Proteomic Analysis of the Carbonate Insoluble Outer Membrane

Outer Membrane Proteome of Burkholderia pseudomallei and Burkholderia mallei From Diverse Growth Conditions. Mark A. Schell , Peng Zhao , and Lance ...
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Proteomic Analysis of the Carbonate Insoluble Outer Membrane Fraction of the Soft-Rot Pathogen Dickeya dadantii (syn. Erwinia chrysanthemi) Strain 3937 Lavanya Babujee,§ Balakrishnan Venkatesh,§ Akihiro Yamazaki, and Shinji Tsuyumu* Laboratory of Plant Pathology, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan Received August 20, 2006

We present results of the first comprehensive proteomic analysis of the outer membrane of the bacterial phytopathogen Dickeya dadantii strain 3937 and its response to virulence-contributing factors such as host plant extract, acidic stress, and iron starvation. We analyzed the carbonate-insoluble membrane fractions, which are highly enriched for outer membrane proteins, using two-dimensional electrophoresis and identified the proteins by MALDI-TOF MS. Forty unique proteins were identified, some of which were differentially expressed under the above conditions. Keywords: Dickeya dadantii • outer membrane • proteome • virulence factors • plant extract • acidic stress • iron starvation • post-translational modifications

Introduction The Gram-negative bacteria differ from the Gram-positive bacteria in that the former possess an outer membrane (OM) that effectively controls permeation as well as export of substances. The OM is an asymmetric bilayer consisting of lipopolysaccharides (LPS), phospholipids, lipoproteins, and proteins. LPS are amphipathic molecules that line the outer leaflet of the OM and protect the cell from hostile environments, and in the case of pathogens, they play a direct role in interactions with eukaryotic host cells.1,2,3 Phospholipids line the inner leaflet and serve mainly as a permeability barrier. They also provide the environment with many enzyme and transporter proteins, and influence membrane-related processes.4 A majority of the proteins at the OM are structural proteins, porins, and high affinity receptors. In addition, some enzymes are also present. Structural proteins bind covalently to the peptidoglycan and serve to bind the OM to the rest of the cell wall. Porins (β-barrel proteins) form hydrophilic channels that function in active ion transport, passive nutrient intake, membrane anchoring, defense against attacking proteins, and adhesion during the early steps of the infection process.5 The receptor proteins transport molecules such as ferric iron, vitamin B12, and fatty acids, which are too large to get through the channels formed by the porins. Many of these receptors are also used by bacteriophages and colicins. The predicted number of outer membrane proteins (OMPs) differs among different bacterial genera (eg., 63 in Pasteurella multocida,6 86 in Eschericia coli (the E. coli cell envelope protein data collection (ecce) http://www.cf.ac.uk/biosi/staff/ehrmann/ * Corresponding author. Mailing address: Laboratory of Plant Pathology, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka 422-8529, Japan. Phone/fax: (81)-54-238-4823. E-mail: tsuyumu@ agr.shizuoka.ac.jp. § Both authors contributed equally to this work.

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Journal of Proteome Research 2007, 6, 62-69

Published on Web 12/07/2006

tools/ecce/ecce.htm), 140 in Caulobacter crescentus,7 and 177 in Pseudomonas aeruginosa (Pseudomonas aeruginosa Community Annotation Project). Some OMPs are constitutively expressed and present in high copy numbers such as the trimeric protein LamB (up to ≈30 000 copies8) and the OmpA protein (>105 copies per cell9). Others, such as the solutespecific channels for carbohydrates, nucleosides, and so forth, are induced under special growth conditions.10-16 The expression of OMPs may be modulated by several factors including temperature,17,18 pH and osmolarity,19 availability of nutrients,20-23 and the presence of toxic substances.24 Phytopathogens are extremely important because of their economic impact in the field of agriculture. Although plant pathogenic bacteria share many features with animal and human bacterial pathogens, the fact that none of the bacterial plant pathogens (barring a few exceptions) are true pathogens to humans or other animals indicates a high specificity at the kingdom level.25 The soft-rot group Erwinia is an important plant pathogen for which Dickeya dadantii and Erwinia carotovora (Eca) are the major species. It has recently been shown that Erwinia can also infect insects.26,27 D. dadantii is associated with systemic infections, vascular disorders, foliar necroses, and latent infections in growing plants of many species resulting in huge economic losses.28 The pathogenicity of soft-rot erwiniae is mainly due to the secretion of cell-wall degrading enzymes occurring after their entry into the plant.29 Their ability to acquire iron and to adapt to apoplastic pH are also crucial for virulence besides other surface structures such as pili, flagella, LPS, and exopolysaccharides.30 It has been hypothesized that the OM properties of environmental and pathogenic groups of bacteria are different and reflect the adaptation of the latter to pathogenicity.31 Thus, altered global expression patterns may be expected for OMPs as the bacteria tailor their gene expression to adapt themselves inside the host plant.32 The role of OMPs in virulence has been demonstrated in several 10.1021/pr060423l CCC: $37.00

 2007 American Chemical Society

Proteomic Analysis of the OM Fraction D. dadantii Strain 3937

human pathogens. There are, however, few such reports from plant pathogens. OMPs that have been implicated in virulence in D. dadantii are the high-affinity receptors for siderophores, Fct and FepA,33 and KdgM, a voltage-dependent, slightly anion selective porin.34 In addition, a green fluorescent protein-based IVET (in vivo expression technology) approach detected changes in OMP encoding genes such as the homologue of yhhA (ID 19164) during infection of spinach by D. dadantii 3937.35 HrcC, an OM porin required for a functional type III secretion system, has also been implicated in virulence of D. dadantii.36 HecA, a member of a class of adhesins, is important in the virulence of D. dadantii on Nicotiana clevelandii seedlings and contributes to attachment, aggregation, and epidermal cell killing. Proteomics-based approaches are useful to define the composition of, and to track dynamic changes at, the OM during specific stages of growth or in response to diverse environmental conditions. Although OM proteomes of several Gram-negative bacteria have been described in recent years,21,37-44 there has been no such report as yet for phytopathogenic Gram-negative bacteria, probably as a consequence of underrepresentation of phytopathogens in genome sequencing projects. D. dadantii strain 3937 and Eca strain SCRI1043 (ATCC BAA-672) have been adopted as model organisms to understand the molecular biology and pathogenicity of the soft-rot erwiniae. The availability of the genome sequence of these phytopathogens (http://www.tigr.org/ tdb/mdb/mdbinprogress.html, http://asap.ahabs.wisc.edu/ annotation/php/ASAP1.htm), together with developments in protein analytical techniques such as two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), now enables the rapid and comprehensive analysis of their OM proteome. We carried out a proteomic investigation of the OM of D. dadantii 3937 to identify its constituent proteins and to investigate the modulation of expression of some of these proteins in the presence of host plant extract, in acidic conditions, and during iron starvation.

Experimental Section Bioinformatic Analyses. The predicted proteome of D. dadantii 3937 (version 6.0) was used for all bioinformatic analyses. The subcellular localization prediction tool PSORTbv.2.0 (http://www.psort.org/psortb/) was used to predict the OM subproteome from the total proteome of D. dadantii. For prediction of β-barrel OMPs, the β-barrel OMP predictor (BOMP) (http://www.bioinfo.no/tools/bomp) was used. Lipoproteins were predicted using LipoP 1.0 (www. cbs.dtu.dk/services/LipoP/), and Lipo (http://www.bioinfo.no/ tools/lipo). To check for the presence of signal peptide, the program SignalP (http://www.cbs.dtu.dk/services/SignalP/) was used. The Pfam server (http://pfam.wustl.edu/ hmmsearch.shtml) was used to assign the proteins to the families. The pred-TMBB server (http://bioinformatics.biol. uoa.gr/PRED-TMBB/) was used to predict the presence of transmembrane β-barrels. Growth and Harvest of D. dadantii. D. dadantii 3937 was grown in M63 minimal medium.45 A 2% (v/v) inoculum was then transferred into a larger volume of the same medium and incubated at 27 °C overnight. Cells were harvested at lateexponential phase by centrifugation at 6000g, transferred to fresh medium containing 0.4% (v/v) hot water extract (pH 5.3) of Saintpaulia ionantha (African violet) prepared as described before,46 and incubated for further 6 h before the cells were harvested. Acid stress was applied by transferring cells at the

research articles exponential phase to minimal medium of pH 5.8 for 6 h. Ironlimiting conditions were provided by transferring cells at the exponential phase to minimal medium that lacked iron and contained 30 µM of the iron chelator EDDHA (ethylenediamine-N,N′bis(o-hydroxyphenyl)acetic acid) and incubating for 6 h. Preparation of D. dadantii Outer Membrane. OMs were prepared by the alkaline sodium carbonate method47 as follows. Harvested cells were washed twice in 50 mM Tris, pH 7.3. The cells were then resuspended in the same buffer containing 2 mM PMSF and sonicated on ice for 5 min at 60% power and a duty cycle of 4 in an ultrasonic disrupter UD-201 (Tommy Japan, Inc.). Then, 0.1 mg DNase (Promega) was added, and the suspension was incubated for 15 min on ice. The supernatant was diluted and, after the addition of 1 M Na2CO3 (pH 11) to a final concentration of 0.1 M, shaken on ice for 1 h. The OM was pelleted by centrifugation at 115 000g, washed twice with buffer, and stored at -70 °C until use. Protein Estimation. Protein content in the OM fractions was estimated according to the Bradford assay (Bio-Rad Laboratories) using bovine serum albumin as the standard. 2-DE. Immobilized pH gradients (IPGs) (18 cm, pH 3-10NL; Amersham-Pharmacia, Uppsala, Sweden) were used for the first dimension. For analytical purposes, 50 µg of protein were resolved. Gels were obtained using 300 µg of protein if proteins were to be subsequently analyzed by mass spectrometry. Protein pellets were resolved in solubilization/rehydration solution containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate), 0.5% (w/v) dithiothreitol, and 0.5% (v/v) corresponding IPG buffer (Amersham-Pharmacia, Uppsala, Sweden). Isoelectric focusing was performed with an IPGphor system (Amersham-Pharmacia, Uppsala, Sweden) at 20 °C. IPG strips were rehydrated for 12 h at 30 V in 350 mL of sample in rehydration solution. Proteins were focused with the following program: 30 V for 12 h (active rehydration), 200 V for 1 h, 1000 V for 1 h, 3000 V for 1 h, 3000-8000 V for 1 h, and finally at 8000 V for a total of 65 kVh. After focusing, the IPG strips were reduced and alkylated in equilibration buffer. Second dimension SDS-PAGE was accomplished on 12.5% polyacrylamide gels using Ettan DALTsix electrophoresis unit (Amersham Biosciences) at 20 °C with 2 W/gel for 30 min followed by 15 W/gel until the dye front migrated nearly to the end of the gel. Analytical gels were fixed overnight and stained with silver.48 Colloidal Coomassie blue staining was carried out for preparative gels.49 Gel images were captured using a digital camera (Nikon). Image manipulation and analysis were performed using Adobe Photoshop CS2. Spots that were present on gels from three independent preparations were selected for further analysis by tryptic digestion and peptide mass fingerprinting via MALDI-TOF-MS. Phospho- and Glycoprotein Staining. Phospho- and glycoprotein staining were performed using Pro-Q Diamond phosphoprotein gel stain and Pro-Q Emerald 488 glycoprotein gel stain, respectively (Invitrogen detection technologies) according to the manufacturer’s instructions. Preparation of Samples for MALDI-TOF-MS. Spots were excised from the gels and subjected to in-gel digestion protocols described by Shevchenko et al.50 After 16 h, the supernatant was recovered, and the remaining peptides were extracted three times with 50% acetonitrile/1% trifluoroacetic acid. The supernatants were pooled, and the peptides were concentrated and purified using ZipTips. The purified peptides were eluted Journal of Proteome Research • Vol. 6, No. 1, 2007 63

research articles with 0.5 µL of matrix solution (2 mg/mL R-cyano-4-hydroxycinnamic acid (Bruker Daltonics GmBH, Germany) in 50% acetonitrile/0.5% trifluoroaceticacid) directly on a polished stainless steel target plate (MTP 384 target plate, Bruker Daltonics GmBH, Germany) and dried in ambient air. MALDI-TOF-MS and Peptide Mass Fingerprinting (PMF). The MALDI-TOF-MS analyses were performed using the AutoFlex (Bruker Daltonics, GmbH, Germany) mass spectrometer fitted with a nitrogen UV laser (20 Hz, 337 nm) incorporating a TOF mass analyzer. Spectra were acquired in a positiveion mode at 20 Hz laser frequency. Data were collected in the positive ion mode over the mass/charge (m/z) range of 5002500 and processed using the FlexAnalysis software, generating a monoisotopic peak list from the spectrum. Spectra were externally calibrated with peptide calibration standards (Bruker Daltonics GmBH, Germany). Between 1000 and 2000 single scans were accumulated for each mass spectrum. All spectra were noise-filtered and deisotoped using the Data Explorer Version 4.0 software (Applied Biosystems). Deisotoped peaks were automatically labeled from the software, and the peaks were used for database search. Autolytic tryptic peptides were used for internal calibration. Protein Identification. For identification of proteins, the D. dadantii protein database of theoretical masses (available at http://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm) was searched with the data obtained from the MS analysis with a peptide mass tolerance of (60 ppm including a maximum of one missed cleavage utilizing the 5.01 version of GPMAW (Lighthouse data, Odense, Denmark) installed on a local server. For confident spot identification, parameters including the number of peptides matched against the nonmatching number, sequence coverage, molecular mass, and pI were used. The theoretical pI, molecular mass, and grand average of hydrophobicity (GRAVY) value of the identified proteins were predicted using the ProtParam tool available at www.expasy.org.

Results and Discussion The D. dadantii 3937 Outer Membrane Proteome Bioinformatic Predictions. We used several programs to obtain an overview of the OMPs in D. dadantii 3937 whose predicted proteome constitutes 4637 protein sequences. The usage of programs in combination is generally recommended to increase the reliability of the predictions and helps to obtain a better coverage.42,51 PSORTb v.2.0 predicted about 76 proteins (including hypotheticals) to be located at the OM. For these proteins, the prediction scores were g9.5 (Supplementary Table 2, Supporting Information). BOMP predicted 68 possible β-barrel OMPs (1.47%), a lower percentage than that predicted for other Gram-negative proteomes (1.8-3%).66 Of these, 8 sequences were found by BLAST to have significant similarity to well-known non-OM β-barrel proteins. These proteins with BOMP categories 3 (1 protein) and 1 (7 proteins) may be considered as false positives. Thirteen sequences, not predicted by BOMP, were found by BLAST to have significant similarity to known β-barrel OMPs. About 47% (28 sequences) of the predicted BOMPs in D. dadantii have no significant hit in the Swiss-Prot or TrEMBL databases (Supplemetary Table 3, Supporting Information). Of these, 7 were neither recognized by PSORT as OMPs nor were they assigned to any family by Pfam. 64

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Lipoproteins. In addition to the integral β-barrel OMPs, the lipoproteins anchored to the OM are also categorized as OMPs. When the predicted proteome of D. dadantii was analyzed with lipoprotein prediction programs, Lipo found 114 (2.5%) sequences that satisfied the criteria for OM lipoproteins given by this program, 85 (1.8%) sequences were predicted as lipoproteins by LipoP, whereas 68 sequences were predicted as lipoproteins by both programs. Of the 31 sequences predicted by Lipo as having a low probability to be considered as lipoproteins, only 2 were predicted as lipoproteins by LipoP. By comparison, the percentage of sequences encoding lipoproteins in some closely related enterobacteria as predicted by DOLOP, a database for bacterial lipoproteins (http://www. mrc-lmb.cam.ac.uk/genomes/dolop/), is as follows: E. carotovora (2.5%), E. coli (1.6-2.2%), Pseudomonas (1.8-2.2%), Shigella (2.1-2.5%), Xanthomonas (1.3-2.4%), and Yersinia (1.3-1.9%). Characterization of D. dadanti OMPs by 2-DE and MS. For proteome studies, two methods are commonly employed to prepare OMs from Gram-negative bacterial cells, the sodium carbonate method and the lauroyl sarcosine method. We preferred the former to prepare OMs from D. dadantii strain 3937 because, besides being relatively simple, a survey of literature showed that OMs obtained using this method are relatively pure and contain fewer proteins from other subcellular compartments (Supplemetary Table 1, Supporting Information). Our rehydration solution that contained urea, thiourea, and CHAPS was efficient in solubilizing the membrane proteins as indicated by the percentage of recovery on SDS-PAGE (data not shown). Although the inclusion of the detergent ASB-14 (amido sulfobetaine-14) has been beneficial for solubilizing membrane proteins from other bacteria,7,38,39,43,44 in our case, it did not have any specific advantage (data not shown). 2D Analysis. On Coomassie blue-stained gels of OMs from untreated samples, about 100 spots were resolved, consistently, of which 85 were analyzed by mass spectrometry. Seventy-eight spots could be unambiguously identified, and these corresponded to 63 unique proteins (Table 1). Of these, 40 (64%) are predicted to have OM localization by various prediction programs. Further, some of the identified proteins are thought to be associated with the OM (see below). A majority of the identified OMPs had isoelectric points between 4 and 7 and molecular mass between 30 and 80 kDa similar to those of other Gram-negative bacteria.37,38 Good correlation was seen between the theoretical and experimental coordinates for pI and molecular weight for the identified proteins (Supplementary Figure 1, Supporting Information). All of the identified proteins except one (ID 15522; GRAVY 0.005) had negative GRAVY values indicating that they are not very hydrophobic. In this respect, they are similar to the OMPs identified from P. aeruginosa.38 In general, the OM proteome map of D. dadantii showed an abundance of proteins that function either as transporters or as receptors. In total, 31 out of 52 (60%) spots representing 18 different proteins belonged to this group. This is not surprising, since the OM serves primarily as a permeability barrier. Ten different porins were identified from the 2D gels [OmpA, (ID 18822), OmpF (ID 20081), OmpX (ID 18909), KdgN (ID 15523), KdgM (ID 19629), ScrY (ID 16548), ID 20079, ID 15522, and ID 16479]. Of these, the most abundant is an unnamed porin (ID 20079) of the OmpF/OmpC family that is slightly cation-selective. Thus, this porin may serve as the major general purpose porin in D. dadantii. OmpA, an abundant

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Proteomic Analysis of the OM Fraction D. dadantii Strain 3937

Table 1. Outer Membrane Proteins of D. dadantii 3937 Identified in This Study by PMF Using MALDI-TOF/MS peptides matched

coverage

score

GRAVY

cal. mol. mass

6 7 7 9 8 4 6 6 19

20 20 23 26 23 31 30 30 29

133 164 153 195 173 142 168 168 285

-0.267 -0.267 -0.267 -0.463 -0.463 -0.1 -0.879 -0.879 -0.463

37.6 37.6 37.6 40.0 40.0 18.1 26.5 26.5 78.7

8.4 8 7.5 4.4 4.2 3.8 6.5 7.1 5.3

om/10 om/10 om/10 om/10 om/10 om/10 om/10 om/10 om/10

18864 acr

10

16

156

-0.463

78.7 5.7 109.9 5.2

om/10

6a 6b 7a 7b 7c 7d 8a

15732 15732 18200 18200 18200 18200 15907

tolC tolC lamB lamB lamB lamB unknown

13 11 11 10 10 9 8

38 29 44 33 30 26 12

274 200 245 177 210 192 126

-0.313 -0.313 -0.466 -0.466 -0.466 -0.466 -0.566

51.0 51.0 44.3 44.3 44.3 44.3 81.2

om/10 om/10 om/10 om/10 om/10 om/10 om/10

8b

15907 unknown

8

16

158

-0.566

81.2 6.8 126.3 6.9

om/10

9a 9b 10a 10b 11 12 13

20079 20079 15394 15394 16754 17464 16712

8 8 6 4 4 22 15

26 27 16 10 5 31 19

178 139 130 68 54 306 224

-0.41 -0.41 -0.298 -0.298 -0.694 -0.354 -0.569

39.8 39.8 47.0 47.0 96.9 89.0 89.8

om/10 om/10 om/10 om/10 om/9.52 om/10 om/9.52

14 15

20338 yieC 20864 hrcC

2 3

4 5

36 45

-0.425 -0.212

59.6 7.7 74.9 6.3

16 17

17614 yhjL 20427 btuB

6 14

6 23

68 231

-0.441 139.7 7.2 150.4 6.4 -0.491 71.7 7.2 60.7 6

om/9.52 om/10

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32a 32b 33 34 35 36

16548 17619 17128 15522 18361 20470 15021 46580 46916 16852 19681 17309 16479 19543 19555 19555 18822 19117 15508 20157

3 3 3 3 3 2 9 2 7 2 8 2 3 2 13 13 7 3 3 3

5 7 7 22 10 4 37 5 11 5 11 12 8 12 21 21 22 5 15 14

43 45 3 103 65 42 230 45 104 72 89 74 30 81 188 188 161 32 43 55

-0.474 59.2 5.5 56.2 5.75 -0.357 54.2 6.0 53.0 6.3 -0.588 53.0 6.9 50.6 6.25 0.005 23.1 6.8 33.8 5.8 -0.482 38.1 6.5 34.8 6.1 -0.127 41.9 5.8 39.3 5.65 -0.56 34.3 6.1 32.0 6.1 -0.101 94.4 6.7 120.6 6.9 -0.42 87.6 6.1 120.6 5.65 -0.334 32.7 8.7 32.4 8.7 -0.096 112.5 5.6 133.9 5.35 -0.073 19.9 5.8 27.5 6 -0.032 51.8 7.0 56.9 6.3 0.153 19.3 6.4 22.4 6.4 -0.428 69.2 5.3 61.8 4.9 -0.428 69.2 5.3 61.8 5 -0.267 37.6 8.8 34.0 8 -0.405 84.4 6.8 126.3 6.9 -0.427 18.4 6.9 18.0 7.1 -0.307 21.6 9.3 23.0 8.5

om/10 om/9.52 om/10 om/10 uk om/9.92 om/9.93 om/9.49 om/10 om/9.49 om/ ex om/9.52 om/10 uk om/9.49 om/9.49 om/10 om/10 om/10 uk

37 38

14551 hemM 46527 fepA

2 2

11 4

37 24

-0.659 -0.501

23.7 9.0 24.8 8.5 84.7 8.0 126.3 8.0

uk om/9.99

39 40

16056 unknown 19629 kdgM

3 7

27 37

88 180

-0.265 -0.652

16.4 9.4 26.7 8.8

om om/10

spot no.

ID

1a 1b 1c 2a 2b 3 4a 4b 5a

18822 18822 18822 20081 20081 18909 15523 15523 18864

5b

a

gene

ompA ompA ompA ompF ompF ompX kdgN kdgN acr

porin porin fadL fadL yddB yaeT imp

scrY yhjJ prtF porin nlpB wza pldA yebT unknown unknown unknown yfaz porin ybaY ppiD ppiD ompA porin pal yeaY

cal. pI

expt. mol. mass

8.8 41.9 8.8 37.3 8.8 33.2 5.3 46.1 5.3 46.1 5.2 16.8 6.9 31.9 6.9 32.0 5.7 104.9

7.8 57.2 7.8 55.3 5.2 47.2 5.2 51.2 5.2 47.2 5.2 47.2 6.8 126.3

expt. pI

6.9 5.75 4.7 4.8 4.8 5 6.4

6.3 42.7 5.3 6.3 42.4 5.6 5.8 47.2 5.6 5.8 44.0 5.65 5.5 137.0 5.3 5.5 120.6 5.1 5.8 119.2 5.6 63.2 5.65 65.5 6.4

17.0 9.0 32.0 8.0

PSORT-B localization/ probabilitya

om/10 om/10

product

outer membrane protein A outer membrane protein A outer membrane protein A outer membrane protein 1a (Ia;b;F) outer membrane protein 1a (Ia;b;F) outer membrane protein X oligogalacturonate specific porin oligogalacturonate specific porin TonB dependent outer membrane receptor for ferric achromobactin TonB dependent outer membrane receptor for ferric achromobactin outer membrane protein outer membrane protein phage lambda receptor protein phage lambda receptor protein phage lambda receptor protein phage lambda receptor protein unknown protein outer membrane hemin receptor unknown protein outer membrane hemin receptor porin porin outer membrane protein FadL outer membrane protein FadL unknown protein outer membrane protein organic solvent tolerance protein (precursor) outer membrane protein type III secretory pathway, porin component cellulose synthase subunit C outer membrane receptor for transport of vitamin B12, E colicins, and bacteriophage BF23 porin invoved in sucrose transport Zn dep peptidase protease outer membrane protein lipoprotein-34 outer membrane protein outer membrane phospholipase A unknown ferrichrome receptor, put. unknown protein CHP similar to serine proteases unknown protein outer membrane exporter lipoprotein peptidyl-prolyl cis-trans isomerase D peptidyl-prolyl cis-trans isomerase D outer membrane protein A TonB-dependent receptor peptidoglycan-associated lipoprotein starvation-inducible outer membrane lipoprotein outer-membrane lipoprotein Ton B dependent enterobactin receptor unknown protein oligogalacturonate specific porin

Om, outer membrane; ex, extracellular; uk, unknown.

porin in many Gram-negative bacteria was also present at significant levels. Five (IDs 15907, 18864, 46527, 46916, and 19117) of the eight Ton-B-dependent OM receptors were expressed under our

experimental conditions, namely, growth in minimal medium. It has been reported that cells grown in minimal medium express higher number of OM receptors than when grown in nutrient-rich media such as LB.42 Judging by the spot intensity, Journal of Proteome Research • Vol. 6, No. 1, 2007 65

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Babujee et al.

Table 2. Non-Outer Membrane Proteins of D. dadantii 3937 Identified in This Study by PMF Using MALDI-TOF-MS spot no.

D. dadantii ID

41a 41b 41c 42 43 44 45

17130 17130 17130 14785 16955 20398 17358

rspA rspA rspA atpD sdhA glpK hypB

46a 46b 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

14951 14951 19864 14506 19949 15461 18920 46975 17955 17665 19391 19735 17941 46981 18455 14948 16152 18196 47057

ilvC ilvC pncB cueO opgG fkpA unknown unknown fliG unknown unknown fliC flgK gnd unknown unknown unknown unknown ybdH

a

gene name

product

starvation sensing protein starvation sensing protein starvation sensing protein F0F1-type ATP synthase, beta subunit succinate dehydrogenase, flavoprotein subunit glycerol kinase Ni2+-binding GTPase involved in regulation of expression and maturation of hydrogenase ketol-acid reductoisomerase ketol-acid reductoisomerase nicotinic acid phosphoribosyl transferase multicopper oxidase osmoregulated periplasmic glucan synthesis protein FKBP-type peptidyl-prolyl cis-trans isomerase acid phosphatase TolB protein flagellum component methyl accepting chemotaxis protein pectate lyase flagellin, filament structural protein flagellar biosynthesis, hook-filament junction protein unknown phage major capsid protein, P2 family unknown unknown unknown protein, cons. Hyp. Prod. oxidoreductase

exp. pI

localizationa

secretome P-score

48.3 48.9 51.5 52.7 64.0 55.6 30.6

5.4 5.7 6.2 4.6 5.5 5.7 5.9

C C C C C/P C C

0.0952 0.0952 0.0952 0.0708 0.1001 0.0759 0.398

55.3 55.3 45.5 51.2 57.9 35.6 44.5 48.3 44.0 62.5 42.9 35.2 65.9 53.0 39.6 30.6 48.3 46.6 37.8

5.35 5.3 6.7 6.4 5.5 5.3 6.2 5.5 4.2 6.3 6.3 4.8 5.6 5.1 4.8 4.8 6.1 6 6.2

C C P/C P P P P P IM IM EX EX EX uk uk uk uk uk uk

0.0795 0.0795 0.071 0.767 0.8639 0.953 0.783 0.952 0.1577 0.1309 0.949 0.917 0.962 0.107 0.442 0.834 0.946 0.357 0.099

C, cytoplasm; P, periplasm; IM, inner membrane; EX, extracellular; uk, unknown.

the most abundant of these is a hemin receptor (ID 15907) that shows about 40% similarity to other known Ton-B-dependent heme receptors. Although it is thought that four of them specifically transport iron (Glasener et al., unpublished results), it is possible that some may have multiple cargos since minimal medium is not particularly restrictive with regard to iron. The other receptors included the OM efflux protein TolC, LamB (ID 18200), the maltose-inducible general transmembrane diffusion channel protein, FadL (ID 15394), involved in the uptake of long-chain fatty acids (LCFAs) and other hydrophobic compounds,52-54 and BtuB, the vitamin B12 receptor. The abundance of cell surface receptors for the infection of bacterial viruses (phages), for the import of bacterial toxins (colicins), and for the upake of Fe3+ complexed by siderophores and heme reflects the major role of the OM in protecting the cell from the environment and to permit and control the exchange and communication between the environment in which the bacteria live and the interior of the cell. The enzymes that were detected were PldA, PpiD, PrtF, YhjJ, and YhjL. Among other OMPs detected, one (ID 16056) has no homologous equivalent in another organism and therefore may be unique to D. dadantii. The function of seven other proteins, including one hypothetical protein, is currently unknown. No known domains were detected for these proteins by Pfam. Very few lipoproteins were detected on the 2D gels (HemM, NlpB, Pal, YeaY, and YbaY). It is widely acknowledged that due to their poor solubility and tendency to aggregate, lipoproteins are difficult to detect on 2D gels. Other analytical methods may prove useful to study lipoproteins. Proteins of Other Subcellular Compartments. Twenty-three proteins with supposed location in subcellular compartments other than the OM were detected on our 2D gels (extracellular, 3; periplasmic, 6; innermembrane, 2; cytoplasmic, 6; and unknown location, 6, Table 2). Since none of the abundant cytoplasmic marker proteins were detected, the presence of 66

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these non-OM proteins is less likely due to contamination with other cellular compartments. Two different reasons could account for the presence of these proteins in our OM preparations. First, very abundant proteins are difficult to eliminate from OM preparations. This may be the case with flagellumrelated proteins such as FliC, FliG, and FlgK that have frequently been found in OM preparations of several Gramnegative bacteria.47 Second, some of these proteins may be genuinely associated with the OM as may be the case for SdhA and AtpD that have been found in OM preparations from E. coli,37 Bartonella,55 and Caulobacter.7 In fact, Lipo predicts Sdh as an OM lipoprotein. Interestingly, high scores for nonclassical protein secretion (http://www.cbs.dtu.dk/services/SecretomeP/) were obtained for 5 of the 6 proteins predicted to be localized in the periplasm. Of these, the TolB protein was also identified in OMs of E. coli.47 Post-Translational Modifications (PTM). A number of proteins were resolved in multiple spots on 2D gels as shown in Figure 1. This is most likely due to post-translational modifications. It is also suggested that the trains of spots arise due to equilibrium between structural isoforms of the polypeptides generated during the 2-DE procedure.42 Highly modified spots represented Imp, KdgN, LamB, OmpA, OmpF, OmpX, TolC, and YaeT, which are encoded by single genes in D. dadantii 3937. Since the pattern of these multiple spots indicated possible phosphorylation (same MW but different pI), we stained the gels using the phosphorylation-specific fluorescent stain Pro-Q Diamond. We also stained the gels for possible glycosylation, since there is evidence for glycosylation of some OMPs in P. aeruginosa.38 Phosphorylation. A comparison of the staining pattern showed that the LamB protein may be modified by phosphorylation (Figure 2 A). The LamB channel facilitates the influx of a wide variety of carbohydrates when they exist in low concentrations in the environment and has been well-

Proteomic Analysis of the OM Fraction D. dadantii Strain 3937

Figure 1. Carbonate insoluble membrane proteins of D. dadantii strain 3937 were separated by 2D electrophoresis using pH 3-10 nonlinear IPG strips and 12% SDS-PAGE. Proteins were stained with Colloidal Coomassie blue. The identified proteins are numbered, and the protein IDs are provided in Table 1.

Figure 2. 1D SDS-PAGE of outer membrane proteins from D. dadantii 3937. Proteins were stained with Phospho (A) and Glyco (B) stains. Lanes 1, 3, 5, and 7, MW-Marker; lane 1, 2, 5, and 6 total protein stain; lanes 3and 4, Phosphostain; lanes 7 and 8, Glycoprotein staining. (Arrow in panel A, LamB; arrows in panel B, Pal.)

characterized from a number of bacteria. In Aeromonas, a LamB-like protein functions as an adhesion factor.56 Glycosylation. Our results indicate that the peptidoglycanassociated lipoprotein (Pal, ID15508) may be modified by glycosylation (Figure 2B). Pal, a member of the OmpA family of proteins, is highly conserved in Gram-negative bacteria. It is anchored to the OM through an N-terminal lipid attachment and stabilizes it by providing a noncovalent link to the peptidoglycan (PG) layer through a periplasmic domain. D. dadantii pal mutants are attenuated in virulence, lack flagella, and are sensitive to high ionic strength and low osmolarity.57 The identity of yet another glycosylated protein could not be ascertained.

research articles The nature of modifications leading to multiple spots for other proteins could not be ascertained. OmpA was found in multiple spots on 2D gels of E. coli,37 and Shigella flexneri.58 Multiple spots for YaeT have been previously seen on 2D gels of OMPs from S. flexneri, although the nature of modification was not reported.58 Several of these proteins have proposed roles in virulence. For example, YaeT is part of the YaeT complex comprised of the lipoproteins NlpB, YfgL, and YfiO that functions in OM assembly and is thought to play an important role in the infection and immunoreaction.58 OmpA plays a role in adherence and colonization in Pasteurella multocida, Haemophilus influenzae, Chlamydia spp., and E. coli. A 38-kDa OMP of Rahnella aquatilis is involved in the adhesion of this bacterium to wheat roots.59 Therefore, the nature and biological significance of PTM of these proteins may be interesting to investigate. Differential Expression of OMPs under a Variety of Conditions. Using proteomic tools, we examined how the expression of OMPs of D. dadantii 3937 is modified during specific environmental conditions (Figure 3). We chose to examine OMP modulation in the presence of host plant extract, in acidic pH and during iron limitation because the soft-rot diseases caused by D. dadantii and E. carotovora represent the “common cold” of plants in their ubiquity and have greater dependence on environmental factors rather than host genotype. Some proteins appeared to be induced or repressed irrespective of the treatments. One of them is the TolC protein which was induced in all three conditions. This may be because a variety of exporters share TolC as the OM channel tunnel of an energy-driven efflux complex. The D. dadantii TolC protein may thus be functionally similar to the E. coli TolC where it is part of the AcrAB-TolC efflux pump, which is the major efflux system involved in resistance to a variety of toxic molecules, including antibiotics, dyes, and detergents. It has been reported that the D. dadantii tolC plays an important role in the survival and colonization of the pathogen in plant tissue.65 Another protein that was similarly induced is a zinc-dependent peptidase encoded yhjJ (ID 17619), whose role is not yet known. The identity of yet another protein (MW 22 kDa, pI 6.5) could not be established. Presence of Host Plant Extract. The multifunctional outer membrane protein OmpA was highly induced in the presence of host plant extract. Evidence from other pathogens indicate that OmpA is involved in binding to specific host cell receptor molecules. Although the role of OmpA protein in the interaction of D. dadantii with its host has not yet been reported, it is likely that the protein has an important role. Interestingly, it appeared that at least part of the OmpA protein population had undergone an acidic shift (Figure 3B) in the presence of host extract, although the nature of the modification was not investigated in this study. While it is possible that the acidic shift indicates a post-translational modification, it is also likely that the OmpA protein is denatured in the presence of host extract. Another differentially expressed protein was the Hrp pathway protein, HrcC, a member of the PulD/pIV superfamily of proteins that function in OM translocation of type II and type III secretion pathways.60 It has been shown that HrcC is required by D. dadantii to elicit HR.61 A similar role has also been postulated for the protein in Pseudomonas syringae.62 Also induced were the oligogalacturonate-specific porins KdgM and KdgN. It has been reported that the synthesis of KdgM is strongly induced in the presence of pectic derivatives.63 Since D. dadantii deploys Journal of Proteome Research • Vol. 6, No. 1, 2007 67

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Babujee et al.

Figure 3. Representative enlarged partial 2D gels of outer membrane proteins of D. dadantii 3937. Cultures were grown in M63 minimal medium, harvested at the mid-exponential phase, and transferred to fresh medium to provide the conditions indicated: (A) Control; (B) plant extract; (C) acidic pH; (D) iron limitation. Proteins were resolved by 2DE and stained with silver nitrate. Arrows and boxes indicate differentially expressed proteins (1, OmpA; 3, YhjJ; 4, TolC; 5, HrcC; 6, Hemin receptor (ID-15907); 7, FkpA; unknown proteins, (2, 8-10).

pectinolytic enzymes to attack host plants, it may be expected that KdgM and KdgN are induced in the presence of host extract. Acidic Conditions. D. dadantii 3937 lacks the acid survival responses found in other enteric microorganisms probably because phytopathogens do not encounter radical changes in pH in their environment as compared to E. coli and other animal pathogenic enterobacteria. However, in order to survive the acidic milieu in the plant apoplast, erwiniae must deploy several adaptive responses. The pattern of OMP modulation during growth in acid pH was much similar to that due to the presence of host extract (Figure 3C), although we did not use organic acids to maintain the acidic pH. This suggests that a common mechanism of acid tolerance is deployed by D. dadantii 3937 in response to both inorganic and organic acids, at least with respect to modulations of the OMPs. Some acidic proteins that were induced specifically during acid stress could not be identified because of the poor quality of spectra obtained. It is known that acidic proteins in general yield poor spectra that hinder their subsequent identification. Iron Starvation. Iron is important for D. dadantii virulence, and about 2% of its genome encodes for genes with iron-related functions (Glasner et al., unpublished results). It is thought that the remarkable adaptability of the bacterium to diverse environments is, in part, due to the great capacity of its genome to encode high-affinity iron uptake systems. Surprisingly, we did not find proteins specifically induced during the iron starvation conditions used in this experiment. However, the level of an OM heme receptor was slightly higher when the bacterial cells were transferred to iron-depleted medium (Figure 3D). Iron restriction, in general, does not seem to significantly alter expression of OMPs as evidenced by studies carried out for E. 68

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coli47 and Neisseria.64 While in these organisms some of the iron receptors could be detected only upon iron restriction, in D. dadantii, iron receptors were expressed even during growth in minimal medium.

Conclusion In conclusion, computer predictions indicated that the protein composition of the D. dadantii OM subproteome is similar to that of other Gram-negative bacteria studied in a similar manner. In all, we identified 40 OMPs on 2D gels of OMs from D. dadantii 3937. Several proteins exhibited isoelectric heterogeneity, of which at least 1, namely, LamB appears to be phosphorylated, and two proteins, namely, pal and an unknown protein appear to be glycosylated. The nature of modifications leading to multiple spots of several other proteins could not be ascertained. We also examined how the OM protein composition changes with respect to the presence of host plant extract, change in pH, and iron status. We found that several proteins which show altered expression levels appear when the cells are exposed to host plant extract, acid environment, or iron starvation. Thus, proteomic and bioinformatic tools can be useful to identify interesting proteins and to get an idea of their function prior to launching functional studies.

Acknowledgment. This work was supported by grants from the Japan Society for Promotion of Science (JSPS) in the form of a postdoctoral fellowship (no. P04196) and a grant-inaid (no. 03203) awarded to B.V. and in part by a grant-in-aid (no. 17108001) and a grant for promotion in science (no. 13073) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to S.T..

research articles

Proteomic Analysis of the OM Fraction D. dadantii Strain 3937

Supporting Information Available: Tables showing a comparison of sodium carbonate and lauroyl sarcosine methods; lists of D. dadantii’s 76 OMPs prdicted by PSORT, 68 BOMPs predicted by BOMP, 85 sequences with signal peptidase II cleavage site predicted by LipoP, and 119 lipoproteins predicted by Lipo. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Tufano, M. A.; Romano, C.; Sommese, L.; Bentivoglio, C.; Galdiero, F. Microbiologica 1985, 8, 181-190. (2) Galdiero, F.; Gorga, F.; Bentivoglio, C.; Mancuso, R.; Galdiero, E.; Tufano, M. A. Infection 1988, 16, 349-353. (3) Venkatesh, B.; Rudolph, K. Phytopathology 2001, 91, S 91. (4) Cronan , J. E. Annu. Rev. Microbiol. 2003, 57, 203-224. (5) Schulz, G. E. Curr. Opin. Struct. Biol. 2000, 10, 443-447. (6) Boyce, J. D.; Cullen, P. A.; Nguyen, V.; Wilkie, I.; Adler, B. Proteomics 2006, 6, 870-880. (7) Phadke, N. D.; Molloy, M. P.; Steinhoff, S. A.; Ulintz, P. J.; Andrews, P. C.; Maddock, J. R. Proteomics 2001, 1, 705-720. (8) Cellular and Molecular Biology; Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M.; Umbarger, H. E., Eds.; American Society for Microbiology: Washington, DC, 1987. (9) Hancock, R. E. W. ASM News 1991, 57, 175-182. (10) Ferenci, T.; Schwentorat, M.; Ullrich, S.; Vilmart, J. J. Bacteriol. 1980, 142, 521-526. (11) Benz, R.; Hancock, R. E. W. J. Gen. Physiol. 1987, 89, 275-295. (12) Maier, C.; Bremer, E.; Schmid, A.; Benz, R. J. Biol. Chem. 1988, 263, 2493-2499. (13) Szmelcman, S.; Schwartz, M.; Silhavy, T. J.; Boos, W. Eur. J. Biochem. 1976, 65, 13-19. (14) Tommassen, J.; Lugtenberg, B. J. Bacteriol. 1980, 143, 151-157. (15) Hancock, R. E. J. Antimicrob. Chemother. 1981, 8, 249-276. (16) Brass, J. M.; Bauer, K.; Ehmann, U.; Boos, W. J. Bacteriol. 1985, 161, 720-726. (17) Bolin, I.; Norlander, L.; Wolf-Watz, H. Infect. Immun. 1982, 37, 506-512. (18) Ozkanca, R.; Flint, K. P. Lett. Appl. Microbiol. 2002, 35, 533-537. (19) Sato, M.; Machida, K.; Arikado, E.; Saito, H.; Kakegawa, T.; Kobayashi, H. Appl. Environ. Microbiol. 2000, 66, 943-947. (20) Hottes, A. K.; Meewan, M.; Yang, D.; Arana, N.; Romero, P.; McAdams, H. H.; Stephens, C. J. Bacteriol. 2004, 186, 1448-1461. (21) Phadke, N. D.; Molloy, M. P.; Steinhoff, S. A.; Ulintz, P. J.; Andrews, P. C.; Maddock, J. R. Proteomics 2001, 1, 705-720. (22) Vos-Scheperkeuter, G. H.; Hofnung, M.; Witholt, B. J. Bacteriol. 1984, 159, 435-439. (23) Boos, W.; Shuman, H. Microbiol. Mol. Biol. Rev. 1998, 62, 204229. (24) Cha, J. S.; Cooksey, D. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 15, 8915-8919. (25) Montesinos, E. Int. Microbiol. 2000, 3, 69-70. (26) Venkatesh, B.; Babujee, L.; Liu, H.; Hedley, P.; Fujikawa, T.; Birch, P.; Toth, I.; Tsuyumu, S. J. Bacteriol. 2006, 188, 3088-3098. (27) Grenier, A. M.; Duport, G.; Pages. S.; Condemine. G.; Rahbe, Y. Appl. Environ. Microbiol. 2006, 72, 1956-1965. (28) Permbelon, M. C. M.; Kelman, A. Annu. Rev. Phytopathol. 1980, 18, 361-387. (29) Perombelon, M. C. M. Ecology and pathogenicity of soft rot Erwinias: an overview. In Plant Pathogenic Bacteria; Klement, Z, Ed.; Akademia Kiado: Budapest, Hungary, 1990; pp 745-751. (30) Hugouvieux-Cotte-Pattat, N.; Condemine, G.; Nasser, W.; Reverchon. S. Annu. Rev. Microbiol. 1996, 50, 213-257. (31) Bengoechea, J. A.; Diaz, R.; Moriyon, I. Infec. Immun. 1996, 64, 4891-4899. (32) Okinaka, Y.; Yang, C. H.; Perna, N. T.; Keen, N. T. Mol. PlantMicrobe Interact. 2002, 15, 619-629. (33) Expert, D.; Toussaint, A. J. Bacteriol. 1985, 163, 221-227. (34) Condemine, G.; Berrier, C.; Plumbridge, J.; Ghazi, A. J. Bacteriol. 2005, 187, 1959-1965. (35) Yang, S.; Perna, N. T.; Cooksey, D. A.; Okinaka, Y.; Lindow, S. E.; Ibekwe, A. M.; Keen. N. T.; Yang, C.-H. Mol. Plant-Microbe Interact 2004, 17, 999-1008.

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