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Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor ^,

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210

Received 3 March 2007/ Accepted 20 April 2007

	ABSTRACT

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Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium. Little is known about the gene regulatory mechanisms for this bacterium. A gene encoding a putative transcription factor, tr1, upstream of three tandem genes encoding outer membrane proteins, including the major outer membrane protein P44, is driven by a strong promoter. In the present study, gel mobility shift assays revealed the presence of A. phagocytophilum proteins that interact with the promoter region of tr1. These proteins interacting with the tr1 promoter region were purified by biotin-labeled DNA affinity chromatography from a large amount of host cell-free bacteria. Mass spectrometry identified the major protein as an A. phagocytophilum 12.5-kDa hypothetical protein, which was named ApxR. In a DNase I protection assay, recombinant ApxR (rApxR) bound cooperatively to four 24- or 25-bp sites within 235 bp upstream of tr1: regions III and IV proximal to tr1 had higher affinity than regions I and II did. Deletion assays showed that regions III and IV were essential for rApxR binding, whereas regions I and II upstream of regions III and IV were not. The primary cis-acting region was region IV, since region IV alone was sufficient for rApxR to strongly transactivate the downstream gene in a lacZ reporter assay. Addition of regions I, II, and III did not enhance transactivation. These results show that ApxR is a novel transcriptional regulator that directly regulates tr1.

	INTRODUCTION

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Human granulocytic anaplasmosis (HGA) (formerly called human granulocytic ehrlichiosis) has recently been recognized as a zoonotic disease of public health importance and is currently one of the most common tick-borne zoonoses in the United States and Europe (4, 18). HGA is an acute febrile systemic illness accompanied by hematologic and liver enzyme abnormalities. It can cause severe and potentially fatal illness, especially in immunocompromised and elderly people (6). The etiologic agent of HGA, Anaplasma phagocytophilum, is a gram-negative, obligatory intracellular bacterium, which was initially determined to have a tropism for granulocytes (2, 9) and currently is also known to infect endothelial cells (11, 16).

A fundamental question in obligatory intracellular parasitism is how intracellular bacteria coordinate and finely tune their development and proliferation with the conditions of their host cells, as these bacteria are absolutely dependent on their host cells. In sequenced genomes of obligatory intracellular bacteria, fewer putative DNA binding proteins are detected than in free-living bacteria due to the relatively homeostatic host cell environment. Yet, very little experimental data are available on these genes or gene products. In the sequenced genome of A. phagocytophilum (7), there are eight putative DNA interacting proteins: two-component system response regulators CtrA and NtrX that contain a DNA binding motif (3), a putative transcriptional regulator encoded by tr1 that has a winged helix-turn-helix motif (13), a basic histone-like protein HU (GenBank accession no. YP_505361.1) (5), a putative transcription factor of the Baf family (GenBank accession no. YP_505575.1), a transcriptional regulator of the MerB family (GenBank accession no. YP_505799.1), an integration host factor (GenBank accession no. YP_505798.1), and an integration host factor ß (GenBank accession no. YP_504885.1). Of these, tr1, which is upstream of tandem genes encoding outer membrane proteins, including the polymorphic major outer membrane protein P44 (13), has been the most studied. The transcriptional start site was previously found at 21 bp upstream of tr1 with a typical –35/–10 ⁷⁰ promoter (13). The upstream region of tr1 has strong promoter activity in Escherichia coli (1) and after transposition into the A. phagocytophilum genome (8). A gene in the A. phagocytophilum genome encodes a primary DNA-dependent RNA polymerase that is homologous to ⁷⁰ factor responsible for constitutive gene expression (7). This suggests that transcriptional activators or repressors play a role in tr1 expression. The objectives of the present study were to investigate A. phagocytophilum proteins that may be involved in the transcriptional regulation of tr1. We performed electrophoretic mobility shift assays (EMSAs), using A. phagocytophilum lysate and a DNA probe within the potential regulatory region upstream of tr1. Using this approach, we isolated a novel A. phagocytophilum protein that we named ApxR that binds to a cis element involved in tr1 regulation. A DNase I protection assay and a lacZ reporter assay were carried out to characterize the cis element and ApxR regulation. Our results indicate that ApxR is the first functionally characterized transcription factor of a member of the order Rickettsiales.

	MATERIALS AND METHODS

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A. phagocytophilum and host cell-free bacteria. Host cell-free A. phagocytophilum HZ strain was prepared by sonication of infected HL-60 cells as previously described (19). A bacterial suspension was passed sequentially through syringe-driven 5-µm nylon (Whatman, Clifton, NJ) and 0.8-µm glass-fiber membrane filters (Millipore, Billerica, MA) for EMSA analysis or through the 5-µm filter only for isolation of the DNA binding protein. The filtrate was centrifuged at 10,000 x g for 10 min, and the pellet enriched with A. phagocytophilum was washed with 2x phosphate-buffered saline (2x phosphate-buffered saline consists of 274 mM NaCl, 20 mM Na₂HPO₄, 5.4 mM KCl, and 3.6 mM KH₂PO₄ [pH 7.2]).

EMSA. The DNA fragments upstream of tr1 shown in Fig. 1, and DNA fragments R3-1 and R3-2 with various deletions, were amplified with the primer pairs shown in Table S1 in the supplemental material. Approximately 5 pmol each of the purified PCR product was biotinylated with a biotin 3'-end DNA labeling kit (Pierce Biotechnology, Rockford, IL). The biotinylated probes were purified and denatured at 94°C for 3 min and then slowly cooled to room temperature to allow proper annealing. The bacterial pellet (approximately 100 µg) was lysed by sonication in 100 µl buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol (DTT), 0.4% NP-40, and 1% protease inhibitor cocktail (Calbiochem, San Diego, CA) and centrifuged at 18,000 x g for 30 min at 4°C. The supernatant of bacterial lysate, bovine serum albumin (BSA), uninfected HL-60 cell lysate (5 µg of each protein, unless otherwise stated), purified recombinant ApxR (rApxR), or purified recombinant GroES (rGroES) was incubated with biotinylated DNA probes (0.1 pmol each) for 30 min at 4°C in 20 µl of binding buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, 2.5% glycerol, 0.1% NP-40, 50 ng/µl of poly(dI-dC) or salmon sperm DNA]. As a control, the sample was incubated with an excess (15x) of unlabeled probe. A 6% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA (TBE) (0.44 M Tris base, 0.44 M boric acid, and 0.01 M EDTA [pH 8.0]) had been prerun for 1 h, and then samples were loaded onto the gel and electrophoresed at 100 V for 1.5 h at 4°C. The biotinylated probes were transferred to a nylon membrane (Amersham Biosciences, Piscataway, NJ) at 380 mA for 45 min. The transferred DNA was cross-linked to the membrane with UV light. The biotin-labeled DNA was detected with a LightShift chemiluminescence EMSA kit (Pierce).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Gel mobility shift assay of native A. phagocytophilum proteins bound to regulatory regions upstream of tr1. (A) Schematic representation of probe locations upstream of the tr1 gene in the p44 expression locus of A. phagocytophilum. The bent arrow indicates the transcriptional start site. (B) Native A. phagocytophilum proteins (AP) bound to the R3, R3-1, and R3-2 probes, but not R2. In each gel, the position of unshifted DNA is seen in the leftmost lane, which contained no protein. Shifted bands are indicated by black arrowheads. Comp., 15-fold excess amount of the unlabeled probe as a competitor. BSA and HL-60, BSA or uninfected HL-60 cell lysate was added instead of AP. The figure is representative of three independent experiments. (C) Isolated A. phagocytophilum proteins bound to the biotinylated DNA probe in streptavidin affinity chromatography subjected to 15% SDS-PAGE and Gel-Code blue staining. Lane 1, A. phagocytophilum lysate flowthrough; lane 2, bound proteins analyzed by proteomics (arrows). The positions of molecular mass standards in lane M (in kilodaltons) are shown to the left of the gel.

Mass spectrometry. After electrophoresis, the gel was fixed in 50% methanol and 10% acetic acid overnight and stained with GelCode Blue (Pierce). After the gel was destained in water, the bands were cut out and digested with trypsin in the gel (Promega, Madison, WI). Capillary liquid chromatography-nanospray tandem mass spectrometry (LC/MS/MS) was performed on a Thermo Finnigan linear quadrupole mass spectrometer (Thermo, San Jose, CA) equipped with a nanospray source operated in positive-ion mode. The LC system was an UltiMate Plus system (LC-Packings A; Dionex Co., Sunnyvale, CA). Sequence information from the MS/MS data was processed using the MASCOTT MA/MA search engine and Turbo SEQUEST algorithm in BioWorks 3.1 software.

Cloning and expression of rApxR and rGroES. apxR and groES were amplified using A. phagocytophilum genomic DNA as a template and directionally cloned into the NdeI and XhoI sites of the pET29a(+) vector (Novagen Inc., Madison, WI). The resulting plasmids (pApxR and pGroES) were amplified in E. coli Novablue cells (Novagen) and expressed in E. coli BL21(DE3) cells (Novagen) as previously described (21). The soluble fraction of E. coli lysate was Ni affinity purified with a His-Select cartridge (Sigma, St. Louis, MO). Purified rApxR and rGroES were dialyzed in EMSA binding buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, and 1 mM DTT.

DNase I footprint analysis. A 314-bp DNA fragment upstream of the tr1 translational start site, including 79 bp upstream of R3 and 235 bp of R3 (20 bp less than the R3 probe used for EMSA at the 3' end) was amplified and cloned into a pCRII vector of a TA cloning kit (Invitrogen) to make the probe template. The probe was generated by PCR with primer set 5 (see Table S1 in the supplemental material), except that the 5' part of the 5'-end primer was labeled with 6-carboxyfluorescein (FAM) at Applied Biosystems, Inc. (ABI) (Foster City, CA). The FAM-labeled probe at 300 ng was incubated with 5 or 1.25 µM rApxR or BSA as a control under the conditions described previously (22). Based on the results of optimization experiments using 10-fold dilutions (from 0.05 to 5 units) of DNase I (Worthington Biochemicals, Lakewood, NJ), 0.05 unit of DNase I was added to the reaction mixture and incubated for 5 min at 25°C. The reaction was terminated by heating at 75°C for 10 min. The digested DNA fragments were purified using a QIAquick PCR purification kit (QIAGEN) and eluted in 40 µl H₂O. Nondigested DNA was used for sequencing reactions with the 5' FAM-labeled forward primer of primer set 5 (see Table S1 in the supplemental material) and a Thermo Sequenase dye primer manual cycle sequencing kit (USB, Inc., Cleveland, OH). The digested DNA and sequencing reaction products were analyzed with a 3730 DNA analyzer (ABI) (22). The sequences were then analyzed with GeneMapper software (ABI) to convert the DNase I digestion map into sequencing data to identify the exact sequences that were protected.

tr1 promoter-lacZ reporter fusion constructs. The lacZ gene was amplified from plasmid pQF50, which contains full-length lacZ with Pfu DNA polymerase (Stratagene, La Jolla, CA). Three different tr1 promoter regions, the entire R3 fragment (235 bp), the 3' R3 fragment containing regions III and IV (116 bp), and the 3' R3 fragment containing region IV (90 bp), were PCR amplified using A. phagocytophilum genomic DNA as the template. The transcriptional fusion was constructed by placing each tr1 promoter region upstream of the promoter-less lacZ gene in pACYC184 (New England Biolabs, Beverly, MA). E. coli BL21(DE3) pretransformed with pApxR or pET29a(+) without an insert was cotransformed with pACYC184 containing each one of the tr1 promoter-lacZ fusion inserts. After overnight culture, transformants were subcultured in LB medium supplemented with 50 µg/ml kanamycin and 25 µg/ml chloramphenicol at 37°C for 2.5 h, followed by 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) induction for 1 h. ß-Galactosidase activity was measured as described previously (15). Plasmids were extracted from each sample by using a GenScript Quickclean 5 M Miniprep kit (GenScript, Piscataway) and run on a 1% agarose gel to confirm the copy number of each plasmid per bacterium. The E. coli suspension was dissolved in 1x SDS-PAGE gel loading buffer at 95°C for 10 min. Approximately 20 µg of total protein was loaded in each well on a 15% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated with peroxidase-conjugated antipolyhistidine antibody (Sigma) at a 1:500 dilution, and reacting bands were visualized by enhanced chemiluminescence (ECL kit; GE Healthcare, Piscataway, NJ).

Statistical analysis. Statistical analyses were performed by using analysis of variance and Tukey's honestly significant difference test, and a P of <0.05 was considered significant.

	RESULTS

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Gel mobility shift assays with A. phagocytophilum native protein binding. Previously, the transcriptional start site was found 21 bp upstream of the tr1 translational start site (ATG) (13), and the 40-bp upstream region was shown to have strong promoter activity in E. coli (1). To investigate whether there is an A. phagocytophilum protein that binds to a cis element to regulate tr1, the 500-bp region immediately upstream of tr1 was analyzed by an EMSA. To construct DNA probes within the 500-bp region, overlapping DNA fragments R2 (271 bp) and R3 (255 bp) were PCR amplified from A. phagocytophilum genomic DNA and biotinylated (Fig. 1A). Lysate from host cell-free A. phagocytophilum was incubated with the probes, and the mixture was analyzed by an EMSA. The results showed a shift of DNA fragment R3 but not DNA fragment R2 (Fig. 1B). To narrow down the target DNA region, fragment R3 was subdivided into overlapping two fragments, R3-1 (151 bp) and R3-2 (125 bp) (Fig. 1A), and biotinylated. EMSA results revealed the binding of both R3-1 and R3-2 probes to an A. phagocytophilum protein (Fig. 1B). The binding of probe R3-2 to the A. phagocytophilum protein was weaker than the binding of R3 and R3-1 probes (Fig. 1B). The addition of unlabeled R3-1 and R3-2 DNA competitively reduced A. phagocytophilum protein binding to the labeled probe, indicating that the binding was specific to each probe (Fig. 1B). The negative controls, BSA and the uninfected HL-60 cell lysate, did not bind to these DNA fragments (Fig. 1B).

Affinity capture and identification of the DNA binding protein by MS. To identify the A. phagocytophilum protein bound to the R3-1 DNA fragment, the protein bound to biotinylated DNA was affinity purified with streptavidin affinity chromatography. SDS-PAGE of affinity-purified proteins revealed two major bands (12.5 and 10 kDa) and several weak bands of larger molecular sizes (Fig. 1C). MS/MS of the 12.5-kDa band revealed two peptide sequences (VIGVDQPK and VSQIINGK) that matched a 111-amino-acid 12.5-kDa hypothetical protein (GenBank accession no. YP_505110.1). We named this protein A. phagocytophilum expression regulator, ApxR. Another major band contained a peptide sequence (VVSVGPGASNSDGK) that matched a 10-kDa chaperonin, GroES (GenBank accession no. YP_504858.1). Proteins of larger sizes could not be identified by MS due to insufficient amounts of protein.

rApxR and EMSA analysis. To further characterize ApxR binding to the regulatory DNA region, apxR was cloned into the pET29a(+) vector (pApxR) and expressed in E. coli. This expression yielded a 119-amino-acid rApxR (12.5 kDa), including an 8-amino-acid six-His tag in the C terminus derived from the pET29a(+) vector. The Ni-affinity-purified rApxR was detected as a single band of approximately 12.5 kDa by Coomassie brilliant blue staining on SDS-polyacrylamide gels and by Western blot analysis with an anti-His tag antibody (Fig. 2A).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Binding of rApxR to the region upstream of tr1. (A) Three micrograms of affinity-purified rApxR was subjected to SDS-PAGE. Lane 1, Coomassie brilliant blue staining; lane 2, Western blot analysis with an anti-His tag antibody. The positions of molecular mass standards in lane M (in kilodaltons) are shown to the left of the gel. The position of rApxR (12.5 kDa) is indicated to the right of the gel. (B) Dose response of rApxR (5, 0.5, 0.05, and 0.005 µg) binding to probes R3, R3-1, and R3-2 (0.1 pmol each) in an EMSA. In each panel, the position of unshifted DNA is seen in the leftmost lane, which contained no protein.

DNase I footprint analysis. The EMSA results demonstrated that rApxR directly interacts with two DNA fragments, R3-1 and R3-2, in a dose-dependent manner. Therefore, we performed DNase I protection assays to determine the DNA sequences to which rApxR would bind. The protected regions of the sense strand were determined by comparing sequences of DNA samples protected by 5 or 1.25 µM rApxR to the sequence of unprotected DNA samples treated with BSA (Fig. 3A). There were four 24- or 25-bp regions protected in tandem by rApxR from bases –197 to –173 (region I), –163 to –140 (region II), –96 to –71 (region III), and –24 to –1 (region IV) (Fig. 3A and B). These four protected regions were all located within R3 but not within the 79 bp upstream of R3. Regions I, II, and III were located within DNA fragment R3-1, whereas 14 bp of region III and all of region IV were located within DNA fragment R3-2. This result explains why the binding of native ApxR as well as rApxR to DNA fragment R3-2 was less or weaker than binding to DNA fragment R3-1 in EMSA (Fig. 1B and 2B).

View larger version (29K): [in this window] [in a new window]   FIG. 3. Identification of the sequence of the ApxR-protected regions of the tr1 promoter by DNase I protection footprinting. (A) Electropherograms superimposed to show the four regions (regions I to IV) protected by different concentrations of rApxR (5 µM [blue] and 1.25 µM [green]) or BSA (red) within region R3 after digestion with DNase I. (B) DNA sequences of four protected regions (underlined) within region R3. The sequences for the R3-1 and R3-2 probes are overlined in red and green, respectively. (C) EMSA showing that regions III and IV are required for rApxR binding to R3-1 and R3-2 probes, respectively. Probes used were R3-1 probes with deletions in region I (del. I), regions I and II (del. I & II), or region III (del. III), and R3-2 probes with deletions in region IV (del. IV), 14 bp at the 5' end of region III (del. III’), or both (del. III’ & IV). Shifted bands are indicated by black arrows.

Deletion analysis of DNA regions required for ApxR binding by EMSA. DNA footprint analysis showed four rApxR-protected regions. Consequently, we determined which regions are required for rApxR binding to R3-1 and R3-2 fragments. Deletion of region I or both regions I and II from fragment R3-1 had little effect on the binding of rApxR in an EMSA analysis (Fig. 3C). However, rApxR failed to bind when region III in fragment R3-1 or region IV in fragment R3-2 were deleted (Fig. 3C). This indicates regions III and IV were essential for rApxR binding, whereas regions I and II and the 14 bp of region III in fragment R3-2 are not essential for rApxR binding to fragment R3-1 or R3-2.

Regulation of tr1 promoter-lacZ fusion with ApxR. Since ApxR bound four regions within the R3 fragment, we examined whether these regions are involved in tr1 transactivation by ApxR. The lacZ reporter fusions were constructed by placing the entire R3 fragment (R3-lacZ), the 3' R3 fragment containing regions III and IV (III&IV-lacZ), and the 3' R3 fragment containing only region IV (IV-lacZ) upstream of the translational start site of a promoterless lacZ gene in pACYC184. Each one of the lacZ reporter fusions was transformed into pApxR-transformed or pET29a(+)-transformed E. coli. In pApxR-transformed E. coli that was cotransformed with any one of three lacZ reporter constructs, the ß-galactosidase activity was significantly greater when rApxR expression was induced with IPTG than without IPTG induction (Fig. 4). In each of the three lacZ reporter constructs, ß-galactosidase activity with IPTG induction was significantly greater in pApxR-transformed E. coli than in pET29a(+)-transformed E. coli (Fig. 4). The primary cis-acting region was region IV, since rApxR strongly transactivated the lacZ reporter gene downstream of region IV in the region IV-lacZ construct. R3 that contains all four regions and region III&IV-lacZ did not differ significantly from the region IV-lacZ construct in the lacZ reporter assay (Fig. 4). Thus, regions I, II, and III did not enhance the transactivation of region IV by ApxR. The relative copy numbers of pApxR and pACYC184 per bacterium were similar among three lacZ reporter constructs with or without IPTG induction and in each lacZ reporter construct with or without IPTG induction. Expression of the rApxR protein upon IPTG induction was confirmed by Western blot analysis (Fig. 4). Taking all the data together, ApxR can function as a transactivator for tr1.

View larger version (20K): [in this window] [in a new window]   FIG. 4. ApxR activates the transcription of tr1 promoter-lacZ fusion. (A) ß-Galactosidase assays were used to measure the transcriptional activity of the entire R3 fragment, regions III and IV, or region IV-lacZ fusion in E. coli strains containing pApxR or the pET29a(+) vector alone. The activation of three tr1 promoter-lacZ fusion constructs was compared with or without rApxR induction and between E. coli strains containing pET29a(+) vector and pApxR. Data are presented as means ± standard deviations (error bars) of triplicate samples. Values that were significantly different (P < 0.01) from the value for the E. coli strains with pET29a(+) alone or the value for the E. coli strains without IPTG induction by Tukey's honestly significant difference test are indicated by an asterisk. A representative result from four independent experiments is shown. (B) Western blot analysis of the expression of rApxR protein (arrow) upon IPTG induction (+) in the E. coli strain cotransformed with pApxR and pACYC184 encoding the entire R3 fragment. The expression of rApxR was not detected in the absence of IPTG (–).

	DISCUSSION

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The role of GroES coaffinity purified with ApxR in tr1 transcription was not further investigated in the present study, since it did not bind to the tr1 promoter region in the EMSA. GroES is a prominent heat shock chaperon in the cytoplasm of A. phagocytophilum, and as such, it may indirectly modulate ApxR function by stabilizing or sequestrating ApxR.

Functions of Tr1 (estimated molecular mass of 21 kDa) are currently under investigation. Tr1 (a hypothetical transcriptional regulator also known as UN1 [17]) has a helix-turn-helix DNA binding motif and is associated with a multigene family encoding major outer membrane proteins of Ehrlichia and Anaplasma species. Initially, tr1 was found upstream of the 22 tandem omp-1 (p28) and p30 loci of Ehrlichia chaffeensis and Ehrlichia canis, respectively, and these omp-1 genes and p30 genes were shown to be polycistronically transcribed from the tr1 promoter (17). Homologs of tr1 were subsequently detected upstream of the p44/msp2 expression locus of A. phagocytophilum (13), the Anaplasma marginale major surface protein 2 (msp2) operon-associated protein (opag genes) expression locus (14), and the Ehrlichia ruminantium map1 locus (20). omp-1, msp2, p44, p30, and map-1 belong to the outer membrane protein 1/Msp2/P44 superfamily Pfam PF0167 (7). A. marginale, a species closely related to A. phagocytophilum, contains a sequence that is identical to –24 to –1 (region IV) upstream of a gene encoding a hypothetical protein that is a homolog of the tr1 protein of A. phagocytophilum (1). A search of the GenBank database identified apxR homologs in the genomes of A. marginale St. Maries, E. canis Jake, E. chaffeensis Arkansas and Sapulpa, and E. ruminantium Welgevonden and Gardel (E value of 5e–12; Table 1). Therefore, there has been evolutionary pressure to conserve apxR and tr1 among Ehrlichia and Anaplasma species, and the means by which the tr1 of A. phagocytophilum is directly regulated by ApxR may be also used to regulate tr1 homologs among other members of Ehrlichia and Anaplasma species.

	ACKNOWLEDGMENTS

 
We thank Michele Zianni at the Plant Biotechnology Center and Kary Green-Church at the Mass Spectrometry and Proteomics Facility, The Ohio State University, for help with the DNase footprint analysis and proteomic analysis, respectively. We appreciate Kathryn Gibson for reading the manuscript.

This research was supported in part by grant R01 AI47407 from the National Institutes of Health.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093. Phone: (614) 292-5661. Fax: (614) 292-6473. E-mail: rikihisa.1{at}osu.edu

Published ahead of print on 4 May 2007.

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

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