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Journal of Bacteriology, April 2008, p. 2841-2850, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01775-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System in Agrobacterium tumefaciens ^,

Hung-Yi Wu, Pei-Che Chung, Hsiao-Wei Shih, Sy-Ray Wen, and Erh-Min Lai^*

Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan

Received 9 November 2007/ Accepted 29 January 2008

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Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.

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Bacterial pathogenesis relies mainly on the activity of proteins secreted by a variety of protein secretion systems, the evolution of which has been driven in large part by the dual-membrane envelope of gram-negative bacteria. In addition to the general secretory pathway (Sec) and twin-arginine translocation (Tat), which export proteins across the inner membrane into the periplasm, there are at least six distinct protein secretion systems in gram-negative bacteria. These secretion systems are able to secrete proteins from the cytoplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (31) and a recently discovered type VI secretion system (T6SS) (11, 29, 33). Furthermore, animal- and plant-pathogenic bacteria have significant common themes, as almost all types of secretion systems are present and secrete virulence factors in pathogenic bacteria attacking members of both the plant and animal kingdoms. Interestingly, bacteria that cause different disease symptoms seem to exploit different types of protein secretion systems.

Agrobacterium tumefaciens causes crown gall disease in a wide range of plants by transforming plants through transfer and integration of its transferred DNA into the host genome (6, 13, 28). This process is activated when A. tumefaciens senses phenolic compounds, such as acetosyringone (AS), released from wounded plant cells. This leads to the expression of virulence (vir) genes transduced by a VirA/VirG two-component system. The T-complex (single-stranded transferred DNA associated with VirD2 pilot protein) and virulence (Vir) proteins, such as VirE2 (single-stranded DNA binding protein) (45), VirF (F-box protein) (45), VirE3 (21, 46), and VirD5 (47), are then transported through a T-pilus-associated T4SS (5, 24) into host plant cells. The T4SS is generally thought to originate from the bacterial conjugation machinery and is assembled as a translocation channel for transmitting protein and DNA among bacteria or to fungi, plants, and mammalian cells. In addition to VirB/D4 T4SS, Sec, Tat, T1SS, and T5SS are encoded in A. tumefaciens (31). One of these systems, Tat, has been shown to be important for A. tumefaciens virulence but also has pleiotropic effects, such as causing defects in the growth rate, motility, and chemotaxis (9). The functions of the other secretion systems in A. tumefaciens have been explored less.

T6SS is highly conserved in animal- and plant-associated Proteobacteria. T6SS was discovered in a non-O1/non-O139 Vibrio cholerae strain, V52, in which the vas (virulence-associated secretion) gene cluster-encoded T6SS is responsible for the loss of cytotoxicity for amoebae and the secretion of two proteins lacking an identifiable N-terminal signal peptide, Hcp (hemolysin-coregulated protein) and VgrG (valine-glycine repeats G) (33). The involvement of T6SS in virulence and Hcp secretion has also been demonstrated for several animal pathogens, such as Pseudomonas aeruginosa (29), Edwardsiella tarda (35), Burkholderia mallei (39), enteroaggregative Escherichia coli (EAEC) (10), and Francisella tularensis (7). Interestingly, both Hcp and VgrG are also part of the T6SS machinery. Mutation in V. cholerae hcp blocks the secretion of VgrG-1, VgrG-2, and VgrG-3 (33). Conversely, both VgrG-1 and VgrG-2 are also required for Hcp and their own secretion in V. cholerae (32). A crystallography study revealed that P. aeruginosa Hcp1 forms a hexamer ring with a 40-Å internal pore, suggesting a possible channel through which substrates are secreted (29). Based on a combination of structure modeling and protein-protein interaction studies, VgrG proteins are thought to assemble into a phage tail spike-like structure for secretion of proteins into the host cell (32). VgrG-1 possesses a C-terminal actin cross-linking domain which was identified by transient expression in mammalian cells (40) and was later shown to possess ATP-dependent actin cross-linking activity in vitro and in vivo (32). Together, Hcp and VgrG may serve as an extracellular T6SS translocon, but this possibility remains to be demonstrated.

T6SS is highly regulated. Hcp was first shown to be regulated coordinately with the hemolysin HlyA by the HlyU regulatory system in V. cholerae O17 (49). Microarray analysis of a deletion mutant with a mutation in vasH, located in the t6ss cluster in V. cholerae strain V52, suggested that hcp expression is also transcriptionally regulated by the predicted activator ⁵⁴ encoded by vasH (33). Even more strikingly, P. aeruginosa Hcp1 was not detectable in the wild type but was expressed and secreted in a mutant with retS deleted, which encodes the T3SS and exopolysaccharide regulator RetS (29). The expression level of Hcp is maximal at a late stage of biofilm development in P. aeruginosa (37), and it has been suggested that this protein plays a role in biofilm development (12). In EAEC, the expression of AaiC/Hcp is upregulated by an AraC family transcriptional regulator, AggR, which plays a central role in modulating adherence of EAEC strain 042 (10). Hcp was also recently identified in the plant extract-induced secretome of the plant-pathogenic bacterium Pectobacterium atrosepticum, and its plant-induced expression was upregulated at the mRNA level (27). Furthermore, recent work also demonstrated that posttranslational regulation of Hcp secretion occurs via threonine phosphorylation (30).

In this study, we used a proteomics approach to analyze the Agrobacterium secretome, which led to identification of 1 known and 11 newly discovered potential secretory proteins. Among these proteins, an unknown protein with limited amino acid sequence homology to Hcp was found to be localized mainly in cytosol but also to be secreted via a T6SS in A. tumefaciens. Tumorigenesis analysis revealed that A. tumefaciens Hcp may play a positive role in facilitating efficient tumorigenesis. This work expands our knowledge of the newly identified T6SS from animal pathogens to plant pathogens.

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Bacterial strains and growth conditions. The strains, plasmids, and primer sequences used in this study are described in Table 1 and Table S1 in the supplemental material. For vir gene induction, A. tumefaciens cells grown overnight in 523 broth (18) with appropriate antibiotics were harvested by centrifugation (7,000 x g, 10 min) and resuspended in fresh induction medium (AB-MES) (pH 5.5) (23) without antibiotics at an A₆₀₀ of 0.1. After growth at 28°C to an A₆₀₀ of 0.2, the cells were further cultured at 19°C for 40 h in the presence of 200 µM AS (Sigma-Aldrich, St. Louis, MO) (0.1% of a 200 mM stock solution dissolved in 100% dimethyl sulfoxide [DMSO]) until they were harvested. Controls were grown under the same conditions without treatment (0.1% H₂O) or in the presence of 14.1 mM (0.1%) DMSO. To investigate Hcp secretion in other growth conditions, A. tumefaciens cells were grown as described above for vir gene induction with the changes in growth medium and temperature indicated below. The concentrations of antibiotics used were 100 µg/ml ampicillin and 10 µg/ml gentamicin for Escherichia coli and 50 µg/ml erythromycin, 50 µg/ml rifampin, 250 µg/ml spectinomycin, and 50 µg/ml gentamicin for A. tumefaciens.

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TABLE 1. Bacterial strains and plasmids

Isolation and analysis of secretory proteins. Secretory proteins were isolated using methods described previously (22), with modifications. For secretome analysis by mass spectrometry, a 250-ml bacterial culture of A. tumefaciens strain C58 grown in AB-MES (pH 5.5) in the absence or presence of AS at 19°C for 40 h was harvested by centrifugation at 10,000 x g and 4°C for 10 min. The resulting supernatant (S1 fraction), containing the proteins secreted into the culture medium, was filtered through a 0.45-µm membrane to remove contaminating bacterial cells. For each 100 ml of the S1 fraction, 15 ml of 100% trichloroacetic acid (TCA) and 3 ml of 1% sodium deoxycholate were added, and the fractions were incubated on ice overnight before centrifugation at 17,000 x g and 4°C for 10 min. The resulting protein pellet was washed with 100% ice-cold acetone three times before shotgun proteomics analysis (see below). For small-scale secretory protein isolation for Western blot analysis, 1 ml of cell culture harvested to obtain the total proteins and the resulting supernatant (S1 fraction) was concentrated by TCA precipitation and resuspended in an appropriate volume (10 µl) of 1 M Tris base (original pH) and an equal volume of 2x sodium dodecyl sulfate (SDS) solubilization buffer (36) prior to SDS-polyacrylamide gel electrophoresis (PAGE) analysis. The total proteins were prepared by dissolving the cell pellet with 1x SDS solubilization buffer to obtain an A₆₀₀ of 5. Ten microliters of the total proteins or the S1 fraction collected from 1 ml of culture medium was loaded into each lane.

Shotgun proteomics LC-MS/MS analysis. To identify the proteins in the S1 fraction, the concentrated proteins were subjected to shotgun trypsin digestion, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The concentrated protein pellet collected from 100 ml of a culture was solubilized in 25 mM ammonium bicarbonate (pH 8.5), reduced with 55 mM dithioerythritol in 25 mM ammonium bicarbonate (pH 8.5) for 1 h at 37°C, and subsequently alkylated with 100 mM iodoacetamide in 25 mM ammonium bicarbonate (pH 8.5) for 1 h in the dark at room temperature. Alkylated samples were digested with 1.5 µg of sequencing-grade, modified trypsin (Promega, Madison, WI) at 37°C overnight. The tryptic peptide mixtures were further purified by use of a ZipTip_µ-C18 pipette tip (Millipore) by following the manufacturer's instructions. The eluted peptides were transferred into a microvial for autosampler injection into a nano-LC-MS/MS system (Q-TOF Ultima API mass spectrometer; Micromass) as described previously (25).

Protein identification. The m/z ratios of the precursor ion and MS/MS-fragmented ions obtained by LC-MS/MS were used to search the annotated A. tumefaciens strain C58 genome in the most recent NCBInr database with the Mascot search engine (http://www.matrixscience.com/search_form_select.html), using a maximum of one missed trypsin cleavage, variable modification including carbamidomethylation, and mass accuracy of 0.25 Da (for both precursor and MS/MS ions). The proteins were identified by peptide mass fingerprinting or MS/MS ion searching, and results with P values of <0.05 were considered high-confidence hits.

Genomic DNA isolation, PCR, and colony PCR. A. tumefaciens cells grown overnight in 523 broth with appropriate antibiotics were used for isolation of genomic DNA with a Qiagen genomic DNA kit (Qiagen Inc., Valencia, CA). PCR analysis was performed by using the Advantage 2 PCR enzyme system (BD Biosciences Clontech, Palo Alto, CA) and following the manufacturer's instructions; 10 ng of genomic DNA was used as the template with appropriate specific primers. Unless indicated otherwise, the PCR protocol consisted of 95°C for 2 min, followed by 95°C for 1 min, 58 to 65°C for 1 min, and 72°C for 1 min/kb for a 30 cycles and then 72°C for 10 min. For colony PCR, approximately one-half of a freshly grown colony was resuspended directly in sterile water and used as the PCR template.

Plasmid construction. The techniques used for DNA cloning have been described previously (36). Plasmid DNA isolation involved use of the Mini-M plasmid DNA extraction system (Viogene, Taipei, Taiwan). To generate the spectinomycin resistance gene (aadA) cassette, aadA was PCR amplified from pART27 with primers Sp-BamHI-F and Sp-BamHI-R and cloned into the TA cloning vector pGEM-T Easy (Promega, Madison, MI), which resulted in plasmid pGEMT-Sp. All other DNA fragments were amplified by performing PCR with specific primers for A. tumefaciens strain C58 genomic DNA or for genomic DNA of the C58-derived strain NT1RE(pJK270). For cloning convenience, each 5' and 3' primer was designed to contain a restriction enzyme site at the 5' end (see Table S1 in the supplemental material). The resulting PCR products were purified with a PCR clean-up kit (Viogene), digested with selected restriction enzymes, and inserted into the appropriate enzyme sites in the cloning vector.

To overexpress His-tagged Hcp protein in E. coli, a DNA fragment containing an hcp open reading frame (ORF) without the stop codon was amplified by performing PCR with primers Hcp-NdeI-F and Hcp-XhoI-R, digested with NdeI and XhoI, and inserted into the NdeI/XhoI site of pET-22b(+) to create plasmid pET-22b(+)-Hcp. To construct a plasmid expressing hcp under the control of the heterologous promoter lacZp, the 1,104-bp SpeI/EcoRI DNA fragment containing the hcp ORF was inserted into SpeI/EcoRI sites of the broad-host-range vector pBBR1MCS-5, resulting in plasmid pHcp. Similarly, the icmF ORF was PCR amplified by using primers IcmF-XhoI-F and IcmF-XbaI-R, digested with the XhoI and XbaI enzymes, and then ligated into the XhoI/XbaI sites of pBBR1MCS-2-derived broad-host-range vector pRL662, resulting in plasmid pIcmF. Plasmid pJQ200SK-hcp::Sp^r, used to generate the hcp deletion mutant, was constructed by ligating an XmaI/BamHI-digested Hcp1 PCR product (1,039-bp DNA fragment upstream of the hcp ORF), the BamHI-digested Sp^r gene cassette, and an XbaI/BamHI-digested Hcp2 PCR product (1,283-bp DNA fragment downstream of the hcp ORF) into the XmaI/XbaI sites of the suicide vector pJQ200SK. Plasmid pJQ200SK-t6ss::Sp^r, used to generate the t6ss operon deletion mutant, was constructed by ligating an XbaI/BamHI-digested T6SS1 PCR product (1,158-bp DNA fragment upstream of the putative t6ss operon), the BamHI-digested Sp^r gene cassette, and the PstI/BamHI-digested T6SS2 PCR product (1,194-bp DNA fragment downstream of the putative t6ss operon) into the XbaI/PstI sites of pJQ200SK. Plasmid pJQ200SK-icmF::Sp^r was constructed by ligating the XbaI/BamHI-digested IcmF1 PCR product (1,079-bp DNA fragment upstream of the icmF ORF), the BamHI-digested Sp^r gene cassette, and the XmaI/BamHI-digested IcmF2 PCR product (1,094-bp DNA fragment downstream of the icmF ORF) into XmaI/XbaI sites of pJQ200SK. The plasmid constructs obtained were confirmed by restriction mapping and DNA sequencing.

Gene replacement. The procedure used for gene replacement is a procedure that was described previously (17), with minor modifications. Five microliters of an overnight culture (grown in LB broth without antibiotics) of E. coli strain S-17 containing pJQ200SK-derived constructs and a culture of A. tumefaciens strain NT1RE were mixed and incubated at 28°C on LB agar overnight. The bacterial cells were then streaked on LB agar containing erythromycin, rifampin, and gentamicin and incubated at 28°C for 2 days to obtain the first crossover events. Three colonies were randomly selected and streaked on the same selection medium for further colony purification. Three independent colonies were each grown in 5 ml of LB broth without antibiotics at 28°C overnight, and serial dilutions (up to 10^–4) were plated onto 523 agar containing 5% sucrose, erythromycin, and rifampin and grown at 28°C for 2 days. Routinely, a few dozen colonies were selected and streaked again on the same selection medium to ensure that the population was homogeneous. Colonies were then selected to obtain Sp^r Suc^r Gm^s colonies, which had undergone a second crossover to replace the corresponding wild-type genes with the Sp^r cassette. The resulting mutants were confirmed by colony PCR and Southern hybridization (data not shown). The Ti plasmid pJK270 was then transferred into the mutants by conjugation.

Overexpression and purification of His-tagged Hcp protein in E. coli. For overexpression of Hcp-His we followed the manufacturer's instructions (Novagen, EMD Biosciences, Inc., Germany) and used previously described methods (36), with modifications. E. coli BL21(DE3) containing pET-22b-Hcp was grown in 200 ml of LB broth with antibiotics. After growth at 37°C to mid-log phase (A₆₀₀, 0.4 to 0.6), the cells were induced by adding 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and cultured for 3 h at 28°C. The cells were centrifuged, and the pellet was washed in 10 ml of wash buffer (20 mM Tris-HCl [pH 8.0], 20 mM NaCl, 0.1 mM EDTA) per g of pellet. The cell pellet was gently resuspended in lysis buffer (10 ml/g pellet) containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.05% NP-40, 0.5 mg/ml lysozyme, and proteinase inhibitors, including 2.0 µg/ml leupeptin, 2 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 3 µg/ml pepstatin A) and then incubated on ice with occasional vortexing for 10 to 15 min until the suspension became viscous. The cell suspension was mixed with 25 µg/ml DNase, 2.5 mM MgCl₂, and 14.3 mM 2-mercaptoethanol and incubated further on ice with occasional vortexing for 5 to 15 min until the viscosity decreased. The cells were broken by sonication on ice until the suspension was translucent. The soluble protein fraction containing Hcp-His was recovered by pelleting the unbroken cells and debris by centrifugation at 15,000 x g for 30 min at 4°C. Hcp-His was purified by use of Ni-nitrilotriacetic acid His Bind resins (Novagen) by following the manufacturer's instructions. One milligram of purified His-tagged protein was separated by 15% glycine-SDS-PAGE, which was followed by Coomassie brilliant blue R-250 staining (36). The major 18-kDa protein band, corresponding to the putative Hcp-His, was cut out and used for polyclonal antibody production by rabbit immunization (GlycoNex Inc., Taipei, Taiwan).

SDS-PAGE and immunoblot analysis. Proteins were fractionated by glycine-SDS-PAGE (36) or Tricine-SDS-PAGE (38). An immunoblot analysis was performed as described previously (23) using primary polyclonal antibodies against Hcp (1:2,500 dilution), NptII (neomycin phosphotransferase II; 1:50,000 dilution; Sigma-Aldrich), Bacillus subtilis GroEL (1:5,000 dilution) (3), VirB9 (1:5,000 dilution), and VirB2 (1:5,000 dilution) (41), followed by treatment with secondary antibody with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Chemichem) and detection by use of the Western Lightning system (Perkin Elmer, Boston, MA). Chemiluminescent bands were visualized on X-ray film (Kodak).

Biochemical fractionation. Cellular fractions were isolated as described previously (8), with minor modifications. Briefly, 250 ml of a cell culture was harvested by centrifugation at 10,000 x g and 4°C for 10 min and resuspended in lysis buffer A (50 mM Tris-HCl [pH 7.5], 20% sucrose, 0.2 M KCl, 0.2 mM dithiothreitol, 0.2 mg/ml DNase, 0.2 mg/ml RNase A, 1 mM PMSF) at an A₆₀₀ of 10. The cells were disrupted by two passes through a chilled French pressure cell (Aminco, Silver Spring, MD) at 16,000 lb/in². The lysate was treated with lysozyme (0.5 mg/ml) on ice for 30 min and centrifuged at 10,000 x g and 4°C for 10 min twice to remove the unbroken cells. The supernatant, referred as the total protein fraction, was centrifuged at 150,000 x g at 4°C for 1 h to separate a soluble fraction containing both cytoplasmic and periplasmic proteins and an insoluble fraction enriched with inner and outer membrane proteins. The resulting insoluble pellet was washed briefly and resuspended in the same volume of 50 mM Tris-HCl (pH 7.5) containing 1 mM PMSF.

Protein sequence analysis. For prediction of the subcellular localization and signal peptides we used PSORTb (http://www.psort.org/psortb/index.html) and SignalP (http://www.cbs.dtu.dk/services/SignalP/). A comparative sequence analysis of proteins encoded by the t6ss gene cluster was performed using BLASTP against the genome sequences of selected bacterial species.

Tumor assay with potato tuber disks. A quantitative tumorigenesis assay with potato tuber disks was performed as described previously (42), with minor modifications. Briefly, A. tumefaciens strains grown in 523 medium supplemented with the appropriate antibiotics at 28°C to an A₆₀₀ of 0.8 to 1.0 were washed with 0.9% sodium chloride and resuspended in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 2 mM KH₂PO₄, 10 mM Na₂HPO₄; pH 7.4) at concentrations of 10⁸ and 10⁶ CFU/ml. Potato tubers were surface sterilized with 1.05% sodium hypochlorite for 5 min and rinsed in sterile water three times. One-centimeter-diameter cores of the potato tubers were obtained using a cork borer, and the cores were cut into 2- to 3-mm thick sections. The potato tuber disks were placed on water agar, infected with 10-µl portions of bacterial cultures, and incubated at 24°C for 2 days. Disks were then placed on water agar supplemented with timentin (100 µg/ml, equivalent to 96.8 µg/ml ticarcillin and 3.3 µg/ml clavulanic acid) and incubated at 24°C for 3 weeks before tumors were scored.

Immunoelectron microscopy. A. tumefaciens strains grown in AB-MES (pH 5.5) at 25°C to an A₆₀₀ of 0.6 to 0.8 were analyzed by immunoelectron microscopy using Hcp antisera. The bacterial cells were frozen by using nitrogen slush, freeze-substituted in ethanol, embedded in LR Gold resin (London Resin Company), sectioned (thickness, 110 to 120 nm) with a microtome (EM UC6; Lecia), and deposited on 100-mesh grids with Formvar supporting film. The samples were incubated first in blocking solution A (0.5% blocking reagent [Roche], 100 mM Tris-HCl [pH 7.5], 15 mM NaCl, 0.3% Tween 20) for 30 min and then in blocking solution B (1% bovine serum albumin, 0.3% Triton X-100, 100 mM Tris-HCl [pH 7.5], 15 mM NaCl, 0.3% Tween 20) for 40 min. Grids were incubated with anti-Hcp primary antibody (1:25 dilution in blocking solution B) for 60 min, washed with buffer I (100 mM Tris-HCl [pH 7.5], 15 mM NaCl, 0.3% Tween 20) five times (5 min each), and then incubated in secondary antibody (1:20 dilution of goat anti-rabbit immunoglobulin G conjugated with 18-nm gold particles in blocking solution B) for 30 min. The samples were washed with buffer I (three times for 5 min each) and double-distilled H₂O (three times for 5 min each), stained with 5% uranyl acetate-0.5% lead citrate, and examined using a PHILIPS-CM100 transmission electron microscope at 80 kV.

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Identification of secretory proteins. The Agrobacterium secretome was analyzed by identifying the proteins present in the culture medium (S1 fraction) of A. tumefaciens strain C58 grown in the presence or absence of AS using shotgun proteomics. Two independent S1 fractions were collected, precipitated with TCA and acetone, digested with trypsin, and subjected to LC-MS/MS analysis, followed by protein identification. Twelve proteins were consistently identified in both experiments (Table 2). Except for the known AS-induced protein VirB1* (1), the identified secretome proteins were detected when cells were grown in either the absence or the presence of AS. Identification of VirB1* (1), the secreted protein known to be involved in T-pilus assembly (50), validated our proteomics approach as a method for identifying secretory proteins. Interestingly, except for an unknown protein (Atu4345; AGR_L_1037), all identified secretome proteins have known or putative Sec-dependent signal peptides or are predicted to be exported across the cytoplasmic membrane (Table 2). Because many effector proteins secreted via T3SS or T4SS also do not have a signal peptide (31), we predicted that Atu4345 may be an effector protein secreted via a protein channel. Indeed, Atu4345 is a small protein with limited sequence similarity (maximum level of identity, 30%) to Hcp, a known secretory protein secreted via the recently identified T6SS (33). Therefore, Atu4345 was designated Hcp.

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TABLE 2. Identification of secretome proteins by shotgun proteomic analysis

Hcp is expressed and secreted under various growth conditions. To determine the expression and secretion patterns of Hcp in A. tumefaciens, we first performed a Western blot analysis to confirm the mass spectrometry identification of Hcp in the S1 fraction. A 17-kDa Hcp protein was detected in both the total cell lysate and the S1 fraction of either A. tumefaciens strain C58 or the virulent strain NT1RE(pJK270) derived from C58 grown in acidic minimal medium AB-MES (pH 5.5) in the absence or presence of AS at 19°C for 40 h (Fig. 1A). Successful vir gene induction by AS was demonstrated by detection of VirB2, the T-pilin subunit, which was also detected in its secreted form in the S1 fraction of NT1RE(pJK270) (Fig. 1). Hcp secretion was not limited to growth in acidic minimal medium or at a low temperature as Hcp was also expressed and secreted when the organisms were grown in AB-MES (pH 7.0) or in rich medium 523 (pH 7.0) at 25°C (Fig. 1B). Hcp was also secreted at wild-type levels in the Ti plasmid-less strain NT1RE, indicating that secretion of Hcp does not require genes encoded by the Ti plasmid (data not shown). The absence of the NptII protein, encoded by a Tn5 transposon inserted into the Ti plasmid pJK270, in the S1 fraction indicated that the detection of secreted Hcp was unlikely to be due to contamination by Hcp protein released from lysed cells. Taken together, the data showed that Hcp was expressed and secreted when A. tumefaciens was grown in either minimal or rich medium and that its secretion was independent of the Ti plasmid.

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FIG. 1. A. tumefaciens Hcp is expressed and secreted under various growth conditions. (A) A. tumefaciens strain C58 was grown in AB-MES (pH 5.5) at 19°C for 40 h in the absence (–) or presence (+) of AS. A. tumefaciens strain NT1RE(pJK270) was grown in the same conditions as C58 except that a DMSO treatment was added. (B) A. tumefaciens strain NT1RE(pJK270) was grown in different culture media at 25°C to an A600 of 0.6 to 0.8. The total proteins (lanes T) and secreted proteins present in culture medium (lanes S1) were analyzed by 16.5% Tricine-SDS-PAGE, followed by immunoblotting with antibodies against Hcp, VirB2, and NptII.

Hcp is localized in both the cytosol and the membrane. Although an appreciable amount of Hcp was secreted into the culture medium, most of the Hcp protein was located in the intact bacterial cells (Fig. 1). Biochemical fractionation indicated that most of the Hcp was in the soluble fraction and that only a small amount was in the insoluble fraction (Fig. 2). The quality of fractionation was verified by using the cytosolic marker GroEL and the membrane marker VirB9 (Fig. 2). Because the soluble fraction contained both cytoplasmic and periplasmic proteins, immunoelectron microscopy was performed to localize Hcp at the cellular level. The data obtained for 63 rod-shaped cells of NT1RE(pJK270) with a mean of 4.7 gold particles/cell demonstrated that the ratio of the amount in the cytoplasm to the amount in the membrane was 10:1 (Fig. 3 and Table 3), which corresponded well with the biochemical fractionation data (Fig. 2). The gold particle signal detected in NT1RE(pJK270) was specific for Hcp, as a significant number of gold particles was not observed in the hcp bacterial cells (mean, 0.3 gold particle/cell). Interestingly, a 14-fold increase in the number of Hcp-specific gold particles was detected in the hcp complementation strain hcp(pHcp) (mean, 66 gold particles/cell) (Fig. 3 and Table 3), in which hcp expression was driven by the heterologous promoter lacZp in the medium-copy-number plasmid pBBR1MCS-5, which suggests that there was overexpression of Hcp in this strain. The absence of Hcp in the hcp strain and the overexpression of Hcp in the hcp(pHcp) strain were also confirmed by Western blot analysis (Fig. 4). Taken together, the data suggest that Hcp is localized mostly in the cytoplasm and that a small portion is located in the membrane system (both inner and outer membranes) without distinct clusters or spatial localization.

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FIG. 2. Hcp is localized in both soluble and insoluble fractions. Equal volumes of total proteins (lanes T), the soluble fraction (lanes S), and the insoluble fraction (lanes IS) of A. tumefaciens strain NT1RE(pJK270) grown in AB-MES (pH 5.5) in the absence (–AS) or presence (+AS) of AS at 25°C to an A600 of 0.6 to 0.8 were resolved by 16.5% Tricine-SDS-PAGE. The proteins were analyzed by immunoblotting with antibodies against Hcp, VirB9, and GroEL.

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FIG. 3. Immunolocalization of Hcp. A. tumefaciens strains grown in AB-MES (pH 5.5) at 25°C to an A600 of 0.6 to 0.8 were analyzed by immunoelectron microscopy with Hcp antisera. The strains used are indicated, and gold particles located in the membrane system are indicated by arrows. WT, wild type. Scale bars = 200 nm.

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TABLE 3. Immunoelectron microscopy detection of Hcp signal

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FIG. 4. Hcp is expressed but not secreted in both icmF and t6ss operon deletion mutants. (A) Total proteins (lanes T) and secreted proteins present in the culture medium (lanes S1) of A. tumefaciens strains grown in AB-MES (pH 5.5) at 25°C to an A600 of 0.6 to 0.8 were analyzed by 16.5% Tricine-SDS-PAGE, followed by immunoblotting with antibodies against Hcp and NptII. (B) Analysis of secreted proteins present in S1 fractions by silver staining. The strains analyzed were wild-type virulent strain NT1RE(pJK270) (WT), hcp deletion mutant NT1RE::hcp(pJK270) (hcp), an hcp complementation strain [hcp(pHcp)], t6ss operon deletion mutant NT1RE::t6ss(pJK270) (t6ss), icmF deletion mutant NT1RE:: icmF(pJK270) (icmF), and an icmF complementation strain [icmF(pIcmF)]. The position of silver-stained Hcp is indicated by an arrow.

Comparative analysis of the t6ss gene cluster of A. tumefaciens strain C58 performed with selected pathogenic and symbiotic bacteria. In view of the conservation of T6SS among pathogenic bacteria, we performed a BLASTP analysis to identify a potential t6ss gene cluster in A. tumefaciens. This proposed t6ss gene cluster comprises two putative operons: an operon which we designated the t6ss operon, consisting of 14 genes (Atu4343 to Atu4330 [see Table S2 in the supplemental material]), and an operon which we designated the hcp operon, consisting of 9 genes (Atu4344 to Atu4352 [see Table S3 in the supplemental material) (Fig. 5). By performing a comparative analysis of selected t6ss operon-containing animal- and plant-associated Proteobacteria, we observed both variations in synteny and conservation of the t6ss gene cluster. There are at least three common features of the t6ss gene cluster. First, 10 proteins (Atu4332 [IcmF], Atu4333 [OmpA], Atu4334, Atu4335 [FhaI], Atu4337, Atu4340/Atu4341 [duplicated gene products], and Atu4342 encoded by the putative t6ss operon; Atu4344 [ClpB], Atu4345 [Hcp], and Atu4338 [VgrG-1]) encoded by the putative hcp operon) are conserved among the bacterial strains analyzed and are likely conserved in the majority of gram-negative bacterial species (Fig. 5; see Tables S2 and S3 in the supplemental material). Second, the close physical proximity of hcp and vgrG is conserved; the members of at least one set of hcp and vgrG genes are adjacent (in V. cholerae, P. aeruginosa, and Pseudomonas syringae) or in the same operon (in A. tumefaciens and Rhizobium leguminosarum). The close physical positions ensure temporally regulated translation for possible physical and functional interactions. Third, gene duplications occur for several conserved components in most, if not all, of the bacterial strains analyzed, and near duplications of an entire t6ss gene cluster occur in P. aeruginosa and P. syringae. The hcp gene is duplicated in V. cholerae, P. aeruginosa, and P. syringae, whereas the vgrG gene is duplicated in all bacterial species analyzed except R. leguminosarum. Interestingly, we found significant gene shuffling in the t6ss gene cluster among the bacterial pathogens and symbionts analyzed. In the future use of phylogenomic analysis may provide insights into the evolutionary paths and the evolutionary significance of the gene duplications and shuffling in the t6ss gene cluster.

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FIG. 5. Comparative analysis of the t6ss gene cluster of A. tumefaciens strain C58 and the genes of selected pathogenic and symbiotic bacteria. The members of the t6ss gene cluster of A. tumefaciens strain C58 are indicated by filled colored arrows (putative t6ss operon encoding 14 proteins) and open colored arrows (putative hcp operon encoding 9 proteins). The putative amino acid sequence of each protein encoded by the t6ss gene cluster of A. tumefaciens strain C58 was used as a query for a BLASTP search against the genomes of selected bacteria, including the human bacterial pathogens V. cholerae O1 bv. eltor strain N16961 and P. aeruginosa PAO1, the plant bacterial pathogen P. syringae pv. tomato DC3000, and the plant symbiotic bacterium R. leguminosarum bv. viciae 3841. Orthologs were recognized (indicated by the same colors) based on a BLASTP E value of <e–10 or based on the annotated or previously published data (29, 33). The ClpB ortholog of R. leguminosarum bv. viciae 3841 was annotated as a pseudogene but identified as a truncated ClpB ortholog in this study (indicated by an asterisk). The genes adjacent to or encoded by the t6ss gene clusters but not recognized as orthologs are indicated by gray arrows. The locus number and published or annotated gene designation are indicated above and below each gene, respectively. The conserved orthologs detected in all five bacterial species analyzed are indicated by bold type.

Hcp is expressed but not secreted in both icmF and t6ss operon deletion mutants. To determine whether Hcp secretion occurs via T6SS in A. tumefaciens, we generated a mutant with a deletion in the putative t6ss operon. Indeed, the Hcp protein accumulated only in the cells and was not secreted into the culture medium in the t6ss deletion mutant, compared with the secretion of Hcp in the wild type (Fig. 4). Thus, Hcp is a bona fide secretory protein, and its secretion depends on the presence of an intact t6ss operon in A. tumefaciens. The requirement for T6SS for Hcp secretion was further supported by the Hcp secretion deficiency in the icmF mutant (Fig. 4), in agreement with previous reports that icmF is essential for Hcp secretion (29, 33). The Hcp secretion deficiency in the icmF mutant was complemented when icmF was expressed under the control of lacZp on a plasmid, demonstrating that icmF is required for Hcp secretion. As expected, the SDS-PAGE analysis of S1 fractions with silver staining clearly revealed that an Hcp protein band was not produced by the icmF and t6ss mutants but was produced by the icmF-complemented strain (Fig. 4). However, proteins other than Hcp were detected in S1 fractions of all strains analyzed, suggesting that they are secreted independent of T6SS.

Immunoelectron microscopy to localize intracellular Hcp in both icmF and t6ss operon mutants revealed that Hcp is localized mainly in the cytosol and that a small portion is in the membrane, but there were slightly higher numbers of gold particles per cell in the icmF mutant (mean, 7.8 gold particles/cell) and the t6ss operon mutant (mean, 12.2 gold particles/cell) than in the wild type (mean, 4.7 gold particles/cell) (Fig. 3 and Table 3). The higher intracellular Hcp signal quantified by immunoelectron microscopy agrees well with the slightly higher level of intracellular Hcp protein detected by Western blotting (Fig. 4), which suggests that the increased intracellular Hcp protein level was due to the loss of a functional T6SS for Hcp secretion in the icmF and t6ss operon mutants.

Loss of Hcp resulted in reduced tumorigenesis efficiency with potato tuber disks. To determine whether Hcp is involved in the virulence of A. tumefaciens, we carried out both qualitative and quantitative tumorigenesis assays to determine the effect of Hcp on tumorigenesis. Deletion of hcp did not block the ability of A. tumefaciens to produce tumors on the stems of host plants such as tomato, tobacco, and white radish (data not shown). Therefore, we performed quantitative tumorigenesis assays with potato tuber disks to determine the effect of Hcp on tumorigenesis efficiency. While no consistent difference in tumorigenesis efficiency was found between the wild type and the hcp mutant when disks were inoculated with a suspension containing 10⁸ CFU/ml of bacterial cells (data not shown), we detected a clear reduction in the tumorigenesis efficiency to about 70 to 80% of the wild-type efficiency with a lower concentration of the hcp mutant (10⁶ CFU/ml) (Fig. 6). Furthermore, complementation of the hcp mutant by expression of hcp driven by lacZp on a plasmid restored tumorigenesis to wild-type or nearly wild-type levels (Fig. 6), indicating that the reduction in tumorigenesis of the hcp mutant on potato tuber disks was due to loss of hcp.

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FIG. 6. Quantitative tumorigenesis assay. Various A. tumefaciens strains were examined for their tumorigenesis efficiency with potato tuber disks. Potato tuber disks were infected with 106 CFU/ml A. tumefaciens for 2 days and then placed on water agar with timentin (100 µg/ml). Tumorigenesis efficiency is expressed in the number of tumors per disk, and the standard errors are averages from results for 60 potato tuber disks for each strain analyzed in each independent experiment. No tumors were detected with the Ti plasmid-less strain NT1RE (data not shown). Representative results are shown, and the data were analyzed by analysis of variance for statistical classification, which revealed four groups (groups a, b, c, and d) of strains based on tumorigenesis efficiency. At least four independent experiments in which similar results were obtained were carried out. WT, wild type.

To further understand whether the icmF gene or the putative t6ss operon is important in A. tumefaciens virulence, we determined the tumorigenesis efficiency of the icmF and t6ss mutants on potato tuber disks. Surprisingly, no reduction in tumorigenesis efficiency was observed (Fig. 6). The data suggest that hcp is important for wild-type virulence, likely via intracellular Hcp rather than via secreted Hcp.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We utilized a proteomics approach to analyze the A. tumefaciens secretome, which led to identification of an Hcp-family protein that is involved in A. tumefaciens virulence and is secreted via a newly identified T6SS. The identification of a T6SS in this plant-pathogenic bacterium confirms that this protein secretion system is conserved across animal and plant pathogens, but the biological functions of the systems may be distinct and require further analysis.

The identification of the known secretory protein VirB1* (1), which is required for T-pilus assembly (50), supports the conclusion that our proteomics approach was successful (Table 2). We also identified ChvE, a sugar-binding protein previously detected in both the periplasm and extracellular space (4), in our secretome analysis; however, whether ChvE is actively secreted must still be determined. In addition, RopB/AopB (Atu1131) was previously identified as an acid-inducible outer membrane protein on the surface of A. tumefaciens (16). A transposon insertion mutation in ropB/aopB resulted in attenuated tumors on Kalanchoe leaves with a low level infection (5 x 10⁷ cells/ml). The identification of RopB/AopB in the S1 fraction indicates that this protein is either secreted by bacterial cells or loosely associated with the bacterial surface. Interestingly, all identified S1 fraction proteins except Hcp possess a putative Sec-dependent signal peptide or are predicted to be localized in either the periplasmic space or membrane systems (Table 2), which suggests that they are exported across the inner membrane before they are secreted extracellularly. Further biochemical and genetic studies are needed to confirm whether they are indeed bona fide secretory proteins. In contrast, the Hcp protein lacks a recognizable signal peptide and transmembrane domain, features typical of many known effector proteins secreted via T3SS or T4SS (31). Our evidence, along with several recent reports of Hcp secretion (10, 27, 29, 33, 35, 39), revealed that T6SS-mediated Hcp secretion is a common theme in plant- and animal-pathogenic bacteria.

The expression and secretion of T3SS- and T4SS-secreted proteins in plant-pathogenic bacteria are usually silenced but are induced in an apoplast-mimicking minimal medium or when the bacteria are in contact with host cells (31). All A. tumefaciens VirB/D4 T4SS effector proteins identified to date (VirD2, VirE2, VirE3, VirD5, and VirF) are encoded by the vir regulon of the Ti plasmid and are expressed and secreted only when a phenolic compound released by the plant is sensed. In contrast, our data indicate that Hcp is expressed and secreted when bacteria are grown in the presence or absence of AS or at various temperatures and pH values (Fig. 1). It is possible, however, that T6SS expression and Hcp secretion are regulated by an environmental cue yet to be determined, as suggested for another system (30). Because current data suggest that hcp may be required for wild-type virulence of A. tumefaciens (Fig. 6), further in planta investigations may uncover potential regulatory mechanisms of T6SS.

Although Hcp is not absolutely required for A. tumefaciens virulence, our quantitative data for potato tuber disks indicate that Hcp likely plays a positive role in facilitating the tumorigenesis efficiency of A. tumefaciens (Fig. 6). Unexpectedly, deletion of either the icmF gene or the entire putative t6ss operon did not result in a significant reduction in the tumorigenesis efficiency compared with that of the wild type (Fig. 6). This result is in contrast to the results for several animal pathogens, in which either a defect in the T6SS machinery (Hcp was not secreted) or deletion of the hcp gene resulted in loss or attenuation of virulence (10, 29, 33, 39). It is possible that the intracellular Hcp that is not secreted is responsible for wild-type virulence of A. tumefaciens. Alternatively, T6SS may act differently in plant-associated bacteria. Indeed, deletion of an hcp gene in the plant-pathogenic bacterium P. atrosepticum did not affect its virulence, but an increase in virulence was detected when hcp was overexpressed (27). In addition, mutations in the imp/t6ss gene cluster in the plant symbiont R. leguminosarum bv. trifolli strain RBL5523, a strain capable of nodule formation but ineffective in nitrogen fixation, allowed this strain to form effective nodules on pea (2). Therefore, the imp/t6ss gene cluster of R. leguminosarum bv. trifolli strain RBL5523 might play a negative role in nitrogen fixation. The role(s) of T6SS and its secreted substrates in A. tumefaciens requires further analysis.

Hcp is a hydrophilic small protein without a recognizable N-terminal signal peptide and transmembrane domain. Western blot analysis revealed that most Hcp is intracellular, even though appreciable amounts of Hcp are secreted into the culture medium (Fig. 1). Our data obtained from biochemical fractionation and immunoelectron microscopy analyses further revealed that Hcp is located in the cytosol in a soluble form (Fig. 2 and 3). Mougous et al. (29) showed that ClpV1, a protein belonging to the AAA+ ClpB family, is required for Hcp secretion and that the functional ClpV1-green fluorescent protein fusion protein is localized to single discrete foci in P. aeruginosa. Interestingly, this punctate localization disappears and the localization pattern becomes diffuse after deletion of hcp-1 (29), suggesting that intracellular Hcp has a structural role in T6SS assembly. The role of Hcp in the functional T6SS machinery is also supported by the requirement for Hcp for VgrG secretion (33). In the future, it would be of great interest to determine the roles of intracellular and secreted Hcp in the function of T6SS.

The conservation of the t6ss gene cluster among many gram-negative proteobacterial pathogens and symbionts strongly suggests that T6SS has an important function in intimate host-microbe interactions. Except for a few characterized components, most proteins encoded by this gene cluster are hypothetical proteins (see Tables S2 and S3 in the supplemental material). Intriguingly, no N-terminal signal peptide could be detected in any protein encoded by the A. tumefaciens t6ss gene cluster, and only 3 of 14 proteins encoded by the putative t6ss operon are likely membrane proteins with predicted transmembrane domains (see Table S2 in the supplemental material). This observation is in striking contrast to observations for T4SS components, virtually all of which integral or peripheral membrane proteins with a detectable N-terminal signal peptide (5). It is possible that only some of the components encoded by the t6ss gene cluster are structural components of T6SS machinery, whereas other proteins encoded are secretory proteins, regulators, or accessory proteins for synthesis or assembly of the T6SS machinery. Systematic mutagenesis studies combined with biochemical analysis and cellular localization analysis should shed light on the biogenesis pathways and biochemical properties of the T6SS machinery.

 
We are grateful to Clarence Kado for providing the A. tumefaciens strains, VirB antibodies, and valuable suggestions. We also thank Ban-Yang Chang for the gift of GroEL antibody, Laysun Ma for providing the icmF construct, Hau-Hsuan Hwang, Yun-Long Tsai, and Jer-Sheng Lin for critically reading the manuscript, and Shu-Hsing Wu, Kuo-Chen Yeh, Stan Gelvin, Lan-Ying Lee, and lab members for discussions. We also acknowledge Wann-Neng Jane and the members of the Plant Cell Biology Center at the Institute of Plant and Microbial Biology at Academia Sinica for technical assistance with electron microscopy and the Core Facility for Proteomics Research located at the Institute of Biological Chemistry, Academia Sinica, which is supported by National Science Council grant NSC 93-3112-B-001-010-Y and the Academia Sinica, for the mass spectrometry analysis.

This work was supported by Academia Sinica.

 
* Corresponding author. Mailing address: 128, Sec. 2, Academia Road, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 11529. Phone: 886-2-2789-2981. Fax: 886-2-2782-7954. E-mail: emlai{at}gate.sinica.edu.tw

Published ahead of print on 8 February 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, April 2008, p. 2841-2850, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01775-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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