Proteomic Analysis of Herbaspirillum seropedicae Cultivated in the

Article pubs.acs.org/jpr

Proteomic Analysis of Herbaspirillum seropedicae Cultivated in the Presence of Sugar Cane Extract Fabio Aparecido Cordeiro, Michelle Zibetti Tadra-Sfeir, Luciano Fernandes Huergo, Fábio de Oliveira Pedrosa, Rose Adele Monteiro, and Emanuel Maltempi de Souza* Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná (UFPR), Centro Politécnico, P.O. Box 19071, Curitiba PR, 81531-990, Brazil S Supporting Information *

ABSTRACT: Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant− H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 downregulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant. KEYWORDS: plant−bacteria interaction, Herbaspirillum seropedicae, sugar cane, diazotroph, proteomic analysis

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induction of protein kinases and two R genes by the plant.10 Together, the results show that the gene expression profiles are altered in both partners of the interaction. Recently, the proteome of the endophytic bacterium Gluconacetobacter diazotrophicus has been analyzed to identify proteins involved in the interaction with sugar cane. Dos Santos et al. (2010)11 found 42 proteins differentially expressed in micropropagated sugar cane 7 days after inoculation with G. diazotrophicus. These proteins were related to membraneassociated structure, redox reactions, transcript and translational regulation, and energy metabolism. One of these proteins, GreA (transcription elongation factor), has also been found in other plant−bacterial associations.12 Studies using metabolic labeling to analyze the differential quantitative proteome of G. diazotrophicus in interaction with micropropagated sugar cane genotypes SP70-1143 and Chunee 24 h after inoculation13 showed genotype-specific molecular response. However, in those studies, the proteome represented a state where the bacterial population was not growing and was probably in equilibrium with the plant roots. To understand the initial stages of the interaction, it is necessary to determine bacterial proteome shortly after exposure to plant-derived compounds. To better understand the molecular mechanisms involved in the establishment of sugar cane−H. seropedicae interaction, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth

INTRODUCTION Endophytic nitrogen-fixing species of the genus Herbaspirillum colonize sugar cane plants and promote growth and productivity.1 Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans are the two species often found associated with sugar cane, being detected in many varieties collected in Brazil, Australia, and India.2 Although both species are diazotrophic and can colonize sugar cane endophytically and stimulate the plant growth, H. rubrisubalbicans can cause a mild disease in some varieties of sugar cane (B-4362 and Taiwang)3 and sorghum, whereas the documented interaction of H. seropedicae is either beneficial or neutral.4 H. seropedicae has also been isolated from rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor),5 banana, and pineapple plants.6 The morphological aspects of the colonization are well studied under laboratory conditions. To gain access to the plant inner tissue, H. seropedicae attaches to the roots, enters through epidermal discontinuities that occur near the sites of lateral root emergence, then invades the intercellular spaces and colonizes the xylem through which it can disseminate throughout the plant.7 However, the molecular mechanisms that mediate plant−H. seropedicae interaction are poorly understood. Rootexudates such as the flavonoids naringenin and daidzein promote Arabidopsis thaliana lateral root crack colonization by H. seropedicae, suggesting that these compounds are important in the colonization process.8 Naringenin can also regulate expression of a variety of genes in H. seropedicae, such as genes involved in cell wall biosynthesis.9 Conversely, inoculation of sugar cane with Herbaspirillum spp. leads to © 2013 American Chemical Society

Received: August 7, 2012 Published: January 21, 2013 1142

dx.doi.org/10.1021/pr300746j | J. Proteome Res. 2013, 12, 1142−1150

Journal of Proteome Research

Article

Data Analysis

phase of cultures grown in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR.

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Gel images were captured using the ImageScannner (GE Healthcare). For each condition, nine gels from three biological replicates and three technical replicates of each sample were analyzed using the ImageMaster 2D Platinum 6.0 (GE healthcare). The detected spots were normalized according to their relative volumes. To identify differential expression between the two growth conditions, statistical analyses (Student t test, 95% confidence interval) were performed, and only protein spots showing a statiscally significant fold change of 1.5 or higher in spot volume were analyzed by mass spectrometry. Fold change was calculated using the averaged means of relative spot volumes from the nine 2D gels of each condition.

MATERIALS AND METHODS

Bacteria, Media, and Growth Conditions

H. seropedicae strain SmR1 (SmR, Nif+)14 was grown at 30 °C at 120 rpm in JNFb-malate medium,15 supplemented with 20 mmol/L of NH4Cl and 80 μg/mL of streptomycin, in the presence or absence of 5% v/v sugar cane extract. Sugar Cane Extract Preparation

The extract was prepared by grinding freshly collected stems of sugar cane var. B-4362. The extract was centrifuged at 4 °C at 18 800g for 30 min. This procedure was repeated three times to remove all of the remaining cell debris. The supernatant was filtered through a 0.22 μm membrane and stored at −70 °C. The sterilized sugar cane extract was added to JNFb-malate to a final concentration of 5% v/v just before inoculation.

Protein Digestion and Mass Spectrometric Analysis and Database Search for Protein Identification

Protein spots were excised, destained with 50% acetonitrile in ammonium bicarbonate 25 mmol/L pH 8.0, dehydrated with 100% acetonitrile, and dried in a speed vac. The spots were rehydrated with 20 μL of a solution of trypsin (final concentration 20 ng/μL) in 40 mmol/L of ammonium bicarbonate plus 10% acetonitrile and digested 16−24 h at 37 °C. The digested material was mixed with a saturated solution of α-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile and 0.1% TFA. Mass spectra were obtained on a MALDI-TOFTOF Autoflex spectrometer (Bruker Daltonics, Bremen, Germany) in a positive ion reflection/delayed extraction mode with external calibration (mass range 800−3200 Da) using an accelerating voltage of 20 kV. Peak lists were created using FlexAnalysis 3.0.

Sample Preparation for Two-Dimensional Electrophoresis

Forty five milliliters of H. seropedicae culture in late exponential growth phase (OD600 of 0.6; 7−8 h incubation at 30 °C, 120 rpm) was centrifuged for 1 min at 12 000g and 4 °C. Cells were washed with 30 mL of PBS (3.0 mmol/L KCl, 1.5 mmol/L NaCl, 68 mmol/L KH2PO4, and 9.0 mmol/L NaH2PO4, pH 7.4). The pellet was resuspended in 600 μL of lysis buffer (7.0 mol/L urea, 2 mol/L thiourea, 4% w/v CHAPS, 1% v/v IPG buffer pH 4−7, and 20 mmol/L DTT).16 The samples were stored in a freezer at −70 °C. One hundred and fifty microliters of the H. seropedicae cell suspension was mixed with 450 μL of lysis buffer, followed by homogenization and incubation for 30 min at room temperature. The samples were lysed by sonication (3 × 10 s with 1 min intervals on an icebath; Ultrasonic Processor XL Heat Systems) and then centrifuged for 30 min at 20 000g and 4 °C. The supernatant was again centrifuged 15 min at 20 000g and 4 °C. The proteins were assayed according to Bradford (1976), and the samples were stored at −70 °C.

Protein Identification

Protein identification was performed using an in-house MASCOT 2.3 server and a database of the H. seropedicae protein sequences (accession number CP002039.1).20 Both Peptide Mass Fingerprinting and MS/MS ion search were used. Carbamidomethylation of cysteines was set as fixed and oxidation of methionine as variable modifications. Error tolerance was 100 ppm for peptide mass fingerprint (PMF) and 0.3 Da for the MS/MS search. Only peptides and proteins with scores higher than the Mascot threshold scores (p < 0.05) were considered for positive identifications.

Two-Dimensional Electrophoresis

Protein isoelectric focusing was performed using IPG strips (13 cm) with a linear pH range (4−7) in an Ettan IPGphor Isoelectric Focusing System (GE Healthcare, Uppsala, Sweden). 400 μg of protein was mixed with rehydration buffer (8.5 mol/L urea, 2% v/v TWEEN 20, 2% w/v CHAPS, 1% v/v IPG buffer pH 4−7, 20 mmol/L DTT, and 0.002% w/v bromophenol blue)17 to a final volume of 250 μL. The rehydration step of 16 h at 50 V was followed by isoeletric focusing and conducted to the final accumulated voltage of 26 400 V. The IPG gel strips were stored at −70 °C. Prior to the second dimension, strips were equilibrated for 30 min in 5 mL of equilibrium buffer (6 mol/L urea, 30% w/v glycerol, 2% w/v SDS, 0.002% w/v bromophenol blue in 50 mmol/L Tris-HCl pH 8.8) containing 50 mg of DTT, followed by 30 min of incubation in 5 mL of equilibrium buffer containing 200 mg of iodoacetamide. The strips were washed with distilled water, and equilibrated in running buffer (3 g of Tris base, 14 g of glycine, 1 g of SDS per liter, pH 8.3).18 The second dimension (SDS-PAGE) was performed in polyacrylamide gels (12.5%) on a Hoefer SE 600 Ruby (GE Healthcare). Gels were stained using colloidal coomassie blue.19

RNA Extraction and Quantitative Real Time PCR (qRT-PCR)

Total RNA from H. seropedicae SmR1 cells grown in the presence or absence of 5% sugar cane extract was purified with RiboPure Bacteria kit (Applied Biosystems). Quantification and purity of RNA samples were performed spectrophotometrically using a spectrophotometer NanoDrop 2000 (THERMO Scientifics), and the integrity of the RNA samples was confirmed by agarose gel electrophoresis. For RT-qPCR, cDNA was obtained using the high-capacity cDNA reverse transcription kit (Applied Biosystems), and amplification was performed using Power SYBR-Green PCR Master Mix on a Step One Plus Real Time-PCR System (Applied Biosystems). RQ (relative gene expression) values were determined by the 2−Δ ΔCt method.21 Reactions using purified RNA before the treatment with reverse transcriptase were used as no template control, and all samples were run in triplicate. Calibration curves for all primer sets were linear over at least 4 orders of magnitude (R2 = 0.97−0.99) (Table S2). 1143



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Figure 1. Differential proteome of Herbaspirillum seropedicae in the presence of sugar cane extract. (A) 2-DE gels of proteins extracted from Herbaspirillum seropedicae cells grown in the absence (C) or in the presence of 5% sugar cane extract (SE). Protein spots overexpressed in the presence of sugar cane extract are 2211, 2663, 2692, 2887, and 2875; protein spots underexpressed in the presence of 5% sugar cane extract are 2430, 2954, and 2897; protein spot 2810 is present exclusively in the presence of 5% sugar cane extract; and protein spots 2708, 2947, 2733, 2937, 2766, 2719, and 2713 are absent in the presence of 5% sugar cane extract. (B) Bar plot of spot volume percentages calculated from master 2D gels of each condition using Imagemaster 6.0 software.

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RESULTS AND DISCUSSION

(SE) are shown in Figure 1A. Analyses using Imagemaster (GE Healthcare) showed an average of 396 spots in each gel, 30 of which represented proteins differentially expressed (1.5-fold difference) as determined by Student’s test (p < 0.05), and were selected for MS analysis. These proteins are probably involved in the adaptation of H. seropedicae to survive in the presence of the plant. A total of 16 differential spots, representing 1 exclusively expressed spot, 7 absent, 5 up- and 3 downregulated in the presence of 5% sugar cane extract (Table 1 and Figure 1B), were identified, and thus the host extract is able to induce and repress specific genes of H. seropedicae. The differential expression of all of them was validated by RT qPCR (Figure 2). We divided these proteins into two groups: proteins related to metabolic pathways and proteins probably involved in plant bacterial interaction. Twelve H. seropedicae proteins differentially expressed in the presence of sugar cane extract are involved in metabolism of

To verify if H. seropedicae gene expression is controlled by sugar cane extract components, we grew the bacteria in the presence of 5% extract and performed proteome analysis. The growth profile of H. seropedicae in the absence and presence of the extract was very similar (Figure S1) with maximum growth rate of μmax = 0.558 ± 0.083 h−1 and μmax = 0.597 ± 0.023 h−1, respectively, although the cultures in the presence of sugar cane extract exhibited a shorter lag phase (Figure S1). The samples for proteome analyses were collected at late exponential growth phase (OD600 = 0.6). Comparison of the 2D-PAGE pattern of H. seropedicae cultivated in the presence or absence of sugar cane extract allowed identification of differentially expressed H. seropedicae proteins. The 2-D electropherograms of H. seropedicae cultivated in the absence (C) or presence of sugar cane extract 1144



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Table 1. Differentially Expressed Proteins of H. seropedicae Strain SmR1 Grown in the Presence or Absence of Sugar Cane Extract pI/Mr (kDa) spot ID

exp

protein score

theor

acession no.

2692

5.5/37.3

5.7/43

110

Hsero_2070

2810

5.2/37

5.3/36

78

Hsero_4662

2875 2211

6.0/31.6 5.3/78

6.6/30 5.1/77

54 78

2430

5.6/36.2

6.0/45

2713

5.9/56.4

2719

protein description

spot fold changea

test t (p value)

seq. peptides identified MS/MS (ion score)

1.55

Hsero_3276 Hsero_0111

FliC flagellin protein; filament structural Protein MreB actin-like ATPase involved in cell morphogenesis protein DapA dihydrodipicolinate synthase protein FusA elongation factor G1 (EF-G1) protein

2.09 2.52

0.05 0.05

158

Hsero_3499

Qor NADPH:quinone oxidoreductase Protein

−3.82

0.05

6.4/59

100

Hsero_0415

absentc

6.3/53.6

6.6/53

86

Hsero_3923

absent

−

2766

5.8/47.4

6.1/52

86

Hsero_4423

absent

−

2937

5.9/27.7

6.0/28

74

Hsero_0163

absent

−

2954 2733 2897 2947

6.7/26.5 5.8/55.6 6.2/35.4 5.6/23

6.7/26 6.2/57 6.6/32 5.9/27

72 50 NDd ND

Hsero_2998 Hsero_0147 Hsero_2976 Hsero_0430

2663 2887

5.2/63 5.5/28

5.1/64 5.6/33

ND ND

Hsero_3689 Hsero_1723

2708

5.4/43

5.6/53

ND

Hsero_0097

PurH bifunctional enzyme: phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase transmembrane protein GltD glutamate synthase (small subunit) oxidoreductase protein GlmU UDP-N-acetylglucosamine pyrophosphorylase protein GpmA 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase protein PhbB acetoacetyl-CoA reductase protein NAD-dependent dehydrogenase protein Mdh malate dehydrogenase protein ThiE thiamine monophosphate synthase protein RpsA 30s ribosomal subunit S1 protein SuhB inositol monophosphatase (extragenic suppressor) protein TufB EF-Tu elongation factor protein

LPLAPLGAAYHDTVR (26) VTLFFGSYHDVDSNENAFR (51) VHAVGLNPPDWYLR (64) AYAQYVSVAASEIALKPQR (35) −

0.01

−e

presentb

−5.09 absent −2.14 absent 2.58 2.09 absent

TLNDEYTQLSQEVFR (119)

0.05 0.05

0.01 0.02

− − IVQGLEIDEFSQER (35) DGASYVAFGGFYPSR (24) GAVIQLTDEVEGYLR (30) GIITQAVVYDPTR (30) LLDQGQAGDNVGVLLR (50)

a

Spot fold change was calculated from the averaged means of normalized spot volumes of at least three 2D-gels of each condition; positive values indicate increased protein spot volume in cells grown in the presence of sugar cane extract, and negative volumes indicate decreased spot volume. b Present exclusively in cells grown in the presence of sugar cane extract. cPresent exclusively in cells grown in the absence of sugar cane extract. d ND: Protein identified only by MS/MS. eNo peptide yielded MS/MS data for these spots.

The absence of this protein when the cells were grown with 5% sugar cane extract may indicate that purine is abundant in the extract. Genes involved in de novo purine biosynthesis are necessary for optimal virulence in animal and plant pathogens such as Salmonella,31 Streptococcus pneumoniae,32 Vibrio vulnificus,33 Xanthomonas oryzae,34 and Bacillus anthracis.35 Purine metabolism also seems to be important for symbiosis because purine auxotrophy in Rhizobium spp. led to abortion of the infection and pseudonodules formation.36−39 Knockout of purH in Mesorhizobium loti caused accumulation of AICAR and Fix negative nodules,40 whereas AICAR down-regulates f ixNOPQ expression of Rhizobium etli.41 The GltD glutamate synthase smaller subunit was not detected in H. seropedicae cells growing in the presence of sugar cane extracts. GltD glutamate synthase (GOGAT) is involved in the early stages of ammonium assimilation, catalyzing the transamidation reaction from glutamine to α-ketoglutarate to form two molecules of glutamate using NADPH as electron donor.42 The glutamine synthase/glutamate synthetase (GSGOGAT) pathway is responsible for the assimilation of ammonium in Proteobacteria and usually is more active under nitrogen-limited.43 In E. coli, the expression of the gltBD operon is not regulated by ammonium, but repressed in the presence of glutamate. Although the regulation of the gltBD operon has not been studied yet in H. seropedicae, the repression of GltD in the presence of sugar cane extract may

nucleotides, cofactors, and vitamins (PurH bifunctional enzyme: phosphoribosylaminoimidazolecarboxamide formyltransferase/inosine monophosphate cyclohydrolase transmembrane protein and ThiE thiamine monophosphate synthase),22,23 ammonia assimilation (GltD glutamate synthase oxidoreductase),24,25 carbohydrate metabolism (GlmU UDP Nacetylglucosamine pyrophosphorylase, GpmA 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase, and SuhB inositol monophosphatase protein),26 energy metabolism (NAD-dependent dehydrogenase, Mdh malate dehydrogenase, Qor NADPH:quinone oxidoreductase),27 protein metabolism (FusA elongation factor G1), fatty acid metabolism (acetoacetyl-CoA reductase), and amino acid biosynthesis (DapA dihydrodipicolinate synthase),28 suggesting that adaptation to the host leads to a considerable metabolic change probably as result of the availability of a new range of nutrients. All of these proteins were previously identified in the proteome reference map of H. seropedicae SmR1.29 The PurH bifunctional enzyme phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP (inosine monophosphate) cyclohydrolase transmembrane protein was absent in cells grown in the presence of sugar cane extract. PurH catalyzes the two final steps in IMP biosynthesis of de novo purine biosynthesis,30 converting AICAR (5-aminoimidazole-4carboxamide ribonucleotide) to FAICAR (5-phosphoribosyl-5formaminoimidazole-4-carboxamide), and the latter to IMP. 1145



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Figure 2. mRNA relative quantification of differencially expressed genes of Herbaspirillum seropedicae grown in the presence of sugar cane extract. Gene expression values in H. seropedicae grown in the presence of 5% sugar cane extract were normalized to the expression of the reference genes 16S rRNA and rpoC and compared to control H. seropedicae without sugar cane extract. Relative expression was determined by the 2−Δ ΔCt (threshold cycle) method.20 The results represent the average of three replicate experiments. Student’s test t results for each gene are shown. The RT-PCR experiments were performed using Power SYBR green master mix (Applied Biosystems), and reactions were run on a Step One Plus real-time PCR system (Applied Biosystems).

be due to the presence of glutamate in the extract.44 Interestingly, a strain of Rhizobium etli carrying mutation in the gltB gene of the GOGAT large subunit led to increase of ammonium secretion to the plant, transport to leaves, and nitrogen accumulation in seeds.45 This result raises the possibility that down-regulation of GOGAT expression in H. seropedicae exposed to sugar cane extract may also facilitate secretion of fixed nitrogen to the plant by the endophytic bacteria. We observed no difference in spot intensity of the glutamine synthetase (not shown). This result was expected because the ammonium level is the major factor regulating GS expression in H. seropedicae. The presence of high NH4Cl (20 mM) in our assays would cause down-regulation of glnA and inhibition of GS activity by adenylylation.29,46 GlmU (UDP-N-acetylglucosamine pyrophosphorylase), GpmA (2,3-bisphosphoglycerate-dependent phosphoglycerate mutase), and NAD-dependent dehydrogenase were absent in the presence of sugar cane extract. GlmU catalyzes the last two sequential reactions in the de novo biosynthetic pathway for UDP-GlcNAc (conversion of Glc-N-1-P to GlcNAc-1-P and GlcNAc-1-P to UDP-GlcNAc). In bacteria, UDP-GlcNAc is used in the synthesis of peptidoglycan47 and lipopolysaccharides (LPS),48 both of which act as plant innate immunity elicitors, and have been called microbe-associated molecular

patterns (MAMPs).49 LPS play an important role in the attachment of H. seropedicae to maize roots, and alterations in LPS structure inhibit the bacteria attachment to the root surface.50 Furthermore, genes of H. seropedicae involved in LPS and exopolysaccharide biosynthesis were found to be regulated by the flavonoids naringenin.9 Because LPS is necessary for efficient adhesion of H. seropedicae to maize roots,50 we tested the ability of the wild type and rmlB mutant (LPS negative) strains to adhere to sugar cane B-4362 roots. We observed approximately 100-fold less cells of the mutant strain attached to sugar cane roots when the strains were grown in the presence or absence of sugar cane extract (Figure S2), indicating that LPS is important for attachment of H. seropedicae to sugar cane roots and that repression of GlmU by the extract did not abolish LPS synthesis. We hypothesize that repression of GlmU is part of a response of the bacteria to components of sugar cane extract, which lead the modulation of LPS and peptidoglycan synthesis to avoid the elicitation of plant defense. GpmA catalyzes conversion of 2-phosphoglycerate to 3-phosphoglycerate, suggesting that the presence of sugar cane extract is inhibiting the glycolysis. The repression of carbohydrate metabolism was also observed during the interaction between G. diazotrophicus and sugar cane.11 The function of NAD-dependent dehydrogenase is not known. 1146



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type 3 secretion system, which may be involved in plant interaction.20 The finding that MreB is induced by sugar cane extract adds to the possibility that it may be involved in plant interaction through type IV pili. The flagellin protein FliC, the major structural component of the flagellar filament,65 was up-regulated by sugar cane extract. Interestingly, f liC up-regulation (overexpressed 1.5 fold) was observed when H. seropedicae was cultivated in the presence of the plant-derived flavonoid naringenin (Tadra-Sfeir, unpublished). The f liC gene was also up-regulated during tomato infection by Ralstonia solanacearum.66 Flagella have been implicated in plant colonization by the plant-growth promoter Pseudomonas fluorescens,67,68 and by the foodborne pathogen Listeria monocytogenes,69 and in biofilm formation by the plant pathogens Erwinia amylovora70 and Xanthomonas axonopodis pv Citri.71 Moreover, nodule development of sweet clover was delayed by mutation in the flagella genes f liP and f lgH of Sinorhizobium meliloti.72 Interestingly, Mesorhizobium loti and Flg22, a 22 amino acids peptide from the N-terminal domain of flagellin, induce similar defense responses in Lotus japonicus. The defense responses induced by Flg22 caused inhibition of infection and delay of nodule organogenesis.73 Together these results suggest that a fine regulation flagellin synthesis and other bacterial elicitors are necessary for establishment of a successful plant−bacteria interaction. The proteomic approach showed that H. seropedicae translation elongation factor EF-TU is strongly repressed in the presence of sugar cane extract. qPCR, however, indicated a decrease of only 40% of tuf B mRNA (Table 1), suggesting post-transcriptional regulation may also operate to control EFTU levels. Similar to our results, several isoforms of G. diazotrophicus EF-TU were down-regulated and one isoform completely repressed after 7 days of cocultivation with sugar cane plantlets.11 On the other hand, Sinorhizobium meliloti EFTU was induced in Medicago truncatula nodulated roots, indicating that this protein may be important for plant root symbioses.74 EF-TU was also up-regulated in G. diazotrophicus when cocultivated for 24 h with sugar cane Chunee and SP701143.13 Our results suggest that the up-regulation of FliC and down-regulation of EF-TU in H. seropedicae grown in the presence of sugar cane extract may be part of a coordinated regulation of factors affecting elicitation of defense systems to modulate plant response temporally and permit plant colonization.

The acetoacetyl-CoA reductase (fatty acids metabolism) is found in smaller quantities in cells cultivated in the presence of sugar cane extract. This protein belongs to the short-chain dehydrogenases/reductases (SDR) family and is related to the poly hydroxybutyrate biosynthesis.51 In Ralstonia eutropha H16, the phaB3 gene (coding acetoacetyl-CoA reductase) expression seems to be repressed in rich medium and plant oil-minimal medium.52 It is possible that substrate availability in the sugar cane extract may inhibit the production of PHB in H. seropedicae at the log phase. However, the qPCR validation data did not agree with the gel quantification: the former method indicated no difference in expression, whereas spot density indicated a 5-fold repression (Table 1). This discrepancy may be due to post-transcriptional regulation. The Mdh malate dehydrogenase and Qor NADPH:quinone oxidoreductase are found in smaller quantities in cells cultivated in the presence of sugar cane extract. These proteins are involved in energy metabolism, suggesting that in the presence of the plant extract, H. seropedicae adjusts its energy metabolism to adapt to the new environmental condition. Four differentially expressed proteins may be involved in the interaction of H. seropedicae with plants: RpsA, MreB, FliC, and EF-TU. Two of these, EF-TU and FliC, are also microbeassociated molecular patterns (MAMPS). The 30S ribosomal protein S1 (RpsA) was up-regulated in the presence of sugar cane extract. RpsA interacts with mRNA leader sequence during translation initiation complex formation, regulating translation initiation.53 RpsA expression is regulated in other plant-bacteria associations. In G. diazotrophicus, the RpsA expression is down-regulated when the cells were cocultivated for 7 days with sugar cane SP70-1143 plantlets.11 On the other hand, the expression of RpsA increased when G. diazotrophicus cells were cocultivated for only 24 h with sugar cane SP70-1143.13 We also observed upregulation of H. seropedicae RpsA after a short exposure (7−8 h) to sugar cane extract. Together the results suggest that the RpsA expression of endophytic diazotrophs may be differentially regulated depending on the stage of the interaction with sugar cane. Spot 2810, expressed exclusively in the presence of sugar cane extract, was identified as the MreB protein. This protein is a bacterial actin homologue, which is essential for chromosome segregation, cell shape maintenance, polar localization of several bacterial proteins, assembly of new peptidoglycan, and bacterial surface motility.54−58 In Myxococcus xanthus, MreB forms helical cytoskeleton filaments, and the inhibition of MreB polymerization caused an inhibition in M. xanthus motility, suggesting that MreB could be necessary for the cellular localization of motility proteins.59 MreB also appears to play a role in type-IV pili localization and associated pilus-driven cell motility in Pseudomonas aeruginosa.60 Type IV pili of P. aeruginosa are necessary for mammalian host cells adhesion during the initial stages of infection61 and also for biofilm formation.62 A mutation in the type IV pili gene, pilM, of Acidovorax avenae subsp. citrulli reduced the virulence, biofilm formation, and twitching motility of this bacterium.63 The involvement of MreB in pili localization suggests a role of this protein in the interaction between the bacteria and the host cell. For example, the Azospirillum brasilense mreB mutant has altered morphology and cell surface,64 indicating that the A. brasilense MreB-like protein could be involved with plant interaction. H. seropedicae SmR1 has genes encoding type IV pili located in a gene cluster also containing the genes for the

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CONCLUSION The sugar cane extract alters the H. seropedicae proteome, and the majority of the proteins differentially expressed are involved in metabolic pathways, indicating that metabolic changes occur in response to signal molecules and nutrients. Although the plant exudate, to which the bacteria are exposed under natural conditions, does not have the same composition of the extract used in this work, it is likely that they share a considerable number of components, suggesting that similar pathways are regulated when the bacteria is in contact with the plant. The results show that biosynthetic pathways such as purine and aminoacid biosynthesis are down-regulated, probably due to the availability of these nutrients in the sugar cane extract. More importantly, the proteins RpsA, MreB, GlmU, FliC, and EF-TU were differentially expressed and may be involved in the interaction between H. seropedicae and plants. GlmU, MreB, and FliC are involved in cell wall modification, indicating that the bacterial envelope changes when the bacteria contact plant1147



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derived molecules. GlmU catalyzes the synthesis of UDPGlcNAc, a precursor of LPS, and peptydoglycan, which together with the proteins EF-TU and FliC are known as MAMPS, eliciting the plant innate immune system. The results provide evidence that the H. seropedicae modulates the plant immune response. In agreement with this suggestion, it has been shown that the defense-related genes coding for thionins and PBZ1 of rice roots were repressed upon inoculation with H. seropedicae.75 Flagella and LPS may also be involved in bacterial attachment onto root surfaces; thus the changes we observed may suggest that H. seropedicae modifies its cell wall in preparation for interaction with the root surface.

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ASSOCIATED CONTENT

* Supporting Information S

Additional figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 55 41 3361 1667. Fax: 55 41 32622042. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Marshall Geoffrey Yates for critical reading of the manuscript and Valter de Baura, Roseli Prado, Marilza Lamour, and Alexsandro Albani for technical support. This work was supported by the Brazilian Program of National Institutes of Science and Technology-INCT/Brazilian Research CouncilCNPq/MCT and Fundaçaõ Araucária.

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REFERENCES

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Proteomic Analysis of Herbaspirillum seropedicae Cultivated in the

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