentration of 0.3 mg/mL. The suspension was incubated for 16 h at 4 °C with careful shaking. Insoluble membranes were removed by centrifugation at 160 000× g for 1 h, 4 °C. Solubilized proteins were applied to a column containing 3 mL immobilized monomeric avidin-agarose (ImmunoPure, Thermo Scientific, Bonn, Germany), equilibrated with PBS buffer (pH 7.0) containing 0.4% CHAPS (PBS/CHAPS) and protease inhibitors. After an incubation period of 1 h, the column was washed with PBS/CHAPS (approximately 7 column volumes) until the eluate was protein free. Biotinylated proteins were eluted with PBS/CHAPS containing 5 mM D-biotin. Protein containing fractions were pooled and concentrated by ultrafiltration (Micron, 10 kDa cutoff, Millipore, Bad Homburg, Germany) and stored at -20 °C. The total protein yield was 50-100 µg protein from 0.5 g cells (wet mass). Three independent biological experiments were performed for vegetative cells as well as for developmental cells. Isolation of Outer Membrane Vesicles. OMVs were purified from culture supernatants of vegetative cells or from supernatants of developmental cells. Vegetative cells of M. xanthus were grown in CTT medium to an optical density of 5 × 108 cells per mL. Cells were harvested and the culture supernatant was used for the isolation of vesicles (vegetative vesicles). Harvested cells were resuspended in MC7 starvation buffer and were developed for 18 h in polystyrene dishes. Cells were harvested and the supernatant was used for the isolation of membrane vesicles (developmental vesicles). Vesicles were isolated using previously described protocols.41 In brief, culture supernatants (1.1 L) were passed through a 0.2 µm vacuum filter (Millipore). The resulting filtrate was centrifuged at 150 000× g for 2 h at 4 °C to recover membrane vesicles. The supernatant was carefully removed and the vesicle pellet was resuspended in 50 mM Tris-HCl pH 8.0 and centrifuged at 150 000× g for 2 h at 4 °C to concentrate vesicles. Three independent biological experiments were performed for vegetative cells as well as for developmental cells. SDS/PAGE, In-Gel Tryptic Digestion and Liquid Chromatography. Purified OMVs or affinity purified biotinylated proteins were resuspended in SDS-PAGE sample buffer and boiled for 5 min. Proteins were separated on 14% SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue R-250 (Figure S1, Supporting Information). Each gel lane was cut into 10 slices. Each slice was destained with 50% acetonitrile (ACN) (v/v) containing 20 mM NH4HCO3, dehydrated with 100% ACN and dried. Gel pieces were rehydrated in 5 mM NH4HCO3 in 10% ACN (v/v) containing 0.015 g/L sequencing-grade modified trypsin (Promega) and incubated for 10 h at 22 °C. Tryptic peptides were extracted with 0.1% trifluoroacetic acid (TFA) (v/v) in water. The extracted peptides were concentrated under vacuum but not to dryness. The resulting peptide mixture was injected onto a PepMap100 C-18 RP nanocolumn (Dionex, Idstein, Germany) and separated on an UltiMate 3000 liquid chromatography system (Dionex, Idstein, Germany) in a continuous ACN gradient consisting of 0-40% B in 40 min, 40-100% B in 10 min (B ) 80% (v/v) ACN, 0.1% (v/v) TFA). Peptides were eluted at a flow-rate of 300 nL/min. A Probot microfraction collector (Dionex, Idstein, Germany) was used to spot liquid-chromatography separated peptides on a MALDI target with a rate of 8 s/spot. The eluate was mixed with matrix

consisting of 4 mg/mL R-cyano-4-hydroxycinnamic acid in 70% (v/v) ACN and 0.1% (v/v) TFA. MALDI-MS/MS Analysis. MALDI-TOF-TOF analysis was carried out on a 4800 Proteomics Analyzer (Applied Biosystems/ MDS Sciex, Forster City, CA) using the 4800 Series Explorer software version 3.5.1. The mass spectrometer was operated in positive-ion reflector mode in a mass range from 840 to 4200 Da with a focus mass on 1700 Da and a S/N minimum set to 40. For one main spectrum 30 subspectra with 30 spots per subspectrum were averaged. Close external calibration was performed with the calibration standard no. 206195 from BRUKER Daltonics (Bremen, Germany) in a mass range from 1046.54 to 3149.57 Da, spotted onto 13 positions of the MALDItarget. The internal calibration in every sample spot was automatically performed using the matrixpeak at 877.031 Da. In MS/MS positive ion mode, 2500 spectra were averaged, collision energy was 1 kV, collision gas was air and external calibration was set using the Glu1-Fibriono-peptide B ((M+H+) ) 1570.6696 Da). MS/MS data were searched against an in-house M. xanthus protein database (7426 protein sequences were downloaded from http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi) using Mascot v2.2.1 (Matrixscience, United Kingdom) embedded into GPS explorer software version 3.6 (Applied Biosystems/ MDS Sciex). MASCOT search parameters were: trypsin as enzyme, up to one missed cleavage site, MS tolerance of 40 ppm and MS/MS tolerance of 0.8 Da, no fixed modifications, variable modifications included methionine oxidation, deamidation and propionamide. Peptide hits with a score >44 (p < 0.05) were used for further manual validation. A minimum of two peptides per protein was required for protein identification and only proteins identified in at least two out of three independent biological experiments were considered as positively identified. To assess the false-assignment distribution of the database search results, MS/MS data were also searched against an in-house protein database composed of Escherichia coli K12 (4126 protein sequences), Bacillus subtilis 168 (4095 protein sequences), Clostridium tetani E88 (2373 protein sequences), Archaeoglobus fulgidus DSM4304 (2402 protein sequences) genome sequences, all downloaded from http:// cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi. Details of identified proteins and identification evidence, including total ion score and sequences of identified peptides are given in Tables S1-S3 (Supporting Information). Electron Microscopy. Vesicles isolated by ultracentrifugation were resuspended in 50 mM Tris-HCl (pH 8.0). For negative staining, a carbon film, evaporated on a freshly cleaved mica surface, was partially floated off the mica by introduction into a sample drop for 1 min. The carbon-mica sandwich was then transferred to a drop of washing solution (double distilled water) and partially floated off for some seconds. The carbon film was then transferred to a drop of negative staining solution, completely floated off and then adsorbed onto a 400 mesh specimen grid. Negative staining solutions were uranyl acetate, 4% (w/v), or phosphotungstic acid (3%, [w/v], titrated to pH 7.0). The grid was removed from the drop and the staining solution was completely soaked off with filter paper, resulting in a shallowly stained specimen.42,43

Results and Discussion Enrichment and Identification of Biotinylated Proteins. The inner and outer membranes of M. xanthus cells have been reported to be difficult to separate experimentally.37,44 ThereJournal of Proteome Research • Vol. 9, No. 10, 2010 5199

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Table 1. Summary of M. xanthus Proteins Identified in Biotinylation Experiments and in Outer Membrane Vesicles

predicted subcellular localization

a

Integral outer membrane proteins Lipoproteins Periplasmic proteins Integral inner membrane proteins Cytoplasmic proteins Total

vegetative cells

vegetative cells

developmental cells

developmental cells

biotinylation

OMV

biotinylation

OMV

56 68 75 23 61 283

36 24 20 2 25 107

45 44 49 17 43 198

48 24 22 3 27 124

a Subcellular localization of proteins were predicted on the basis of the following criteria: Integral outer membrane proteins, N-terminal signal peptide with a signal peptidase I cleavage site predicted using SignalP376 and TatP77 and a potential beta-barrel structure using TMBB (http:// bioinformatics2.biol.uoa.gr/PRED-TMBB); lipoproteins, N-terminal signal peptide with signal peptidase II cleavage site predicted using LipoP;78 periplasmic proteins, Signal peptidase I cleavage site but no inner membrane or outer membrane protein prediction; integral inner membrane proteins, One or more trans-membrane helices predicted using TMHMM;79 cytoplasmic proteins, no signal peptidase cleavage site and no inner membrane or outer membrane prediction.

fore, to identify M. xanthus outer membrane proteins we adapted a method that was originally developed for Helicobacter pylori.30 In this method, intact cells are treated with the hydrophilic biotinylation reagent S-NHS-LC-biotin (sulfosuccinimidyl-6-(biotinamido)hexanoate). However, some of the reagent enters the periplasm resulting in substantial labeling of periplasmic proteins. The molecular mass of S-NHS-LCbiotin is 557 Da, which is below the exclusion limit of 600-800 Da of outer membrane porin channels in Escherichia coli and Salmonella typhimurium,45-47 and it has been suggested that S-NHS-LC-biotin enters the periplasm through outer membrane porins.30 The characteristics of the outer membrane porins of M. xanthus are not known. In order to reduce potential problems with permeation of the outer membrane by the labeling reagent, we used the highly hydrophilic compound NHS-PEO12-biotin (NHS (N-hydroxysuccinimide)PEO12 (polyethylene oxide)-biotin), which has a molecular mass of 941 Da. This reagent carries an NHS ester group that reacts with primary amine groups to form stable amide bonds in that way coupling PEO-biotin to proteins. Moreover, the hydrophilic PEO12 spacer arm increases water solubility and, thus, reduces permeation of the inner membrane in case the compound enters the periplasm. Light microscopy of cells after exposure to NHS-PEO12-biotin showed that cells were morphologically normal and had not undergone lysis; however, viable counts showed that cell viability was reduced approximately 5-fold (data not shown). Biotinylated membrane proteins were further enriched by cell lysis and membrane isolation. Proteins were solubilized with the nondenaturing detergent CHAPS and applied to a monomeric avidin-agarose column, which binds biotin and biotinylated proteins reversibly. Proteins were eluted with biotin, concentrated by ultrafiltration and separated by 1D SDS/PAGE (Figure S1, Supporting Information). An entire gel lane was excised and cut into 10 bands for trypsin digestion and nano-LC-MALDI-MS/MS analysis. To determine the outer membrane proteome three independent biological experiments were conducted for vegetative cells as well as for cells starved for 18 h. Under the conditions used, starving cells have formed nascent fruiting bodies after 18 h of starvation; however, sporulation has not initiated (data not shown). 283 proteins were identified from vegetative cells in at least two out of the three independent biological experiments. Similarly, 198 proteins were identified from developing cells in at least two out of the three independent biological experiments (Table 1). The set of proteins identified from the two cell types overlapped significantly with 168 proteins detected in both vegetative and developmental cells while 115 5200


were specifically detected in vegetative cells, and 30 specifically detected in developmental cells (Figure 1). Details about all proteins identified are provided in Tables S1 and S2 (Supporting Information). To evaluate the specificity of the labeling procedure for outer membrane proteins, we predicted the subcellular localization of the proteins identified by determining the presence or absence of signal peptides with a signal peptidase I cleavage site or with a signal peptidase II cleavage site, trans-membrane helices, and a potential beta-barrel structure. This analysis allowed us to categorize proteins as integral outer membrane proteins (signal peptide with a signal peptidase I cleavage site and a potential beta-barrel structure), lipoproteins (signal peptide with signal peptidase II cleavage site), inner membrane proteins (one or more trans-membrane helices), periplasmic proteins (signal peptidase I cleavage site but no inner membrane or outer membrane protein prediction), and cytoplasmic proteins (no signal peptidase cleavage site and no inner membrane or outer membrane prediction). Among the total of 313 proteins identified using the biotinylation approach, 58 (19%) (Table 1; Figure 1; Tables S1, Supporting Information) are predicted integral outer membrane proteins. Among these proteins Oar38 has been shown to localize to the outer membrane. 74 proteins (24%) are predicted lipoproteins and among these Tgl has been shown to localize to the outer membrane,18 CglB has been suggested to localize to the outer membrane17 or the inner membrane37 and FibA has have been shown to localize either to the inner membrane37 or the extracellular matrix.14,44 27 proteins (9%) are predicted inner membrane proteins. 84 proteins (27%) are predicted periplasmic. Finally, 70 proteins (21%) are cytoplasmic. Importantly, among these latter proteins only two ribosomal proteins (MXAN3323 and MXAN3325 identical to ribosomal protein S13 and S4, respectively) and two chaperonins (MXAN4467 and MXAN4895) were detected. Thus, at least 243 of the 313 identified proteins (79%) are predicted to be cell envelope proteins. The under-representation of abundant cytoplasmic proteins such as ribosomal proteins, translation factors and chaperonins among proteins with a predicted cytoplasmic localization in combination with the observation that the biotinylation procedure does not cause cell lysis support the notion that labeling of these proteins is not due to cell lysis. In total, the detection of potential cytoplasmic, periplasmic and inner membrane proteins indicates that the

Outer Membrane Proteome of Myxococcus xanthus

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Figure 1. Venn diagram representation of distributions of proteins identified in the biotinylation method and in outer membrane vesicles as a function of their predicted subcellular localization.

labeling reagent is able to penetrate the outer membrane and to some extent also the inner membrane. Analysis of the Proteome of M. xanthus Membrane Vesicles. Recently it was shown that M. xanthus cells shed membrane vesicles into the extracellular space.48 Because membrane vesicles shed by other Gram-negative bacteria derive from the outer membrane, we speculated that purified native membrane vesicles could be a useful tool to determine the outer membrane proteome in M. xanthus. Membrane vesicles were isolated from culture supernatants of vegetative cells and cells developed for 18 h by ultracentrifugation following a previously described procedure.41 Vesicle preparations negatively stained with either uranyl acetate or phosphotungstic acid were analyzed by electron microscopy (Figure 2). The vesicle diameter ranged from 30 to 100 nm in good agreement with the vesicle diameter of 30 to 60 nm reported by Palsdottir et al.48 Moreover, no cell debris (cell wall fragments, cellular appendages, cytoplasmic contents) was observed (Figure 2). In both preparations (vegetative and developmental cells), but especially in preparations from developmental cells (Figure 2A and B), large vesicles were observed, decorated with characteristic ring-like features on the vesicle surface, identical to the overall structure of outer membrane porins (Figure 2B). The smaller vesicles are likely right-side out vesicles, with a rim of lipopolysaccarides lining the vesicle membrane (Figure 2D, inset). The larger vesicles with visible porins could be either inside-out vesicles or vesicles after loss of LPS. It could be excluded that the rim consists of F1-ATPase particles, as observed for inside-out vesicles of the cytoplasmic membrane (see, for example, ref 49) because staining of the M. xanthus vesicles with either uranyl acetate or phosphotungstic acid shows diffuse rims, consisting of small particles of various size and shape (Figure 2D, inset) whereas the characteristic 10-nm F1-particle could not be observed. These observations strongly suggest that the vesicles derive from the outer membrane. Proteins in vesicles were separated by 1D SDS/PAGE (Figure S1, Supporting Information), an entire

Figure 2. Negatively stained outer membrane vesicle preparations from M. xanthus. (A, B) Preparations from developmental cells, stained with uranyl acetate. Large vesicles show ring-like features on the vesicle surface (indicated by arrows). (C, D) Preparations from vegetative cells, stained with uranyl acetate or phosphotungstic acid (D, inset). In D, a diffuse rim of small particles is marked by arrows.

gel lane excised and cut into 10 bands for trypsin digestion and proteins identified by nano-LC-MALDI-MS/MS analysis. Details about all proteins identified are provided in Tables S1 and S2 (Supporting Information). In total, 162 proteins were identified from the vesicles (Table 1; Figure 1). 107 and 124 proteins were identified in vesicles from vegetative and developmental cells, respectively. 69 proteins were identified in vesicles from both cell types, 38 proteins were only identified in vesicles form vegetative cells, and 55 proteins only identified in vesicles from developmental Journal of Proteome Research • Vol. 9, No. 10, 2010 5201

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Table 2. Summary of the Predicted Localization of M. xanthus Proteins Identified in Biotinylation Experiments and in Outer Membrane Vesicles detected in

a

predicted subcellular localizationa

total proteins detected

biotinylation and OMV

biotinylation (only)

OMV (only)

Integral outer membrane proteins Lipoproteins Periplasmic proteins Integral inner membrane proteins Cytoplasmic proteins Total

64 85 97 28 88 362

45 (70%) 25 (29%) 19 (20%) 2 (7%) 22 (25%) 113 (31%)

13 (20%) 49 (58%) 65 (66%) 25 (89%) 48 (55%) 199 (55%)

6 (10%) 11 (13%) 13 (14%) 1 (3%) 18 (20%) 50 (14%)

See footnote a to Table 1.

cells. Among the 162 identified proteins, 51 (31%) are predicted integral outer membrane proteins including Oar and the outer membrane secretin PilQ of T4P. 36 (22%) are predicted lipoproteins. 32 proteins (20%) are periplasmic; 3 proteins (2%) are predicted inner membrane proteins; and 40 proteins (25%) are predicted to be cytoplasmic. Hence, M. xanthus vesicles are highly enriched for integral outer membrane proteins, lipoproteins and periplasmic proteins. In total, these findings strongly suggest that the vesicles isolated from M. xanthus culture supernatants derive from the outer membrane. Comparative Analysis of the Two Methods. In total, 362 proteins were detected by the two methods (Table 2). Onehundred thirteen proteins were detected by both methods and 249 were detected by one method only. The overlap between proteins detected by both methods strongly depends on their predicted subcellular localization (Table 2; Figure 1). Thus, 70% of all predicted integral outer membrane proteins were detected by both methods whereas only 29% of all predicted lipoproteins, 20% of all predicted periplasmic proteins, 7% of predicted inner membrane and 25% of predicted cytoplasmic proteins were detected by both methods. Generally, proteins identified by both methods were detected in cells treated equally, that is, a protein was detected by both methods in vegetative cells and/or by both methods in developmental cells (Figure 1; Table S2, Supporting Information). Integral Outer Membrane Proteins in Intact Cells and in Membrane Vesicles. Among the total of 64 integral outer membrane proteins detected, six were only detected in OMV and 13 were only detected in the biotinylation experiments. These observations suggest that OMV do not represent the full set of integral outer membrane proteins but are enriched for a subset of these proteins. To assign functions to the 64 integral outer membrane proteins detected, we searched for homologous genes using BlastP50 and conserved domains using the SMART database51 (Table 3). Moreover, orthologous proteins were identified using a pairwise reciprocal best BlastP hit method in which M. xanthus proteins were searched against 973 completely sequenced bacterial genomes (Table 3). 38 (59%) of the integral outer membrane proteins are hypothetical proteins or proteins with conserved domains of unknown function. Among these, 20 are unique to the Myxococcales and five unique to M. xanthus. Among integral outer membrane proteins with functionally characterized domains, TonB-dependent transporters52 comprise the largest group. In total, 11 TonB-dependent transporters out of 20 predicted from the M. xanthus genome were identified. All 11 proteins were detected in vegetative as well as in starving cells (Table 3, Table S2, Supporting Information). Interestingly, 5 of the genes for the identified TonB5202


dependent transporters (MXAN0856, MXAN1450, MXAN4746, MXAN5023 and MXAN7331) are flanked by genes encoding lipoproteins, which were also detected in our studies (Table S1, Supporting Information). Most TonB-dependent transporters characterized in other bacteria mediate the energy-dependent transport of low abundant substrates or substrates with molecular masses exceeding the exclusion limit of the general porins across the outer membrane.52 Mainly iron-siderophore complexes and vitamin B12 have been identified as substrates.53 Synthesis of these transporters is transcriptionally regulated according to the cellular demand and the availability of the substrate.52 Such a classical role could be envisaged for one of the TonB-dependent transporters identified in this study. MXAN6579 is genetically linked to components of an inner membrane iron-transport system (MXAN6576 and MXAN6575). For the remaining TonBdependent transporters, in particular the highly abundant transporters, this classical function appears less likely. A subclass of TonB-dependent transporters, designated as TonB-dependent transducers, is involved in both transport and trans-envelope signal transduction.54,55 One TonB-dependent transducer is known to have a signaling function only.56 These receptors/transducers bind the ligand that they transport, and via a conserved signaling domain at the N-terminus, information about ligand binding is transmitted to an inner membrane antisigma factor that controls the activity of an ECF-type sigma factor that in turn controls the expression of the TonBdependent transporter. Most of the TonB-dependent transporters identified in this study carry N-terminal extensions of up to 300 amino acids. These extensions are, however, not sequence related to the signaling domain of known TonBdependent transducers. Consistently, none of the corresponding receptor genes are genetically linked to genes encoding a sigma factor or an ECF-type sigma factor, a genetic organization typical for TonB-dependent transducers.55 This would also argue against a classical signaling function of M. xanthus TonBdependent transporters. This raises the question about the function of these TonB-dependent transporters. There is accumulating evidence for TonB-dependent transporters being involved in transport of polymeric substrates across the outer membrane.57-60 M. xanthus makes its living mainly on proteins released by the lysis of other microorganisms.61 A strategy that would involve the cleavage of protein substrates into larger polypeptides by extracellular proteases and the subsequent transport of these polypeptides via TonB-dependent transporters into the periplasmic space, where the subsequent hydrolysis to amino acids and oligopeptides would take place, could be advantageous for the cells to avoid loss of amino acids and small peptides by diffusion. Moreover, this strategy would avoid

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Outer Membrane Proteome of Myxococcus xanthus

Table 3. Integral Outer Membrane Protein in M. xanthus Identified by Biotinylation of Intact Cells or by the Analysis of Outer Membrane Vesicles systematic gene name

MXAN0518 MXAN0821 MXAN0856 MXAN1450 MXAN4559 MXAN4746 MXAN5023 MXAN6044 MXAN6579 MXAN6911 MXAN7331 MXAN3905 MXAN4176 MXAN5030 MXAN6487 MXAN7436 MXAN2090 MXAN5042 MXAN6246 MXAN2514 MXAN3106 MXAN5772 MXAN0562 MXAN4727 MXAN4728 MXAN7040 MXAN0162 MXAN0219 MXAN0585 MXAN0620 MXAN0924 MXAN1329 MXAN1424 MXAN1916 MXAN2426 MXAN2466 MXAN2539 MXAN2540 MXAN2659 MXAN2865 MXAN3553 MXAN3667 MXAN3830 MXAN4092 MXAN4400 MXAN4440 MXAN4686 MXAN4866 MXAN4976 MXAN5102 MXAN5375 MXAN5491 MXAN5686 MXAN5809 MXAN5855 MXAN5931 MXAN6090 MXAN6196 MXAN6366 MXAN6751 MXAN7203 MXAN7317 MXAN7340 MXAN7407

gene name

oar

gspD1 gspD2 pilQ yaeT fadL

annotation

phylogenetic distributiona

TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TonB-dependent receptor TolC-like protein TolC-like protein TolC-like protein TolC-like protein TolC-like protein OmpA domain protein OmpA domain protein OmpA domain protein outer membrane secretin outer membrane secretin type IV pilus secretin Porin O/P-like protein OmpH family protein, Omp85 family protein long-chain fatty acid transport protein conserved hypothetical protein hypothetical protein alpha-2-macroglobulin family protein hypothetical protein hypothetical protein conserved hypothetical hypothetical protein hypothetical protein hypothetical protein hypothetical protein conserved hypothetical conserved hypothetical hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein

Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Myxococcales Myxococcales Myxococcales Myxococcales Conserved Myxococcales Conserved Conserved Myxococcales Myxococcales Myxococcales M. xanthus Myxococcales Myxococcales M. xanthus Conserved Myxococcales Myxococcales Myxococcales M. xanthus M. xanthus M. xanthus Myxococcales Conserved Conserved Myxococcales Conserved Conserved Myxococcales Myxococcales Conserved Myxococcales Myxococcales Myxococcales

vegetative cells biotinylation

+ + + + + + + + + + + + + + + + + + + + + + + +

developmental cells

OMV

+ + + + +

+ +

+ + + + + + + + + + + + + + +

+ + +

+ +

+

+

+

+ + + + +

+ +

+ + + + + + + + +

+ +

+

+ + + +

+ + + + +

OMV

+

+ + +

biotinylation

+ + + +

+

+

+ + + + + + + +

+ + + + + + + + + + + + + + + + +

+ + + +

+

+ + +

+ + +

+ + + +

+ + + + +

+ +

+ + + + + + + + + + + + + +

+ + + + + + + + + +

+ + + + + +

+ + + + +

+ + + + +

+ + + + + +

a Orthologous proteins were identified using a pair wise reciprocal best BLASTP hit method50 in combination with an expect score cutoff of 1 × 10-5 and homology over at least one-third of the query sequence length. The phylogenetic distribution of proteins is as follows: Proteins labeled conserved have orthologs in least one species outside the Myxococcales; proteins labeled Myxococcales have orthologs in at least two of the four Myxococcales species with completely sequenced genomes (Anaeromyxobacter dehalogenans,80 Stigmatella aurantiaca (http://www.ncbi.nlm.nih.gov/sites/ entrez?Db)genome&Cmd)ShowDetailView&TermToSearch)5526, Sorangium cellulosum81 and M. xanthus 82); proteins labeled M. xanthus have no orthologs.

the competition for amino acids and short peptides with other microorganisms. Given the large number of predicted periplasmic proteases (see below), the periplasmic space might function as a special compartment for the digestion of such polypeptides.

Alternatively, TonB-dependent transporters may act together with inner membrane ExbB/ExbD/TonB complexes. Such complexes represent ideal systems to transmit energy from the cytoplasmic membrane to the cell surface for purposes other than substrate transport. This could include the delivery of Journal of Proteome Research • Vol. 9, No. 10, 2010 5203

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Kahnt et al.

Table 4. Outer Membrane Vesicle Proteins in M. xanthus with Predicted Hydrolytic Functions systematic gene name

gene name

detected in vegetative cells

predicted subcellular localizationa and annotation

biotinylation

Lipoproteins MXAN0805 MXAN3160 MXAN4534 MXAN5933

peptidase, M10A/M12A subfamilies peptidase, M13 (neprilysin) family Chitinase, class I peptidase, M48 (Ste24 endopeptidase) family

Periplasmic proteins MXAN0220 MXAN1650 MXAN2253 MXAN2661 MXAN2814 MXAN2906 MXAN3564 MXAN5326 MXAN5970

lipase, alpha/beta fold family peptidase, S1A (chymotrypsin) subfamily putative peptidase, S37 family 5-nucleotidase family protein N-acetylmuramoyl-L-alanine amidase penicillin acylase family protein peptidase, M36 (fungalysin) family putative phytase peptidase, S8 (subtilisin) family

+

Cytoplasmic proteins MXAN1389 MXAN2016 MXAN2236 MXAN2382 MXAN4935 MXAN5407 MXAN6266 MXAN6601

putative alkaline phosphatase prolyl endopeptidase precursor Pep putative 5-nucleotidase peptidase, M18 (aminopeptidase I) family putative esterase choloylglycine hydrolase family protein putative 2,3-cyclic-nucleotide 2-phosphodiesterase peptidase, S9C (acylaminoacyl-peptidase) subfamily

+ +

a

+

+ + + + +

+ +

biotinylation

+ + + +

+ +

OMV

+ +

+

+ + + +

+

+ +

+ +

+

+ +

+

+ +

+

+ +

+ + +

+ + + + + + +

+ + + +


energy to the A-motility system22 or the general stabilization of the cell envelope. Also a role in cell division, in analogy to the Tol-Pal system,62 could be envisaged. In this context, the receptor MXAN0856 is an interesting example. MXAN0856 forms a putative transcription unit with two lipoproteins (MXAN0855 and MXAN0857). MXAN0855 contains a peptidoglycan-binding domain conserved in OmpA, MotB and Pal.63 MXAN1450 (Oar) is another example. For this protein, a function in cellular adhesion during fruiting body formation has been suggested.38 The M. xanthus genome encodes 15 TolC paralogs,8 which comprise the outer membrane components of efflux pumps or of type I secretion systems.64 Among the 15 paralogs, 5 were detected in our analyses in both vegetative and developmental cells. Three outer membrane proteins detected in both vegetative and developmental cells carry a C-terminal domain that is conserved in the peptidoglycan-binding domains of OmpA, MotB and Pal.63 All 3 secretin paralogs encoded by the M. xanthus genome8 were also identified. Secretins form outer membrane complexes that are essential for T4P biogenesis and type II and III protein secretion systems.65 Interestingly, the detection profiles of the 3 secretions were different. PilQ, the secretin of T4P system, was only detected in OMV from developing cells. PilQ is localized to the cell poles of the rodshaped M. xanthus cells.19,39 These observations suggest that the sites of OMV biogenesis include the cell poles. In addition, a putative long-chain fatty acid transporter (FadL), two potential porins (MXAN0562 and MXAN4727), and a homologue of Omp85 (YaeT), the central component of the outer membrane assembly machinery,66 were identified. Lipoproteins in Intact Cells and in Membrane Vesicles. Among a total of 85 lipoproteins identified, 25 were detected in both methods whereas 11 were only detected in OMV (Table 2; Tables S1 and S2, Supporting Information; Figure 1). Sixtytwo of the 85 (73%) lipoproteins identified are hypothetical proteins or proteins with conserved domains of unknown function. Among these, 30 are unique to the Myxococcales or M. xanthus. Among lipoproteins with functionally characterized 5204

+

detected in developmental cells

OMV


domains, 15 enzymes with predicted hydrolytic functions comprise the largest group. Four of these proteins were detected in OMV (Table 4). Lipoproteins are either anchored to the inner or in the outer membrane via three fatty acids that are attached to the N-terminal cysteine residue that immediately follows the cleavage site of lipoprotein signal peptides.67 In Escherichia coli, the final destinations of lipoproteins depends on the residue at position +2, which is immediately after the lipid-modified Cys.68 Lipoproteins with an aspartate residue at this position are retained in the inner membrane whereas those having other amino acids are targeted to the outer membrane by the Lol system. For lipoproteins from Pseudomonas aeruginosa, additional inner membrane retention signals have recently been identified including amino acids at positions +3 and +4.69,70 We speculate that M. xanthus also harbors a system for sorting lipoproteins to the inner and outer membranes and that the 49 lipoproteins detected only in the biotinylation experiments are primarily located in the inner membrane and that the lipoproteins identified in OMV are anchored in the outer membrane. The lipoproteins detected in this report represent and invaluable resource for analyzing mechanisms involved in lipoprotein sorting in M. xanthus. Luminal Load of Vesicles. Among a total of 97 periplasmic proteins detected, 19 (20%) were detected by both methods, 65 (66%) only by the biotinylation method and 13 (14%) only in OMV. 56 of the 97 predicted periplasmic proteins are of unknown function. 27 proteins are predicted to have hydrolytic functions (Table S1, Supporting Information). Of these 27 proteins, 9 were detected in OMV (Table 4). These 9 proteins include 4 proteases, a peptidoglycan hydrolase, a lipase, a penicillin amidase, which might serve as a defense against betalactam antibiotics, and a 5-nucleotidase as well as a phytase, which are possibly involved in the mobilization of phosphate from organic sources such as DNA/RNA and phytic acid. Surprisingly, we also detected a significant number of proteins in OMV with a predicted cytoplasmic localization. These proteins may represent a contamination with cytoplas-

research articles

Outer Membrane Proteome of Myxococcus xanthus Table 5. M. xanthus Proteins Only Detected in Developmental Cells systematic gene name

gene name

predicted subcellular localization and annotationa

phylogenetic distributionb

detected in biotinylation

OMV

+

+ + + +

Integral outer membrane proteins MXAN0162 conserved hypothetical protein MXAN1329 conserved hypothetical protein MXAN3667 hypothetical protein MXAN5772 pilQ type IV pilus secretin

Conserved Myxococcales M. xanthus Conserved

Lipoproteins MXAN1063 MXAN1342 MXAN1981 MXAN2183 MXAN2290 MXAN3417 MXAN4653 MXAN4668

putative lipoprotein putative lipoprotein hypothetical protein putative lipoprotein putative lipoprotein putative lipoprotein putative lipoprotein hypothetical protein

Myxococcales Conserved Myxococcales M. xanthus Conserved Conserved Myxococcales Conserved

Periplasmic proteins MXAN0133 hypothetical protein MXAN1020 hypothetical protein MXAN1769 NlpC/P60 family protein MXAN2422 hypothetical protein MXAN2708 putative organic solvent tolerance protein MXAN3285 peptidase, S8A (subtilisin) subfamily MXAN3676 hypothetical protein MXAN3719 Mce family protein MXAN3807 hypothetical protein MXAN4269 conserved hypothetical protein MXAN4433 peptidase, S1C (protease Do) subfamily MXAN4439 hypothetical protein MXAN4915 conserved domain protein MXAN5391 hypothetical protein MXAN5587 hypothetical protein MXAN6116 hypothetical protein

Myxococcales M. xanthus Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Myxococcales Conserved Myxococcales Conserved Myxococcales

+ + + +

Integral inner membrane proteins MXAN1081 NADH dehydrogenase I, M subunit MXAN3319 secY preprotein translocase MXAN3554 M23 peptidase domain protein MXAN4134 putative membrane protein MXAN4691 secD protein-export membrane protein MXAN5716 hypothetical protein

Conserved Conserved Myxococcales Conserved Conserved Myxococcales

+ + +

Cytoplasmic proteins MXAN0094 amidohydrolase family protein MXAN0412 conserved hypothetical protein MXAN0804 hypothetical protein MXAN1896 serine/threonine protein kinase MXAN2236 putative 5-nucleotidase MXAN2787 homogentisate 1,2-dioxygenase MXAN3108 FHA domain/tetratricopeptide repeat protein MXAN3143 putative xanthine dehydrogenase MXAN3182 serine/threonine protein kinase MXAN3556 hypothetical protein MXAN4219 alpha keto acid dehydrogenase complex, E3 component, lipoamide dehydrogenase MXAN4494 putative Phage tail sheath protein MXAN4495 conserved hypothetical protein MXAN4564 2-oxoisovalerate dehydrogenase complex, E1 component, alpha subunit MXAN4565 2-oxoisovalerate dehydrogenase complex, E1 component, beta subunit MXAN5265 Kelch domain protein MXAN5266 hypothetical protein MXAN5407 choloylglycine hydrolase family protein

Conserved Conserved Myxococcales Myxococcales Conserved Conserved Conserved Conserved Myxococcales Conserved Conserved Conserved Conserved Conserved Conserved Conserved M. xanthus Conserved

+

a


b

+

+

+ + + + +

+ + +

+

+ + +

+ + + +

+ + + + + +

+ + +

+

+ + + + + + + + +

+ + + + + +

+ + +


mic proteins. However, OMV are lacking abundant cytoplasmic proteins (e.g., ribosomal proteins, chaperonins and translation factors) which strongly argue against a contamination of the vesicles by cytoplasmic proteins. Among the 40 proteins with a predicted cytoplasmic localization detected in OMV, 8 are predicted to have a hydrolytic function including 3 proteases and 3 enzymes possibly involved in mobilization of phosphate (Table 4). We do not know how cytoplasmic proteins become enriched in OMV; however, the reproducibility with which these proteins were detected may indicate that these proteins are specifically sorted to OMV. Remarkably, two of the cytoplasmic proteins detected in OMV belong to the branched-chain keto acid dehydrogenase (BCKAD) complex (also termed the E-signal),71 namely the

alpha and beta subunit of the 2-oxoisovalerate dehydrogenase (E1 component) (MXAN4564 and MXAN4565) (Table S1, Supporting Information). Interestingly, these two proteins were specifically detected in vesicles from developmental cells (Table S1, Supporting Information). The BCKAD complex is involved in the synthesis of iso- and anteiso-fatty acids and secondary metabolites.72 However, a catalytic role within the vesicles seems unlikely as this would depend on the presence of coenzyme A, lipoamide and NAD+ and would require the recycling of NAD+. An important issue concerns the function of outer membrane vesicles in M. xanthus. It has been shown that membrane vesicles released from P. aeruginosa are able to lyse a variety of Gram-negative and Gram-positive bacteria.73 Given the Journal of Proteome Research • Vol. 9, No. 10, 2010 5205

research articles predatory life style of M. xanthus, lysis of prey cells could be an important function of membrane vesicles. In line with this suggestion, OMV contain a large number of proteins with predicted hydrolytic function (Table 4). Vesicle formation increases approximately 8-fold during starvation (data not shown). This increase in vesicle formation could be a response to increase the predatory activity in order to exploit new sources of nutrients. Alternatively, outer membrane vesicle production could help to remodel the cell envelope during development by removing unwanted outer membrane or periplasmic proteins and peptidoglycan.74 In E. coli vesicle formation increased with the accumulation of misfolded proteins in the periplasmic space and an enrichment of misfolded proteins in the vesicles was observed.75 Finally, vesicles offer the possibility to exchange proteins or low molecular mass compounds between cells. In this context, it is particularly striking that we specifically detected the two cytoplasmic BCKAD-enzymes in OMV from developmental cells. M. xanthus mutants lacking the BCKAD complex have a strong developmental phenotype.71 Development of these mutants is restored by codevelopment with wildtype cells. Because of this extracellular complementation the complex was designated as the E-signal.71 The extracellular complementation of E-signal mutants by wild-type cells could be accomplished via membrane vesicles, either by the transfer of the BCKAD complex present in vesicles or by the transfer of phospholipids containing branched-chain fatty acids. In principle, the incorporation of luminal cargo into OMV could be a selective process for a subset of proteins or could occur by bulk-flow. Intriguingly, among the 84 periplasmic proteins detected by the biotinylation approach, only 19 were also detected in vesicles. We speculate that the significant difference between the two methods in detecting proteins with a predicted periplasmic localization could involve the specific sorting of periplasmic proteins to OMV as has previously been suggested for OMV formation in E. coli and Xanthomonas campestris.35,36 According to this model, the biotinylation method detects a large complement of periplasmic proteins because it penetrates the outer membrane; however, proteins detected in OMV only include those which are sorted to the OMV. Developmentally Regulated Outer Membrane Proteins. To begin to understand how the outer membrane proteome of M. xanthus is remodeled during development, the outer membrane proteome and the proteome of outer membrane vesicles were determined for both vegetative cells and for cells after 18 h of starvation. Since we did not perform a quantitative analysis, we focus here on proteins that were exclusively detected under either vegetative or developmental conditions. In total, 114 proteins were only detected in vegetative cells including 7 integral outer membrane proteins, 29 lipoproteins and 37 periplasmic proteins (Figure 1; Tables S1 and S2, Supporting Information). 51 proteins were only detected in developmental cells (Table 5; Tables S1 and S2, Supporting Information). These 51 proteins include three hypothetical integral outer membrane proteins (MXAN0162, MXAN1329 and MXAN3667). MXAN0162 and MXAN1329 have orthologs in different bacterial species, in particular organisms belong to the Cytophaga-Flavobacterium-Bacteroides group, which use gliding motility for cell movement. MXAN3667 is unique to M. xanthus and has no homologues in current databases. Eight lipoproteins were specifically detected in developmental cells. Four of these proteins are specific to the Myxococcales or M. xanthus whereas the remaining five are conserved in other 5206


Kahnt et al. species outside the Myxococcales. Sixteen periplasmic proteins were specifically detected in developmental cells. Twelve of these are of unknown function, and one is a predicted protease. These 51 developmental specific proteins are candidates for having specific functions in development. Concluding Remarks. Forty percent of M. xanthus genes are predicted to encode proteins of unknown function.8 Among the integral outer membrane proteins, lipoproteins and periplasmic proteins identified in this study, 59, 73 and 58%, respectively, are of unknown function. Moreover, among these proteins, approximately 35% (presently) have orthologs only in the Myxococcales. Thus, among the proteins detected in this study, there is a clear overrepresentation of proteins of unknown function suggesting that M. xanthus in particular and Myxococcales in general may possess many novel mechanisms involved in cell envelope function. This study provides the first global proteomic profile of the outer membrane of M. xanthus and the first proteomic characterization of M. xanthus outer membrane vesicles. The comparison between both data sets clearly shows that the vesicles derive from the outer membrane. Moreover, our data provide evidence that OMV represent a subset of the total outer membrane proteome of M. xanthus, thus supporting the notion that OMV derive from a specific sorting process. We have identified a large number of outer membrane proteins of unknown function and many of which are unique to Myxococcales. The functions of these proteins and the concepts presented will be addressed in future genetic studies.

Acknowledgment. We greatly acknowledge Ralf Koebnik for helping with the analysis of TonB-dependet transporters. This work was supported by the Max Planck Society. Supporting Information Available: Supplementary figure and tables. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 2003, 67 (4), 593–656. (2) Dworkin, M. Recent advances in the social and developmental biology of the Myxobacteria. Microbiol. Rev. 1996, 60, 70–102. (3) Lueders, T.; Kindler, R.; Miltner, A.; Friedrich, M. W.; Kaestner, M. Identification of bacterial micropredators distinctively active in a soil microbial food web. Appl. Environ. Microbiol. 2006, 72, 5342–5348. (4) Reichenbach, H. Biology of the Myxobacteria: Ecology and Taxonomy. In Myxobacteria II; Dworkin, M., Kaiser, D., Eds.; American Society for Microbiology: Washington, D.C., 1993; pp 13-62. (5) Rosenberg, E.; Keller, K. H.; Dworkin, M. Cell density-dependent growth of Myxococcus xanthus on casein. J. Bacteriol. 1977, 129, 770–777. (6) Bode, H. B.; Mu ¨ ller, R. Secondary metabolism in Myxobacteria. In Myxobacteria: Multicellularity and differentiation; Whitworth, D. E., Ed.; ASM Press: Washington, D.C., 2008; pp 259-282. (7) Sudo, S.; Dworkin, M. Bacteriolytic enzymes produced by Myxococcus xanthus. J. Bacteriol. 1972, 110 (1), 236–245. (8) Konovalova, A.; Petters, T.; Søgaard-Andersen, L. Extracellular biology of Myxococcus xanthus. FEMS Microbiol. Rev. 2010, 24, 89–106. (9) Hodgkin, J.; Kaiser, D. Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement. Mol. Gen. Genet. 1979, 171, 177–191. (10) Wu, S. S.; Kaiser, D. Genetic and functional evidence that type IV pili are required for social gliding motility in Myxococcus xanthus. Mol. Microbiol. 1995, 18, 547–558. (11) Clausen, M.; Jakovljevic, V.; Søgaard-Andersen, L.; Maier, B. High force generation is a conserved property of type IV pilus systems. J. Bacteriol. 2009, 191, 4633–4638.

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