NopM and NopD Are Rhizobial Nodulation Outer Proteins

NopM and NopD Are Rhizobial Nodulation Outer Proteins: Identification Using LC-MALDI and LC-ESI with a Monolithic Capillary Column Joaõ A. Rodrigues,†,‡ Francisco Javier Lo´ pez-Baena,†,§ Francisco Javier Ollero,§ Jose´ Marıá Vinardell,§ Marıá del Rosario Espuny,§ Ramon A. Bellogıń,§ Jose´ Enrique Ruiz-Sainz,§ Jerry R. Thomas,| Dave Sumpton,‡ James Ault,‡ and Jane Thomas-Oates*,‡ Department of Chemistry, University of York, Heslington, York, United Kingdom, Departamento de Microbiologıá, Facultad de Biologıá, Universidad de Sevilla, Sevilla, Spain, and Technology Facility, Department of Biology, University of York, Heslington, York, United Kingdom Received October 4, 2006

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLCMS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD. Keywords: nanoLC-MS • electrospray • MALDI • monolithic columns • nodulation outer proteins • type III secretion system • Sinorhizobium fredii HH103 • soybean

Introduction Rhizobia are soil bacteria able to establish symbiotic interactions with leguminous plants that lead to the formation of root nodules where these bacteria fix atmospheric nitrogen. The establishment of a successful symbiotic relationship requires a molecular dialogue between the partners.1-2 This molecular dialogue begins when flavonoids, naturally exuded by legume roots, activate the transcription of bacterial nodulation genes. Some of these genes encode proteins responsible for the biosynthesis and secretion of signal molecules called Nodfactors or lipochitin oligosaccharides (LCOs), which mediate species specificity and induce the development of root nodules in the plant. Flavonoids also activate the secretion to the extracellular environment of bacterial proteins through a type III secretion system (T3SS). These secreted proteins, called nodulation outer proteins (Nops), are involved in bacterial hostrange determination in several legumes.3 The type III secretion apparatus is highly conserved among bacterial pathogens and constitutes a complex that spans the inner and outer bacterial membranes.4 This secretion system is used by plant and animal pathogenic bacteria to deliver effector proteins directly into the eukaryotic cell or secrete * To whom correspondence should be addressed. Professor Jane ThomasOates, Department of Chemistry, University of York, Heslington, YO10 5DD, York, United Kingdom. Tel, +44 1904434459; Fax, +44 1904432516; E-mail, [email protected]. † These two authors contributed equally to this work. ‡ Department of Chemistry, University of York. § Universidad de Sevilla. | Department of Biology, University of York. 10.1021/pr060519f CCC: $37.00

 2007 American Chemical Society

proteins to the extracellular environment.5 The T3SS has also been identified in symbiotic bacteria such as Rhizobium sp. NGR234,6 Sinorhizobium fredii USDA257,7 S. fredii HH103,8-9 Mesorhizobium loti MAFF303099,10 and Bradyrhizobium japonicum USDA110.11 Rhizobium sp. NGR234, a broad host range rhizobial strain that does not nodulate soybean, secretes at least six proteins in a type III-dependent manner: NopA, NopB (formerly NolB), NopC (formerly Fy1), NopL (formerly Y4xL), NopP (formerly Y4xP), and NopX (formerly NolX).12-15 Only NopL and NopP have previously been described as effector proteins.13,16-18 S. fredii USDA257 also secretes a number of proteins,6,19-20 and in B. japonicum USDA110, eight proteins were identified as being type III secreted, using 2D-PAGE and mass spectrometry.21 The role of nodulation outer proteins in cultivar-specificity determination has been studied in the symbiotic interaction between S. fredii strains USDA257 and HH103 and soybean. The main difference between these strains is their differential capacity to nodulate agronomically improved American soybeans. USDA257 only nodulates primitive Asiatic soybean cultivars.22 Mutants of USDA257 affected in Nop secretion gain the capacity to nodulate American soybeans.23 In contrast, HH103 naturally forms nitrogen-fixing nodules in American soybean cultivars, such as Williams, and in Asiatic soybeans such as Peking.24 A so-called geLC-MS/MS25 approach has been employed in this work to identify changes in the secretion proteome of S. fredii HH103 in response to induction by plant-derived flaJournal of Proteome Research 2007, 6, 1029-1037

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research articles vonoids. In geLC-MS, following separation of proteins by molecular weight on a 1D SDS gel, bands are excised and proteolytic digestion of the proteins is performed. Liquid chromatographic separation of the resultant peptides followed by mass spectrometric identification is carried out to determine the proteins present in the gel bands. In this paper, we have employed both online LC-electrospray- and offline LC-MALDIMS and MS/MS to identify the peptides. Combined use of these two approaches for analysis of the same sample has been shown to be complementary, allowing an increase in proteome coverage.26 Here we have performed the LC separations using capillary polystyrene-divinylbenzene (PS-DVB) monolithic stationary phases. These non-particulate polymeric rods of material allow faster, more efficient separations and much reduced sample amounts than conventional packed C18 capillary columns. Our combined use of monolithic stationary phases and complementary mass spectrometric approaches has enabled the identification of novel, low abundance secreted proteins. In this paper, we report the identification of five Nops secreted by HH103 using this newly developed monolithic LCMS/MS proteomic approach. Two of them, NopM and NopD, have not been previously described as type III-secreted proteins in rhizobia, whereas secretion of NopP, NopL, and NopX has been reported in other rhizobial strains. Type III-dependent secretion of NopA, NopC, NopL, NopP, and NopX by HH103 was confirmed by western blot analysis.

Experimental Section Purification and Analysis of Secreted Proteins. Extracellular proteins of SVQ269 ()HH103RifR) and SVQ518 ()SVQ269 rhcJ:: Ω) were extracted following the protocol described by Vinardell et al. (2004)9 with some modifications.9 YM bacterial cultures (50 mL) were grown at 28 °C with shaking for 40 h (approximately 108 bacteria mL-1) and induced with the flavonoid genistein (3.7 µM) when required. Cultures were centrifuged for 20 min at 10 000g at 4 °C. The supernatants were mixed with 3 volumes of cold acetone and incubated at -20 °C for 24 h. The mixtures were centrifuged for 45 min at 22 000g at 4 °C. The precipitates were washed with 70% acetone, dried, and resuspended in 250 µL of buffer containing 8 M urea, 4% wt/ vol CHAPS, 0.75 mM DTT, and 0.001% bromophenol blue. Samples were stored at -80 °C until required. SDS-PAGE was performed on 12% (wt/vol) polyacrylamide gels, and proteins were visualized by Coomassie blue staining. For immunostaining, extracellular proteins were resuspended in 400 µL of sample buffer (2% SDS, 10% glycerol, 0.125 M Tris-HCl, pH 6.8, 0.1% bromophenol blue, and 5% β-mercaptoethanol). Aliquots of proteins were separated on 15% (wt/ vol) SDS polyacrylamide gels and electroblotted to Immun-Blot polyvinylidene difluoride (PVDF) membranes using a Mini Trans-Blot Electrophoretic transfer cell (Bio-Rad, CA). Membranes were blocked with TBS containing 2% (wt/vol) BSA and then incubated with antibodies raised against NopA, NopC, NopL, NopP,12-14 diluted 1:3000 in the same solution. (These antibodies were a gift of Dr. W. J. Deakin, De´partement de Biologie Ve´ge´tale, University of Geneva, Switzerland.) Antirabbit immunoglobulin antibodies (AP-conjugate) were used as secondary antibodies. Reaction results were visualized using NBT-BCIP. MALDI MS. Six bands were excised carefully from the Coomassie blue-stained gel and prepared for sequencing according to Shevchenko et al. (1996).27 Water (50 µL) was 1030

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added to each tube and vortexed briefly; the supernatant was removed and stored on ice. To elute the peptides from the gel pieces, they were washed three times with 50% acetonitrile/ 5% formic acid, removing the supernatant after each wash. All the removed supernatants were combined and stored on ice. After the third wash, the eluted peptides were concentrated. The washing step was repeated, and the solution was evaporated to dryness again. Peptides were redissolved by adding 0.5 µL of formic acid and 10 µL of water and directly spotted onto a MALDI target plate using the dried-droplet method with R-cyano-4-hydroxycinnamic acid (CHCA) as matrix.28 MALDITOF/TOF analysis was performed using the 4700 Proteomics Analyzer (Applied Biosystems, Warrington, UK); both MS and MS/MS spectra were acquired and used in database searching using Mascot (Matrix Science, London, UK) to search both NCBInr and SwissProt databases. Monolithic LC-MALDI-MS/MS Analysis. An aliquot (1 µL) of the tryptic digests was injected directly onto a polystyrenedivinylbenzene polymeric (PS-DVB) monolithic column (200 µm i.d. × 5 cm long) (LC Packings, Amsterdam, The Netherlands). Peptides were separated using a linear gradient of 2-50% (v/v) CH3CN over 20 min. UV-detection was performed at 214 nm and the flow rate was set at 3 µL/min. The Probot microfraction collector (Dionex, Sunnyvale, U.S.A.) was set to collect fractions every 6 s onto a MALDI target plate. The eluent from the HPLC was mixed 1:1 (v/v) postcolumn with matrix (7 mg/mL CHCA) in a solvent consisting of acetonitrile/water (60: 40% v/v) before spotting onto the target plate using a low dead volume mixing tee. The 4700 Proteomics Analyzer (Applied Biosystems, Warrington, UK) was used to analyze the samples; both MS and MS/MS data obtained were submitted to Mascot (Matrix Science, London, UK) using the GPS Explorer software (Applied Biosystems, Warrington, UK) for submitting data for database searching. All searches were performed against the NCBInr (build 120104 (1 591 231 sequences; 521 258 069 residues)) protein sequence database. In the Mascot search, type of instrument was TOF/TOF, peptide mass tolerance was set to 200 ppm, and fragment mass tolerance was limited to (0.15 Da. The maximum number of missed cleavages was set to 1 and the possible variable modifications of methionine oxidation and cysteine carbamidomethylation were used. Monolithic LC-ESI-MS/MS Analysis. An aliquot (1 µL) of the tryptic digests was injected directly onto a polystyrenedivinylbenzene polymeric (PS-DVB) monolithic column (100 µm i.d. × 5 cm long) (LC Packings, Amsterdam, The Netherlands). Peptides were separated using a linear gradient of 2-50% (v/v) CH3CN over 20 min at a flow rate of 1.1 µL/min. Eluting peptides were directly infused into a QSTAR Pulsar i hybrid q-oTOF mass spectrometer (Applied Biosystems, Warrington, UK) fitted with a modified MicroIonSpray head. Data dependent analysis (DDA) was used with two dependent MS/ MS scans of 3 s duration per 0.5 s MS scan. Dynamic exclusion was set at 120 s to preclude reanalysis of precursors already taken for MS/MS. All MS/MS data obtained were submitted to Mascot (Matrix Science, London, UK) for database searching. All searches were performed against the NCBInr (version NCBInr 20050204) protein sequence database. The Mascot search was performed using a maximum number of missed cleavages of 2, instrument type of default, variable modifications of methionine oxidation and cysteine carbamidomethylation, a peptide tolerance of 100 ppm and a fragment mass tolerance of 0.8 Da.

NopM and NopD

Figure 1. Extracellular protein profile of S. fredii HH103. Lanes 1 and 2: SVQ518 ()HH103 RifR rhcJ::Ω); Lanes 3 and 4: SVQ269 ()HH103 RifR). Lanes 2 and 4: extracellular proteins from cultures grown in the presence of genistein 3.7 µM. Molecular masses (kDa) of the marker are indicated on the left. S. fredii HH103 proteins whose secretion depends on the flavonoid genistein and on a functional T3SS are marked with an asterisk. Bands that were taken for proteomic analysis are marked as B1-B6. Protein (25-35 µg) from the supernatant was loaded in each lane.

Results Monolithic Capillary Liquid Chromatography Coupled to Matrix Assisted Laser Desorption/Ionization- or ElectrosprayMass Spectrometry Used to Analyze S. fredii HH103 Extracellular Proteins. In previous studies using 15% polyacrylamide silver stained SDS-PAGE gels, we showed that S. fredii HH103 secreted at least six nodulation outer proteins in response to genistein, one of which was identified as NopA.8-9 Initial attempts to identify the proteins in these silver stained bands using mass spectrometry gave no significant results. Hence we used 12% polyacrylamide SDS-PAGE to achieve a better separation of the secreted proteins, followed by traditional Coomassie blue staining. As a consequence, nine bands were detected in the supernatants of genistein-induced HH103 cultures (Figure 1, bands marked with an *) that are not detected in the supernatants of uninduced cultures. We disregarded the three bands with the lowest molecular masses for MS analysis due to the availability of antibodies raised against NopA and NopC, which cross-reacted only with the bands of about 6 and 12 kDa, respectively. The protein of about 20 kDa was identified as a nodulation outer protein in S. fredii USDA257 and Rhizobium sp. NGR234 and named NopB.15,20 Sequencing of the S. fredii HH103 nopB gene and analysis of its predicted amino acid sequence showed that there was a high degree of identity with the corresponding proteins of S. fredii USDA257 and Rhizobium sp. NGR234,8 consistent with this

research articles protein also corresponding to NopB in S. fredii HH103. We then selected for mass spectrometric analysis only those bands that could potentially give us more information about possible differences in the set of proteins secreted by S. fredii HH103 when compared with those secreted by other rhizobia. The other six bands were thus excised from the gel (Figure 1), the proteins in the bands digested with trypsin and the resulting mixtures of released peptides analyzed using MALDIMS and MALDI-MS/MS. Samples were initially analyzed using a classical MALDI-MS peptide mass fingerprint approach. Although the use of peptide mass fingerprinting failed to match any peptides when searching against either the NCBInr or the SwissProt databases and therefore no protein identifications were made, the use of MS/MS ion searching proved to be successful and allowed single peptide matches for two Nops from S. fredii HH103, NopL from band B3 and NopP from band B5. Two flagellins, flagellin B and flagellin D, were also identified in band B5 by virtue of sequence similarities to flagellins from S. meliloti, again based on single peptide matches (data not shown). However, no protein identifications were obtained from similar analysis of bands B1, B2, B4, and B6. To increase the proteome coverage and attempt to obtain results from the bands failing to yield data using this classical approach, we then considered the application of alternative methods such as liquid chromatographic methods coupled to mass spectrometry (LC-MS). Recent advances in LC-MS together with the continuous development of novel stationary phases we felt offered the potential solution. One of the most promising stationary phases for capillary RP-HPLC-MS is the polystyrene-divinylbenzene polymeric monolithic column (PS-DVB). These columns were first introduced in the early nineties for the study of peptides and proteins,29-30 but only recently have robust, reproducible monolithic PS-DVB columns become commercially available. This type of column, where the stationary phase is a continuous, rigid polymeric rod with a porous structure, presents several advantages when compared to conventional packed columns; the absence of an interparticular void, combined with the high porosity, higher surface to volume ratio and the absence of particles removes diffusion as the rate-limiting step in the separation and brings an inherent gain in chromatographic speed without any apparent loss in separation efficiency. The result is very narrow (a few seconds in width), sharp, well-resolved peaks. We therefore employed monolithic columns in both on-line and off-line capillary LC workflows for the analysis of the proteins secreted by Sinorhizobium fredii HH103; this approach allowed us to obtain much higher quality data from all six bands than was possible on direct analysis of protein band digests. The LC-ESI-MS/MS approach is a rapid, on-line and thus fully automatable technique, although it is its speed that is also a potential disadvantage; since the monolithic columns give such narrow chromatographic peaks, if more than two or three peptides coelute, it is possible that the mass spectrometer may be unable to record product ion spectra on them all before they have eluted and peptides in the next peak are entering the ion source, However, this potential disadvantage is the offline LC-MALDI approach’s strength. It decouples the chromatography from the mass spectrometric measurements so that each can be carried out on their own optimal time scales. This makes full automation more difficult and results in a longer overall experiment, but offers the possibility of yielding more data, especially from very complex peptide mixtures. In addiJournal of Proteome Research • Vol. 6, No. 3, 2007 1031

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Table 1. Identification of Extracellular Proteins from Genistein Induced Cultures of S. fredii HH103 Using LC-MALDI-MS/MS MOWSE score

mol mass (kDa)

gi|27376804 (BAC46958)

236

185.702

Blr1904 (B. japonicum USDA110)

gi|12620608 (BAC47169)

136

63.663

NolX (S. fredii USDA257)

gi|420884 (now replaced in NCBInr by gi|462734) (AAG45732)

323

63.972

B3

Y4xL (S. fredii USDA257)

gi|19749315 (AAL98685)

509

37.051

B4

Y4xL (S. fredii USDA257) Y4yP (S. fredii USDA257)

gi|19749315 AAL98685 gi|19749332 AAL98702

39b

37.051

294

31.055

Flagellin D (S. meliloti 1021)

gi|15964424 NP_384777

195

42.015

Y4yP (S. fredii USDA257)

gi|19749332 AAL98702

107

31.055

AGR_C_958 (Flagellin) (A. tumefaciens C58)

gi|15887889 NP_353570

140

32.844

Flagellin A and/or Flagellin B (S. meliloti 1021)

gi|15964422/ gi|15964423/ gi|17380397 (NP_384775/ NP_384776/ P13119

92

40.693/ 40.717/ 40.695

sample

proteins identified

B1


B2

B5

B6

a

accession number NCBInr (SwissProt)

peptides matcheda

SFSDYLR FVDPLEALR LVQAIDYLR TLNNPQGVAGER QMQEPASPSTAR HDQAPDPGEPDR QQSPDEPMAALAR DYQLQELDLQR LVEAMGEEFQSR LAEHGLVGDVDAER LYGGNIDK YSEYLPK NLSLADFK VNDPSTPPDLK FYTNMTTANR TINSHQELFFGSGDLTR DPLALLPPHMR + M AGSWQVGPSR IRPSLDFDI ATASPAPLTAER SPQPSEQQPHAR SPHPSEQRPHAR ALQVPEYDQDLIWQR VLQDAPEHDQDQHVEAAGPR SPQPSEQQPHAR VLQDAPEHDQDQHVEAAGPR TADVAQQYGIR IDSSSDFHYTQSASK QTDAETQEFADTFAR EITDIADNPQEYSDFVSAK TEGGFSMEEEFESK LVAATEDGVDK LVDADMNEESTR + M VGTASDNAAYWSIATTMR TADVAQQYGIR QTDAETQEFADTFAR IVAATEEGVDK IGLQEDFVSK LSDSIDSGVGR LVDADMNEESTR

+ M ) methionine oxidation. b Below the 95% confidence level, but also identified using ESI (Table 2)

tion, it has been shown that MALDI and ESI processes favor the ionization of different subclasses of peptides, so that the two approaches are truly complementary.31 Our approach using both monolithic-LC-ESI- and monolithicLC-MALDI-MS/MS made it possible to identify several additional proteins that were not identifiable using exactly the same amounts of proteolytically released peptides without separation. Monolithic-LC-MALDI allowed identification of the four proteins identified without chromatography (now all on the basis of two peptides or more) and an additional five proteins that were not previously identified. In addition to the increase in the number of peptides for each protein, the resulting MOWSE scores were also much higher (Tables 1 and 3). Figure 2, panels B and C, show typical MS and MS/MS spectra of one peptide eluting at 7 min and collected as a single spot on the MALDI target plate. The fact that a single peptide 1032


is concentrated into the MALDI spot greatly enhances the quality of the MS data; separation improves mass spectrometric response by reducing competition between peptides in a mixture during the ionization process, enhancing the quality of the MS/MS data produced. Monolithic-LC-ESI (typical data are shown in Figure 3) allowed identification of seven of the nine proteins identified using LC-MALDI (Tables 1, 2, and 3). Although the proteins identified using the two ionization techniques are largely the same (although MALDI allows identification of two additional proteins not identified by ESI), the peptides are generally different. This is very striking in the example of NopX in band 2 where LC-ESI identified 17 peptides and LC-MALDI seven, with five of those peptides commonly identified by both methods. Similarly for NopD in Band 1, one method (this time LC-MALDI) identified eight peptides, whereas LC-ESI identified threesin this case there are no common

NopM and NopD

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Figure 2. (A) UV chromatogram (214 nm) of a tryptic digest of band B2 using a monolithic PS-DVB column; (B) MALDI-MS spectrum from fraction eluting at 7 min from the chromatogram above; (C) CID-MS/MS tandem mass spectrum of tryptic peptide (NLSLADFK) with m/z 907.57 from the spectrum in (B).

peptides. The results make it very clear that the use of both methods together is worthwhile, dramatically increasing the confidence of protein assignment due to the complementarity of the approaches. Examination of the peptides identified by MALDI and ESI in our study follows the same trends as those reported by Stapels and Barofsky31 in favoring the ionization of different subclasses of peptides. For example, our data are clearly consistent with the observation31 that MALDI favors the ionization of Arg-teminated peptides over Lys-terminated (ratio of K/R terminated peptides matched ) 0.35). The peptides matched in our study using ESI (K/R ) 0.69) do not demonstrate the apparent overall preference of ESI for Lys-terminated peptides reported by Stapels and Barofsky,31 although the proportion of Lys-terminated peptides matched from our ESI data is clearly much higher than from our MALDI data. We suggest that, while the trends are consistent, it is unsafe to draw

Figure 3. (A) Total ion chromatogram of a tryptic digest of band B2 using a monolithic PS-DVB column with ES-MS, (B) ESI mass spectrum of material eluting at 7.36 min from the chromatogram in (A), (C) CID-MS/MS tandem mass spectrum of tryptic peptide (RLDGSGFLEQHSNIT) with m/z 961.5 from the spectrum in (B).

any deeper conclusions in this regard, since the number of peptides in our study is, by its nature, very much lower than that in Stapels and Barofsky’s work, and we are studying secreted proteins while their study was of DNA-binding proteins. The mean aliphatic indices and hydrophobicities of the peptides matched in our study by ESI (92.30 and -0.40 respectively) are both higher than those (62.58 and -0.77 respectively) of the peptides matched from the MALDI data, Journal of Proteome Research • Vol. 6, No. 3, 2007 1033

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Table 2. Identification of Extracellular Proteins from Genistein Induced Cultures of S. fredii HH103 Using LC-ESI-MS/MS

sample

proteins identified

accession number NCBInr (SwissProt)

B1


B2



mol mass (kDa)

gi|27376804 (BAC46958)

230

185.702

gi|27377015 (BAC47169)

244

64.149

974

63.972

gi|19749311 (AAG45732)

peptides matcheda

LGAESDVLR SPLYSQDAPLILGLEK QLGSPQGLSPVSAHSDDDALAWLSEELAR LLELR LEALDEIAR LTSLPDALPSR WQPWETVLR LAEHGLVGDVDAER LAIR IGPLASR LYGGNIDK YSEYLPK NLSLADFK MGLADQPDIK + M VNDPSTPPDLK NGGALMHVVDSVLR + M KNGGALMHVVDSVLR + M EDLLPPDAESTLEDLQK TINSHQELFFGSGDLTR QALIEAGIGVAAQAVGLVSGPGVK ITWNGGSLTKPELQIVAVLNR CPPQVIAAAQYFVSHPEEWK + C SASNLLPMISSNPAQFAQASLAK + M AAIEALLQDPELFYAIGSQGDGR AAIESMDQTPQSAVAIDDHYVAPAPIQSSR VNDPSTPPDLK FHVPETEAAQNAVADMK + M TINSHQELFFGSGDLTR THHGFFDFGGGHTVDSGNVSK ITWNGGSLTKPELQIVAVLNR AAIEALLQDPELFYAIGSQGDGR IRPSLDFDI SPQPSEQQPHAR ALQVPEYDQDLIWQR VLQDAPEHDQDQHVEAAGPR LVAATEDGVDK LVDADMNEESTR VGTASDNAAYWSIATTMR

B3


gi|420884 (now replaced by gi|462734) (AAG45732)

387

51.173

B4

Y4xL (S. fredii USDA257)

gi|19749315 (AAL98685)

248

37.051

B5

Flagellin C and/or Flagellin D (S. meliloti 1021)

gi|12057228/ gi|15964424 (AAB81421 CAC45243)

204

40.963/ 42.015

Y4yP (S. fredii and Rhizobium sp. NGR234) FlaB (Sinorhizobium meliloti) and/or Flagellin A (Sinorhizobium meliloti1021)

gi|19749332 (AAL98702)

89

31.055/ 31.230

WTQTSAAEWR DGERPLLNWR

gi|12057230/ gi|15964422 gi|15964423 (AAB81423/ CAC45241/ CAC45242)

166

40.695/ 40.693/ 40.717

LSDSIDSGVGR LVAATEDGVDK VDTAYSGMESAIEVVK

gi|19749332 (AAL98702)

219

31.055

B6

Y4yP (S. fredii)

a

MOWSE score

WTQTSAAEWR DGERPLLNWR YTLDHEPPVVPIDLK EITDIADNPQEYSDFVSAy

+ M ) methionine oxidation, + C ) cysteine carbamidomethylation.

which is also in accord with previously reported observations.31 The mean masses of the matched peptides also follow the same trend in our data as that reported previously,31 with ESI apparently identifying slightly larger peptides than MALDI; mean matched peptide lengths from the ESI data were 14 amino acids long (mean mass 1591.35 Da), whereas from MALDI were 12.25 amino acids in length (mean mass 1378.35 Da). 1034


Identification of Nodulation Outer Proteins Secreted by S. fredii HH103. Three Nops, NopX, NopL, and NopP, previously described as secreted by S. fredii USDA257 and Rhizobium sp. NGR234, were identified in the supernatants of flavonoidinduced cultures of S. fredii HH103 (Tables 1, 2 and 3). These protein bands were not observed in SDS-PAGE gels of supernatants of flavonoid-induced cultures of SVQ518, an HH103 rhcJ mutant which is impaired in Nops secretion (Figure 1).8

research articles

NopM and NopD Table 3. Comparison of LC-MALDI- and LC-ESI- MS/MS Resultsa MALDI band

1 2

3 4 5

6

a

identified proteins

Blr1693 [B. japonicum] NolX [S. fredii] Blr1904 [B. japonicum] NopL [S. fredii] NolX [S. fredii] NopL [S. fredii] FlaC or D [S. meliloti] NopP [S. fredii] Flagellin D [S. meliloti] AGRC958 [Agrobacterium tumefaciens] NopP [S. fredii] Flagellin A or B [S. meliloti]

peptides matched

MOWSE score

electrospray peptides matched

MOWSE score

8

236

3

230

7 2

323 136

17 5

974 244

7 ND 2 ND

509 ND 39 ND

ND 6 4 3

ND 387 248 204

5 3

294 195

2 ND

89 ND

2

140

ND

ND

2 2

107 92

4 3

219 166

ND ) not detected.

LC-MS/MS proteomic analyses (Tables 1, 2 and 3) indicated that band B2 of about 60 kDa contains a protein homologous to NopX of S. fredii USDA257 (AAG45732). This protein is 98 and 72% identical to NopX of Rhizobium sp. NGR234 (AAB91942) and M. loti MAFF303099 (BAB52646), respectively, and 45% identical to HrpF of Xanthomonas campestris pv. vesicatoria (AAB86527). The results of similar analyses of bands B3 and B4, of about 40 and 37 kDa, respectively, showed that both bands contain a protein homologous to the effector NopL of S. fredii USDA257 (formerly Y4xL, AAL98685). This protein showed 95, 34, and 32% identity with the corresponding proteins from Rhizobium sp. NGR234 (AAB91935), B. japonicum USDA110 (BAC47045), and M. loti MAFF303099 (BAB49164), respectively. The shorter ∼37 kDa protein could arise by post-translational modification, represent a shorter isomeric form of the protein, be the product of the degradation of the NopL protein, or simply be an artifact of the gel separation. In any case, the presence of both proteins is common in most of the gels analyzed. The 51 kDa form of NopX was also identified in Band 3 (about 40 kDa), for which we suggest similar possible explanations. A protein that shares homology with the S. fredii USDA257 NopP effector protein (formerly Y4yP AAL98702) was identified in bands B5 and B6. S. fredii USDA257 NopP showed 95% identity with NopP of Rhizobium sp. NGR234 and 43% with the protein ID84 from B. japonicum (AAG60738). A variety of flagellins were also identified in bands B5 and B6 (molecular weights of about 32-34 kDa), by virtue of sequence similarities with flagellins from S. meliloti and A. tumefaciens (Tables 1, 2 and 3). S. fredii HH103 Secretes Two Novel Nodulation Outer Proteins. In addition to the proteins described above, two new proteins whose secretion depends on flavonoids and on a functional T3SS were detected. (1) A protein with a molecular weight of about 180 kDa was detected in band B1. This protein, for which we propose the name NopD, showed homology to the hypothetical protein Blr1693 (BAC46958) which belongs to a small family of hypo-

Figure 4. Western-blot analysis of type III-dependent secretion of S. fredii HH103 nodulation outer proteins NopA, NopC, NopP, and NopL. (A) Sodium dodecyl sulfate polyacrylamide (15%) electrophoresis gels stained with silver nitrate showing the extracellular protein profiles of S. fredii strains. Lanes 1 and 2: SVQ269 ()HH103 RifR); Lanes 3 and 4: SVQ518 ()HH103 RifR rhcJ::Ω). Lanes 2 and 4: extracellular proteins from cultures grown in the presence of genistein 3.7 µM. M: Molecular masses (kDa) of the marker. (B) Corresponding western blots using antibodies raised against NopA, NopC, NopL, and NopP.

thetical proteins (Bll8244, Blr1693, Blr1705) from B. japonicum. Blr1693 is also 50% identical to the M. loti MAFF303099 protein Mlr6316. (2) A protein, for which we propose the name NopM, that is orthologous to the hypothetical B. japonicum USDA110 protein Blr1904 (BAC47169) was identified in band B2. This protein was detected in the same band as NopX. In fact, both proteins have similar predicted molecular weights (64 kDa). Protein Blr1904 shows homologies to Y4fR from Rhizobium sp. NGR234, YopM from Yersinia spp., the SspH proteins of Salmonella typhimurium and members of the IpaH family from Shigella flexneri. All these proteins contain leucine-rich repeat (LLR) sequences that are thought to be involved in protein-protein interactions. To our knowledge, type III-dependent secretion of the proteins NopD and NopM, identified in bands B1 and B2, respectively, has not been previously described in rhizobia. Confirmation of T3SS-Dependent Secretion of Several S. fredii HH103 Nodulation Outer Proteins. Secretion of Nops by S. fredii HH103 has also been studied by western blot analysis using specific antibodies against several Nops secreted by Rhizobium sp. NGR234 (Figure 4). For this purpose, antibodies raised against the effectors NopL, and NopP, already identified in Rhizobium sp. NGR234, were available. In addition, we also used antibodies against NopA, a previously identified S. fredii HH103 nodulation outer protein,8 and NopC, a recently identified nodulation outer protein from Rhizobium sp. NGR234. These antibodies cross-reacted with the corresponding proteins isolated from genistein-induced supernatants of S. fredii HH103. No signal was observed in proteins isolated from uninduced S. fredii HH103 cultures or in culture supernatants of its mutant derivative SVQ518 (Figure 4). Journal of Proteome Research • Vol. 6, No. 3, 2007 1035

research articles Discussion Recently, the use of capillary PS-DVB monolithic columns for on-line RP-HPLC-ESI-MS has been demonstrated for proteomic applications.32-33 In addition, the use of both MALDI and electrospray in a packed column RP-HPLC approach has been shown to yield complementary data that increase proteome coverage when both are used on the same sample. For this reason, we have developed both online LC-electrospray and offline LC-MALDI methods using the capillary PS-DVB monolithic columns. The speed of the separations and the robustness of the system enabled rapid and very sensitive analyses of very small amounts of sample and made possible identification of significantly more proteins than could be identified from the same amount of each digest without fractionation. The LC-ESI method uses a smaller column than the LC-MALDI method, and offers the advantages of a rapid, on-line and thus fully automatable technique. In spite of our reservations that the chromatographic separation might be just too fast and the peaks too narrow for on-line coupling, the system gave really excellent data from these relatively simple peptide mixtures, and in our hands the anticipated potential drawbacks proved unfounded. In contrast, the LC-MALDI method using the larger column allowed decoupling of the chromatographic and mass spectrometric experiments and, together with the speed and high mass accuracy of the MALDI-TOF/TOF mass spectrometer, allowed a slower but perhaps more exhaustive analysis of the separated peptides. The two methods allowed identification of the same proteins but via different peptides, further increasing confidence in the protein assignments. The application of the monolithic combined LC-ESI-MS/MS and LC-MALDI-TOF/TOF proteomic approach used in this work proved to be a fast, robust, easy-to-use and highly sensitive methodology which gave a dramatic increase in proteome coverage. For this reason, it proved to be vital for analyzing the S. fredii HH103 secretome, where extracellular proteins are always expected to be found in very low concentrations. In particular, the use of the monolithic LC-MS/MS approach developed in our laboratory allowed identification of a range of both known and novel proteins, when analysis of unfractionated peptide mixtures using established approaches gave none. Using this proteomic approach we have identified three Nops secreted by S. fredii HH103, NopP, NopL, and NopX, already identified from Rhizobium sp. NGR234 and S. fredii USDA257, but also two proteins not previously described as being type III secreted in rhizobia, NopD and NopM. NopX (NolX) exists in two forms, both of which are identifiable from our data. These two forms (63 972 Da and 51 173 Da) are identical in their first 461 amino acids but differ in their C-terminal regions. In band 2, the ESI data identified more peptides than the MALDI data, critically identifing peptides QALIEAGIGVAAQAVGLVSGPGVK and NGGALMHVVDSVLR which are located in the variable C-terminal region, making it possible to identify unambiguously the ∼64 kDa form of NopX in band 2. The MALDI data identified fewer peptides and those identified are all located in the identical first 461 amino acids, making it impossible to differentiate the two forms of the protein from these data alone, and highlighting the clear advantage of using the two ionisaton methods together for optimal results. In band 3, ESI identified six peptides, all consistent with both forms, but giving a higher score for the shorter form. MALDI 1036


Rodrigues et al.

identified no NolX peptides in band 3. We thus assign the band 3 form of NopX as the 51 kDa form, consistent with both the MS data and the SDS-PAGE migration behavior. NopX shows homologies to the protein HrpF of X. campestris pv. vesicatoria that may be inserted in the plant cell membrane and required for the type III effector proteins to enter the host cells.34 In S. fredii USDA257, NopX is expressed in planta during the early stages of nodule development, being localized in the infection threads of soybean and cowpea.19 Inactivation of nopX of S. fredii USDA257 and HH103 retarded initial nodulation rates in American and Asiatic soybean cultivars.35 NopL and NopP of Rhizobium sp. NGR234 are two effector proteins that can be phosphorylated by plant kinases.13,16 NopL, when delivered into the plant cell, could be involved in the modulation of the activity of signal transduction pathways that culminate in the activation of pathogenicity related (PR) proteins.16 Mutations of both nopL and nopP reduced nodulation efficiency on the tropical legumes Flemingia congesta and Tephrosia vogelli.18 The S. fredii HH103 NopM protein is homologous to the putatively encoded product of the gene y4fr of Rhizobium sp. NGR234 and the YopM protein of Yersinia spp. In Yersinia spp., YopM is secreted via T3SS and migrates to the nucleus of the target cell.36-37 However, its function in pathogenicity remains unknown.38 The S. fredii HH103 NopD protein is homologous to Blr1693, a putative B. japonicum protein whose C-terminal region aligns with the catalytic domain of members of the C48 cysteine peptidase family of proteins that includes the ubiquitin-like protease Ulp1, the T3SS effector protein XopD of X. campestris pv. vesicatoria39 and the M. loti type IV secreted protein Msi059.40 XopD in X. campestris pv. vesicatoria targets SUMO (small ubiquitin-like modifier) conjugated proteins in planta, suggesting that XopD protease mimics a host protease that removes SUMO modifications. This proteolysis could be a mechanism to alter host cell signalling events for the pathogen’s benefit.39 The use of specific antibodies made it possible to further confirm flavonoid dependent type III-dependent secretion of two of the Nops identified mass spectrometrically, NopL and NopP, as well as of NopA, a nodulation outer protein previously identified in S. fredii HH103, and NopC. LC-MS analyses additionally revealed the presence of flagellins in bands B5 and B6. These two bands appeared both in genistein-induced as well as uninduced culture supernatants of both wild-type strain SVQ269 and its mutant derivative SVQ518. The reason for analyzing these bands was that a genistein-induced band migrating between bands B5 and B6 was observed in the extracellular proteins of SVQ269. We thus decided to excise and sequence these bands independently to ensure the induced protein was submitted to MS analysis. This allowed the identification of NopP and some flagellins. The presence of these flagellins in S. fredii HH103 culture supernatants does not apparently depend on either flavonoids or a T3SS. Previous studies carried out in B. japonicum and Rhizobium sp. NGR234 showed that genes that code for Nops are preceded by a tts box, a conserved sequence located upstream of the genes regulated by ttsI, the putative transcriptional regulator of the T3SS.11,41 These tts boxes were also found upstream of the S. fredii HH103 genes nopC (formerly fy1), nopP, and nopX, all of them located within the tts cluster. In Rhizobium sp. NGR234, the gene y4fr, which codes for a putative NopM

research articles

NopM and NopD

orthologue, is also preceded by a tts box but it is localized outside the tts cluster. There are other putative candidates for nodulation outer proteins in S. fredii HH103, such as orthologues of the proteins encoded by genes y4zC or y4lO of Rhizobium sp. NGR234, due to their homology with Avr proteins of plant pathogens and also the presence of a tts box preceding both genes. Southern blots confirmed the presence of a copy of y4zC but not y4lO in the S. fredii HH103 genome (data not shown). Further experiments are necessary to elucidate the possible role of these proteins in the determination of the nodulation range of HH103 and its host legumes. The development of this new proteomics approach provides us with a new tool to identify other putative effector proteins, usually found at very low concentrations, that could be involved in host range determination in rhizobia.

Abbreviations ESI, electrospray; MALDI, matrix-assisted laser desorptionionization, LC-MS, liquid chromatography-mass spectrometry; Nops, nodulation outer proteins; T3SS, Type Three Secretion System; PS-DVB, polystyrene-divinylbenzene; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOF, time-of-flight

Acknowledgment. We acknowledge Ken Cook from Dionex, UK for generously providing the monolithic columns used in this work, the University of York, the European Union (ICA4-CT-2001-10056), and the Spanish MCyT (AGL2002-04188C06-04) for funding JAR and the project. J.T.-O. is grateful to the Analytical Chemistry Trust Fund, the Royal Society of Chemistry Analytical Division and the British Engineering and Physical Sciences Research Council (EPSRC) for financial support. We also thank Dr. W. J. Deakin (De´partement de Biologie Ve´ge´tale, University of Geneva, Switzerland) for the generous gift of the NopA, NopC, NopL, and NopP antibodies. Supporting Information Available: Figure S1. Mascot database search results with LC-MALDI-MS/MS data. Figure S2. Mascot database search results with LC-ESI-MS/MS data. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Geurts, R.; Bisseling, T. Rhizobium Plant Cell supplement 2002, S239-S249. (2) Gage, D. J. Mol. Biol. Rev. 2004, 68, 280-300. (3) Perret, X.; Staehelin, C.; Broughton, W. J. Microbiol. Mol. Biol. Rev. 2000, 64, 180-201. (4) Hueck, C. J. Microbiol. Mol. Biol. Rev. 1998, 62, 373-433. (5) Pallen, M. J.; Chaudhuri, R. R.; Henderson, I. R. Curr. Opin. Microbiol. 2003, 6, 519-527. (6) Viprey, V.; del Greco, A.; Golinowski, W.; Broughton, W. J.; Perret, X. Mol. Microbiol. 1998, 28, 1381-1389. (7) Krishnan, H. B.; Lorio, J.; Kim, W. S.; Jiang, G.; Kim, K. Y.; DeBoer, M.; Pueppke, S. Mol. Plant Microbe. Interact. 2003, 16, 617-625. (8) de Lyra, M. C. C. P.; Lo´pez-Baena, F. J.; Madinabeitia, N.; Vinardell, J. M.; Espuny, M. R.; Cubo, M. T.; Bellogıń, R. A.; RuizSainz, J. E.; Ollero, F. J. Int. Microbiol. 2006, 9, 125-133. (9) Vinardell, J. M.; Ollero, F. J.; Hidalgo, A.; Lo´pez-Baena, F. J.; Medina, C.; Ivanov-Vangelov, K.; Parada, M.; Madinabeitia, N.; Espuny, M. R.; Bellogıń, R. A.; Camacho, M.; Rodrı´guez-Navarro, D. N.; Soria-Dıáz, M. E.; Gil-Serrano, A. M.; Ruiz-Sainz, J. E. Mol. Plant Microbe. Interact. 2004, 17, 676-685.

(10) Kaneko, T.; Nakamura, Y.; Sato, S.; Asamizu, E.; Kato, T.; Sasamoto, S.; Watenabe, A.; Idesawa, K.; Ishikawa, A.; Kawashima, K.; Kimura, T.; Kishida, Y.; Kiyokawa, C.; Kohara, M.; Matsumoto, M.; Matsuno, A.; Mochizuki, Y.; Nakazaki, N.; Shimpo, S.; Sugimoto, M.; Takeuchi, C.; Yamada, M.; Tabata, S. DNA Res. 2000, 7, 331-338. (11) Krause, A.; Doerfel, A.; Go¨ttfert, M. Mol. Plant Microbe. Interact. 2002, 15, 1228-1235. (12) Marie, C.; Deakin, W. J.; Viprey, V.; Kopcinska, J.; Golinowski, W.; Krishnan, H. B.; Perret, X.; Broughton, W. J. Mol. Plant Microbe. Interact. 2003, 16, 743-751. (13) Ausmees, N.; Kobayashi, H.; Deakin, W. J.; Marie, C.; Krishnan, H. B.; Broughton, W. J.; Perret, X. J. Bacteriol. 2004, 186, 47744780. (14) Deakin, W. J.; Marie, C.; Saad, M. M.; Krishnan, H. B.; Broughton, W. J. Mol. Plant Microbe. Interact. 2005, 18, 499-507. (15) Saad, M. M.; Kobayashi, H.; Marie, C.; Brown, I. R.; Mansfield, J. W.; Broughton, W. J.; Deakin, W. J. J. Bacteriol. 2005, 187, 11731181. (16) Bartsev, A. V.; Boukli, N. M.; Deakin, W. J.; Staehelin, C.; Broughton, W. J. FEBS Lett. 2003, 554, 271-274. (17) Bartsev, A. V.; Deakin, W. J.; Boukli, N. M.; McAlvin, C. B.; Stacey, G.; Malnoe, P.; Broughton, W. J.; Staehelin, C. Plant Physiol. 2004, 134, 871-879. (18) Skorpil, P.; Saad, M. M.; Boukli, N. M.; Kobayashi, H.; Ares-Orpel, F.; Broughton, W. J.; Deakin, W. J. Mol. Microbiol. 2005, 57, 13041317. (19) Krishnan, H. B. J. Bacteriol. 2002, 184, 831-839. (20) Lorio, J. C.; Kim, W. S.; Krishnan, H. B. Mol. Plant Microbe. Interact. 2004, 17, 1259-1268. (21) Su ¨ ss, C.; Hempel, J.; Zehner, S.; Krause, A.; Patschkowski, T.; Go¨ttfert, M. J. Biotechnol. 126, 69-77. (22) Keyser, H. H.; Bohlool, B. B.; Hu, T. S.; Weber, D. F. Science 1982, 215, 1631-1632. (23) Balatti, P. A.; Pueppke, S. G. Plant Physiol. 1990, 94, 1276-1281. (24) Buendıá-Claverıá, A. M.; Chamber, M.; Ruiz-Sainz, J. E. Syst. Appl. Bacteriol. 1989, 17, 155-160. (25) Froehlich, J. E.; Wilkerson, C. G.; Ray, W. K.; McAndrew, R. S.; Osteryoung, K. W.; Gage, D. A.; Phinney, B. S. J. Proteome Res. 2003, 2, 413-425. (26) Bodnar, W. M.; Blackburn, R. K.; Krise, J. M.; Moseley, M. A. J. Am. Soc. Mass Spectrom. 2003, 14, 971-979. (27) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (28) Kussmann, M.; Lassing, U.; Stumer, C. A.; Przybylski, M.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 483-493. (29) Wang, Q. C.; Svec, F.; Frećhet, J. M. F. Anal. Chem. 1993, 65, 2243-2248. (30) Wang, Q. C.; Svec, F.; Frećhet, J. M. F. J. Chromatogr. A. 1994, 669, 230-235. (31) Stapels, M. D.; Barofsky, D. F. Anal. Chem. 2004, 76, 54235430. (32) Premstaller, A.; Oberacher, H.; Walcher, W.; Timperio, A. M.; Zolla, L.; Chervet, J. P.; Cavusoglu, N.; van Dorsselaer, A.; Huber, C. G. Anal. Chem. 2001, 73, 2390-2396. (33) Ivanov, A. R.; Zang, L.; Karger, B. L. Anal. Chem. 2003, 75, 53065316. (34) Buttner, D.; Bonas, U. EMBO J. 2002, 21, 5313-5322. (35) Bellato, C.; Krishnan, H. B.; Cubo, T.; Temprano, F.; Pueppke, S. G. Microbiol. 1997, 143, 1381-1388. (36) Skrzypek, E.; Cowan, C.; Straley, S. C. Mol. Microbiol. 1998, 30, 1051-1065. (37) Benabdillah, R.; Mota, L. J.; Lu ¨ tzelschwab, S.; Demoinet, E.; Cornelis, G. R. Microb. Pathog. 2004, 36, 247-261. (38) Cornellis, G. R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8778-8783. (39) Hotson, A.; Chosed, R.; Shu, H.; Orth, K.; Mudgett, M. B. Mol. Microbiol. 2003, 50, 377-389. (40) Hubber, A.; Vergunst, A. C.; Sullivan, J. C.; Hooykaas, P. J.; Ronson, C. W. Mol. Microbiol. 2004, 54, 561-574. (41) Marie, C.; Deakin, W. J.; Ojanen-Reuhs, T.; Diallo, E.; Reuhs, B.; Broughton, W. J.; Perret, X. Mol. Plant Microbe. Interact. 2004, 17, 958-966.

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Journal of Proteome Research • Vol. 6, No. 3, 2007 1037

NopM and NopD Are Rhizobial Nodulation Outer Proteins

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