bacteroides fragilis btga mobilization protein binds to...

JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Sept. 1998, p. 4922–4928 Vol. 180, No. 18

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

The Bacteroides fragilis BtgA Mobilization Protein Bindsto the oriT Region of pBFTM10

LEONID A. SITAILO,1 ALEXANDER M. ZAGARIYA,1† PATRICK J. ARNOLD,1

GAYATRI VEDANTAM,1 AND DAVID W. HECHT1,2*

Department of Medicine, VA Hospital, Hines, Illinois 60141,1 and Department of Medicine,Microbiology and Immunology, Program in Molecular Biology, Loyola

University Chicago, Maywood, Illinois 601532

Received 15 January 1998/Accepted 13 July 1998

The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriTregion necessary for transfer in Bacteroides fragilis and Escherichia coli. The BtgA protein was predicted to con-tain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the regionimmediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriT region. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgA was cloned and overexpressed inE. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific bind-ing of BtgA protein to its cognate oriT. DNase I footprint analysis demonstrated that BtgA binds apparentlyin a single-stranded fashion to the oriT-containing fragment, overlapping inverted repeats I, II, and III and theputative nick site.

Bacterial conjugation is a major mechanism for the transferof chromosomal or plasmid DNA between prokaryotic cells.As a result, dissemination of antibiotic resistance determinantscan occur, significantly influencing the virulence of variouspathogens such as Escherichia coli, agrobacteria, enterococci,staphylococci, streptococci, streptomycetes, and the Bacteroi-des fragilis group (2, 4, 10, 22, 33, 34). The initial reactionsduring conjugal DNA transfer require multiple complex pro-tein-DNA and protein-protein interactions that result in theformation of a relaxosome at the cis-acting origin of transfer(oriT). Relaxosomes are stable nucleoprotein complexes re-sulting in site- and strand-specific nicking at a site (the nic site)in the oriT region (24). The relaxase, or DNA strand trans-ferase, cleaves at this nic site and attaches covalently to the 59terminus. DNA is transmitted unidirectionally as a single strandinto a recipient cell with 59-to-39 polarity in a rolling-circlemanner, followed by complementary-strand synthesis in thenew host (for detailed reviews of conjugative transfer involvingR and F factors, see references 25 and 44).

Much attention has been focused on the biochemical pro-cesses involved in the initiation of DNA transfer of the IncPaplasmid RP4. The generation of the single DNA strand that istransferred requires formation of the initiation complex, orDNA relaxosome, in a cascade-like fashion. The first step inrelaxosome formation is binding of an RP4 protein, TraJ, to a19-bp inverted repeat within the oriT region and a 10-bp pal-indrome, srj (24, 43). In the second step, TraI, another RP4protein, binds TraJ by both protein-protein interactions andthe recognition of a 6-bp sequence, sri, in the nic region. Sub-sequently, site- and strand-specific cleavage of a unique phos-phodiester bond at the nic site of the transfer origin occurs,followed by covalent attachment of TraI to the 59 terminus of

the DNA strand involved in transfer. The binding and nickingevents are thought to be independent processes (16, 26). TraIis also able to induce a second cleavage reaction proposed toterminate the rolling-circle model of transfer DNA replica-tion (27). Following strand transfer to a new host, TraI cata-lyzes the recombination of two single-stranded DNA mole-cules at the RP4 nic site (27). A third protein, TraH, stabilizesthe TraJ and TraI nucleoprotein complex by specific protein-protein interactions and does not bind DNA itself (24, 45). Afourth protein, TraK, interacts with oriT by binding DNA as atetramer over a range of approximately 180 nucleotides, down-stream of nic (46). Binding of TraK increases the formation ofrelaxation complexes in vitro and in vivo, possibly by influenc-ing DNA topology to expose the nic site for more efficientcleavage by TraI (43).

Transferable antibiotic resistance plasmids of the B. fragilisgroup have also been described. They include the clindamycin-resistant plasmids pBFTM10 (12), pBF4 (40), and pBI136 (31)and the metronidazole-resistant plasmids pIP417, pIP419, andpIP421 (11, 37, 38). pBFTM10 is a 14.9-kb plasmid that is trans-ferable within B. fragilis. When pBFTM10 is fused to the E. colireplicon pDG5 to form pGAT400, it can also transfer fromB. fragilis to E. coli. Further, pGAT400 is also mobilizable with-in E. coli when coresident with the IncPb plasmid R751, but itrequires an intact pBFTM10 transfer region (12). Character-ization of the pBFTM10 transfer region has demonstrated thatonly two genes, btgA and btgB, are necessary for transfer withinB. fragilis and mobilization by R751 in E. coli. DNA sequenceanalysis of btgA revealed a helix-turn-helix DNA binding motif,suggesting that it was a DNA binding protein (12). In addition,the identification of three sets of inverted repeats (IRI, IRII,and IRIII) and a putative nic site in the region upstream ofbtgA suggested that this was the pBFTM10 oriT (12, 39).

In this paper, we report the cloning of the pBFTM10 oriTand the expression and purification of the BtgA protein, andwe demonstrate specific binding of BtgA to its cognate oriT asdetermined by mobility shift assays and footprinting analyses.

* Corresponding author. Mailing address: Loyola University Chi-cago, Bldg. 54, Room 101, 2160 S. First Ave., Maywood, IL 60153.Phone: (708) 216-2792. Fax: (708) 216-2269. E-mail: [email protected].

† Present address: Division of Neonatology, Department of Pediat-rics, University of Illinois at Chicago, Chicago, IL 60616.

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MATERIALS AND METHODS

Bacterial strains, plasmids, and media. The bacterial strains and plasmidsused in this study are listed in Table 1. Media, antibiotic concentrations, andE. coli growth conditions were as previously described (1, 20). Bacto Agar andtryptone were obtained from Difco Co. (Detroit, Mich.). For strains requiringantibiotic selection, cells were grown at 37°C overnight in Luria broth (LB)supplemented with 200 mg of ampicillin per ml. E. coli XL1-Blue was used forroutine cloning procedures (29). The putative oriT region was cloned into theBamHI site of pGEM-7Zf(1) (Promega, Madison, Wis.), to give pGEM-7ZT,and into the BamHI site of pACYC184, to form pJAYC1. E. coli BL21(DE3) wasused as the host for btgA cloning and protein expression. A 646-bp fragmentcontaining btgA was cloned into the BamHI site of the expression vector pET-19b(Novagen, Madison, Wis.) to give pDHBA.

Plasmid mobilization experiments. All mating experiments were performed aspreviously described (12, 23), in triplicate, using E. coli HB101 cells containingR751 as donors and E. coli DW1030 cells as recipients (Table 1). Bacterialcultures were grown overnight at 37°C in LB with the appropriate antibiotics.Overnight cultures were diluted 10-fold in LB and grown to early log phase. Ina sterile microcentrifuge tube, 0.15 ml of donor cells was mixed with 1.35 ml ofDW1030 cells and then pelleted by centrifugation at 10,000 3 g for 1 min. Afterthe supernatant was discarded, cell pellets were resuspended in 100 ml of 13MPBS (8 mM Na2HPO4, 2 mM NaH2PO4, 145 mM NaCl [pH 6.9]). Resus-

pended cells were then spotted onto sterile 25-mm, 0.45-mm-pore-size GelmanGN-6 cellulose nitrate filters (Nalge Co., Rochester, N.Y.) on Luria agar platesand incubated at 37°C for 180 min. Transconjugants were enumerated by platingon selective media, and the mobilization frequencies of plasmids were normal-ized to the number of R751 transconjugants from the same experiment. Themobilization frequency of R751 was calculated as the mean 6 standard error ofthe number of R751 transconjugants divided by the number of viable R751 donorcells.

DNA manipulations and enzymes. General DNA techniques were performedas described by Ausubel et al. (1). Purification of DNA for cloning was accom-plished by using Geneclean II (Bio 101 Inc., Vista, Calif.) as recommended by themanufacturer. Competent E. coli BL21(DE3) cells for transformation were pre-pared by the calcium chloride method (3). Restriction endonucleases, T4 polynu-cleotide kinase, and pGEM-7Zf(1) were obtained from Promega, MunI wasobtained from Boehringer Mannheim (Indianapolis, Ind.), and DNase I was ob-tained from Worthington Co. (Freehold, N.J.).

PCR techniques. The GeneAmp core reagent kit (Perkin-Elmer Cetus Co.,Norwalk, Conn.) was used according to the manufacturer’s specifications for allPCR amplifications, which were performed on a Perkin-Elmer Cetus GeneAmpPCR System 2400 thermal cycler with plasmid pGAT400 (12) as the template.Two primers, arot5B (59-GGTGTAGTGGGATCCAGGTTCTTTCTTAGTGCC-39) and arot3B (59-CGCGGATCCGACAGAAGTGGTTGTTTC-39), wereused to introduce BamHI restriction endonuclease sites at both ends of the oriTregion cloned into the pGEM-7Zf(1) (Fig. 1) to give pJA-7ZT. Primer arot5Bis complementary to nucleotides 334 to 351 upstream of the inverted repeats, andprimer arot3B is complementary to nucleotides 621 through 638 immediatelydownstream of the inverted repeats (12). Similarly, primers arba5N (59-CGCGGATCCTATGGATAAAGAAACAACAACC-39) and arba3B (59-CGATGGATCCTACCGCCTCCCTGTATCTTAC-39) were used to introduce a BamHI re-striction endonuclease site at the 59 and 39 ends of the B. fragilis btgA gene forcloning in pET-19b/BamHI to give pDHBA. All PCR products were electropho-resed through 1.3% agarose gels. The appropriate bands (646 bp for btgA and304 bp for the oriT region) were excised and purified by using Geneclean II (Bio101) prior to restriction endonuclease treatment and cloning.

DNA sequence determination. Sequencing was performed by the Sangerdideoxy-chain termination method, using a Sequenase version 2.0 reagent kit asspecified by the manufacturer (Amersham Co., Arlington Heights, Ill.).

Protein expression and purification. The BtgA protein was purified by anaffinity binding procedure using His-Tag resin as specified by the manufacturer(Novagen). In brief, an overnight culture of E. coli BL21(DE3) carrying pDHBAwas diluted 10-fold in fresh LB with ampicillin at a final concentration of 200mg/ml and incubated at 37°C. Isopropyl-b-D-thiogalactopyranoside (IPTG) wasthen added to a final concentration of 1.0 mM, and cells were grown at 37°C withaeration for another 2 h before harvesting. Harvested cells were kept on ice andlysed by sonic disruption in a binding buffer containing 5 mM imidazole, 0.5 MNaCl, and 20 mM Tris-HCl (pH 7.9). Cell debris was removed by centrifugationat 4°C at 10,000 3 g and cell extracts were incubated with His-Tag resin for 30min at 4°C with gentle rotation.

BtgA was eluted from the His-Tag resin in buffer containing 400 mM imida-zole, 6 M urea, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9). Single-step dialysisto native conditions in storage buffer (20 mM Tris-HCl, 100 mM NaCl, 10 mMb-mercaptoethanol, 0.5 mM EDTA, 15% glycerol [pH 7.9]) yielded mostly in-

FIG. 1. Schematic representation of the pBFTM10 transfer region. Base pair numbers correspond to those used by Hecht et al. (12). Open rectangles representthe positions of the DNA oligonucleotides used for PCR amplification of the oriT region and the btgA gene: a, arba5N; b, arba3B; c, arot5B; and d, arot3B. Theexpanded view of the oriT region illustrates the positions of IRI, IRII, and IRIII relative to the start codon (ATG) of the btgA gene.

TABLE 1. E. coli strains and plasmids

Strain orplasmid Descriptiona Reference

or source

StrainsDW1030 E. coli Spr recA 29BL21(DE3) E. coli F2 (r2 m2) (DE3) NovagenHB101 E. coli Smr recA 29

PlasmidspGAT400 Tra1 Ampr Cmr 12R751 Tra1 Tmpr 19pACYC184 Tra2 Cmr Tetr New England BiolabspGEM-7Zf(1) Ampr cloning vector PromegapJA-7ZT pBFTM10 oriT cloned into

pGEM-7Zf(1)This study

pJAYC1 pBFTM10 oriT cloned intoBamHI site of pACYC184

This study

pET-19b Ampr, expression vector NovagenpDHBA btgA in pET-19B BamHI site This study

a Ampr, Clnr, Tmpr, Tetr, Cmr, Spr, and Smr, resistance to ampicillin, clinda-mycin, trimethoprim, tetracycline, chloramphenicol, spectinomycin, and strepto-mycin, respectively; Tra1 and Tra2, transfer-proficient and -deficient, respec-tively.

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soluble protein. Therefore, the following dialysis steps were carried out to reducethe urea, salt, and imidazole concentrations: the first dialysis, to 100 mM imida-zole, 2 M urea, and 200 mM NaCl; the second, to 10 mM imidazole, 0.5 M urea,and 100 mM NaCl; the third, to 100 mM urea and 100 mM NaCl; and the fourth,to storage buffer (20 mM Tris-HCl [pH 7.9], 100 mM NaCl, 10 mM b-mercap-toethanol, 0.1 mM EDTA, 15% glycerol). With this method, approximately 85%of purified protein was recovered in soluble form. The protein concentration wasdetermined with the Bradford reagent (Bio-Rad Co., Hercules, Calif.), aliquot-ed, and stored at 280°C. Aliquots of BtgA were mixed with an equal volume of23 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)sample buffer, boiled for 5 min, and subjected to SDS-PAGE on 10% gels (15).Gels were stained with 0.5% Coomassie brilliant blue (Boehringer Mannheim) in25% methanol–10% acetic acid.

Protein sequencing. An overnight culture of E. coli BL21(DE3) carryingpDHBA was grown under the conditions described above for protein expression.After IPTG induction, final harvesting, and resuspension in water, 100 ml ofwhole cells was mixed with 100 ml of 23 SDS-PAGE sample buffer (15) andboiled for 5 min. An aliquot was then applied directly to an SDS–10% polyacryl-amide gel and electrophoresed at 100 V for 1 h. Proteins from the gel weretransferred to a ProBlott membrane according to the manufacturer’s instruc-tions. After Ponceau-S (Sigma) protein staining, the BtgA protein band wasexcised and sequenced. The first 24 amino-terminal amino acids were sequencedwith an ABI 477A protein sequencer (Applied Biosystems Co., Foster, Calif.)with an on-line ABI 120A phenylthiohydantoin analyzer. This analysis confirmedthe correct fusion between the vector and BtgA protein.

DNA labeling and DNA mobility shift assay. The 304-bp oriT fragment wasobtained by excision from pGEM-7ZT via BamHI restriction or by PCR. Theresulting 304-bp oriT fragment was isolated by electrophoresis through a 1.3%agarose gel, purified by using Geneclean II, and labeled for footprinting assaysusing T4 DNA polynucleotide kinase as described by Sambrook et al. (29), withsome modifications. When BamHI digestion was used, a dephosphorylation stepwas included. The labeling mix contained the 59 terminus of dephosphorylatedDNA (1 to 50 pmol), 103 bacteriophage T4 polynucleotide kinase buffer (5 ml),[g-32P]ATP (3,000 Ci/mmol; 10 mCi/ml; 5 ml; Amersham Co., Arlington Heights,Ill.), bacteriophage T4 polynucleotide kinase (10 U), and water to 50 ml. If a high-er efficiency of labeling was required, the reaction was performed in a volume of20 ml, using 10 mCi of [g-32P]ATP (3,000 Ci/mmol; 10 mCi/ml). After incubationof this mixture for 30 min at 37°C, the enzyme was heat inactivated at 95°C for5 min (29). Labeled fragments were then subjected to restriction endonucleasecleavage (with MunI, to obtain a 271-bp double-stranded DNA fragment specif-ically labeled on the upper strand, and with DdeI, to obtain a 295-bp double-stranded DNA fragment specifically labeled on the lower strand) overnight at37°C. Labeled fragments were purified by electrophoresis through a 5% acryl-amide–0.53 Tris-borate-EDTA gel. The purified DNA band was visualized byautoradiography, excised, eluted overnight with water, and used in DNase Ifootprinting assays.

For electrophoretic mobility shift assays, the 304-bp oriT fragment was labeledby using T4 polynucleotide kinase as described above, but with no subsequentrestriction. The labeled fragment was purified from unincorporated nucleotidesby acrylamide-gel electrophoresis followed by elution. DNA binding reactionswere performed in a 40-ml final volume of the binding buffer [10 mM Tris-HCl(pH 7.9), 5 mM MgCl2, 1 mM CaCl2, 100 mM KCl, 2 mM dithiothreitol, 50 mgof bovine serum albumin per ml, 2 mg of sonicated calf thymus DNA per ml, 1 mgof poly(dI-dC) (Boehringer Mannheim)] (1). Samples were incubated at roomtemperature for 30 min; then an equal volume of stop solution containing glyc-erol (final concentration, 5%) and bromophenol blue (1 mg/ml) was added priorto loading onto a 5% native acrylamide–0.53 Tris-borate-EDTA gel. Gels wereprerun for 1 h, or until the current had stabilized, with buffer in the upper tankreplaced with fresh buffer before loading the samples. Electrophoresis wasstarted at 100 V and, after 10 min, changed to a constant voltage of 200 V at 4°Cfor 2 h. Gels were vacuum dried and exposed to Kodak X-Omat AR-5 film.

DNase I footprinting. DNase I footprinting analysis was performed as de-scribed by Galas and Schmitz (8). Briefly, 10 ng of the oriT fragment (specificallylabeled on the upper or lower strand) was incubated for 30 min at 4°C withvarious concentrations of purified BtgA protein in 13 binding buffer used for thegel shift assay described above, in a total reaction mixture volume of 40 ml. Anequal volume of the mixture (40 ml) containing 2 mM CaCl2 and 15 mM MgCl2was added, and the cleavage reaction was carried out in a total volume of 80 mlby adding DNase I to 2 ng. After 1 min of incubation at room temperature,DNase I was inactivated by adding an equal volume (80 ml) of stop solutioncontaining 10 mM EDTA, 0.5% SDS, 100 mM NaCl, and 75 mg of tRNA per ml.Following phenol extraction and ethanol precipitation, samples were washedwith 70% ethanol, dissolved in formamide dye solution, electrophoresed on an8% polyacrylamide sequencing gel (28), and visualized by autoradiography. Toresolve longer nucleotide sequences, increased electrophoresis time and LongRanger acrylamide (FMC Bioproducts, Rockland, Maine) were used.

Digital imaging and data presentation. Autoradiographs and Coomassie blue-stained gels were scanned with a ScanMaker III (Microtek, Palm Bay, Fla.) orHewlett-Packard ScanJet 4C (Hewlett-Packard, Louisville, Ky.) flatbed scannerat high resolution (1,000 dots/inch), using the software application Adobe Pho-toshop version 3.0.5 (Adobe Systems Inc., Buffalo, N.Y.). Scanned images werethen transferred to the application Microsoft Powerpoint 4.0, text and labels

were added, and images were printed at high resolution (1,440 dots/inch) onan Epson Stylus Color 1520 printer (Epson America Inc., Torrance, Calif.).Hewlett-Packard satin-gloss heavy-weight photographic paper was used for allprints.

Nucleotide sequence accession number. The GenBank accession number forthe pBFTM10 mobilization region is M77806.

RESULTS

Cloning the oriT region of pBFTM10. Previous DNA se-quence analysis of the pBFTM10 mobilization region inpGAT400 revealed three sets of inverted repeats and a possi-ble nic site, based on consensus sequence immediately up-stream of btgA (12). In addition, preliminary DNA relaxosomeisolation studies also demonstrated that single-stranded DNAnicking occurred in or near this region (unpublished data). Todetermine if this area served as a functional oriT and a likelybinding region for btgA, a 304-bp fragment encompassing thethree inverted repeats and the putative nic site was initiallyamplified by PCR using plasmid pGAT400 as a template. Fig-ure 1 illustrates the transfer region of pBFTM10 and the loca-tions of the PCR primers (see Materials and Methods). Fol-lowing cloning into the pGEM-7Zf(1) BamHI site, DNAsequence analysis confirmed that the amplified oriT region wasproperly cloned without any PCR-induced errors. The 304-bporiT fragment was then excised, cloned again into the uniqueBamHI site of pACYC184 to form pJAYC1, and used in bac-terial conjugation experiments.

Demonstration of oriT function by bacterial conjugation.Previously, pGAT400 was shown to mobilize between E. colimating pairs when coresident with R751. pGAT400 containsbtgA and btgB, both necessary for its own transfer, while R751is presumed to provide the machinery necessary for transferbetween E. coli cells (12, 17, 19, 30). To determine if the clonedputative oriT region was functional, pJAYC1 was transformedinto E. coli HB101 containing pGAT400 and R751, or R751alone, and used as a donor in bacterial conjugation experi-ments. HB101 cells containing either pGAT400, R751, pJAYC1,or pACYC184 were used as negative controls.

Donor cells containing pGAT400 and R751 together witheither pJAYC1 or pACYC184 were mixed with E. coli re-cipient DW1030 in mating experiments, and the transfer fre-quencies of R751, pGAT400, pJAYC1, and pACYC184 weredetermined. Throughout all mating experiments, the trans-fer frequencies of R751 ([1.87 6 0.23] 3 1022) and pGAT400([5.72 6 0.64] 3 1022; normalized to the level for R751) weresimilar to previously observed values (12). When coresidentwith both pGAT400 and R751, pJAYC1 was mobilized toDW1030 at a frequency of (4.29 6 1.18) 3 1022 (normalizedto the level for R751), similar to that of pGAT400. However,when coresident with R751 alone or when present with no co-resident plasmid, pJAYC1 was not mobilized, indicating therequirement for pGAT400. In addition, when the donor har-bored pJAYC1 and pGAT400 in the absence of R751, pJAYC1was not mobilized, indicating the requirement for R751 formobilization in E. coli. As expected, pACYC184 did not mo-bilize when coresident with either R751 or pGAT400 or withboth.

To ensure that pJAYC1 transfer was not the result of cointe-grate formation with pGAT400, transconjugants were platedon selective media and assayed for cotransfer of pGAT400 andpJAYC1 (see Materials and Methods). Only 15% of 200 Spr

Cmr pJAYC1 transconjugants contained pGAT400 (Ampr),while only 1% of 100 Spr Ampr pGAT400 transconjugantscontained pJAYC1 (Cmr). Restriction endonuclease treatmentof plasmid DNA isolated from 20 Spr Cmr Amps or Spr Cmr

Ampr pJAYC1 transconjugants confirmed that pJAYC1 and

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pGAT400 were not altered, and cointegration of pJAYC1 witheither pGAT400 or R751 did not occur (data not shown). Inaddition, R751 was found in 100% of pGAT400 transconju-gants but only 85% of pJAYC1 transconjugants. These findingsindicated that pGAT400 and pJAYC1 cotransfer was rareand not the result of cointegrate formation. Thus, the 304-bp pBFTM10 DNA fragment cloned in pJAYC1 containeda functional oriT, and pJAYC1 mobilization required the btgAand btgB genes.

Cloning and expression of BtgA. The predicted amino acidsequence of BtgA revealed a helix-turn-helix DNA bindingmotif (amino acids 118 to 137) (12). This protein, therefore,seemed a likely candidate for binding to the oriT region. Totest this possibility, btgA was cloned into the pET-19b expres-sion vector to give pDHBA and overexpressed in E. coli BL21(DE3). The positions of primers arba5N and arba3B used toamplify btgA by PCR are illustrated in Fig. 1. DNA sequencingof the entire 630-bp insert and part of the pET-19b promotersequence confirmed that the cloned gene contained no se-quence errors. Following IPTG induction of pDHBA, an ap-proximately 27-kDa band was visualized (Fig. 2). The pre-dicted mass of BtgA, as determined from the DNA sequence,was 23.2 kDa, or 26.1 kDa with the leader sequence from thepET-19b expression vector included. To confirm that this bandrepresented expressed BtgA, the first 24 amino acids of theamino terminus of the 26.1-kDa protein band were sequenced,revealing the correct predicted fusion protein.

BtgA was expressed in E. coli at relatively high levels. Scan-ning densitometry using an AMBIOS video imaging system toquantitate protein concentrations in Coomassie brilliant blue-stained SDS-polyacrylamide gels indicated that 10.6% of thetotal amount of E. coli protein expressed was BtgA (data notshown).

Gel mobility shift assay. The BtgA-oriT complex was ana-lyzed in an electrophoretic mobility shift assay to demonstratenoncovalent interaction of BtgA with the oriT (1, 8). PurifiedBtgA protein ranging from 10 to 300 ng and labeled 304-bppBFTM10 oriT fragment were incubated with a binding bufferand electrophoresed in 5% polyacrylamide gels at 4°C (Fig. 3;see Materials and Methods). Increasing concentrations ofBtgA (10, 50, and 100 ng [Fig. 3, lanes 2 to 4, respectively])demonstrated a concentration-dependent manner of bindingwith oriT. At 300 ng (lane 5), BtgA demonstrated the maxi-mum shift of oriT DNA (position A). The presence of poly(dI-dC) at 1 and 3 mg failed to disrupt the interaction between theoriT region and BtgA (lanes 6 and 7, respectively). To demon-strate the specificity of binding, specific competitor pJA-7ZT(containing pBFTM10 oriT) plasmid DNA was added in a50-fold excess, resulting in partial inhibition of BtgA binding,while 100- and 300-fold excess competitor completely inhibitedthe binding reaction (lanes 8 to 10). Of note, storage of BtgA

at 280°C resulted in some loss of binding activity after a fewdays, likely the result of precipitation.

DNase I footprinting. DNase I protection experiments wereused to localize the precise binding sites of BtgA on thepBFTM10 oriT. The oriT fragment was specifically labeled onthe upper or lower strand with [g-32P]ATP and then incubatedin the presence of increasing amounts of purified BtgA. TheseDNA-protein complexes were subjected to partial DNase Idigestion to define the binding site(s) of BtgA. The resultingproducts were separated on 8% polyacrylamide sequencinggels and visualized by autoradiography (Fig. 4). Three distinctregions of protection were visualized on the lower strand. Tworegions, consisting of 36 nucleotides (462 to 498 [Fig. 4A]) and35 nucleotides (501 to 535 [Fig. 4A]), demonstrated the stron-gest protection after the addition of $0.5 mg of BtgA, while athird region, containing three identifiable nucleotides (536 to558 [Fig. 4A]), demonstrated weaker protection at higher pro-tein concentrations. In addition, 3 nucleotides, 498 to 500,showed weaker protection and were located between the strong-ly protected regions. The first protected region overlaps theright half of IRI (which contains a consensus sequence forthe nick site) and the left half of IRII (Fig. 4 and 5), while thesecond protected region overlaps the right half of IRII andthe left half of IRIII. The third, weaker region of protectionoverlaps 10 nucleotides of the right half of IRIII. Bands at 458to 461 and 498 to 500 demonstrated possible minimal protec-tion. No regions of protection were found in the remainingsequence. We detected no regions of protection on the upperstrand that would correspond to the protected zones on thelower strand (Fig. 4B).

DISCUSSIONThe conjugative mechanisms required for transfer of plas-

mids in Bacteroides spp. are not well understood but are pre-sumed to include initiation processes, similar to those ofRP4 or the F plasmid (6, 9, 27, 44). Thus, it is presumed thatrelaxosomes are formed prior to transfer of a single strand ofDNA. For RP4, this requires an oriT region, two DNA bindingproteins, a stabilizing protein, and a nickase (16, 43). pBFTM10has only two transfer genes, both of which are necessary forDNA transfer.

FIG. 2. Overproduction and purification of BtgA. Lane 1, Promega mid-range protein molecular mass marker (masses in kilodaltons are shown at theleft); lane 2, proteins that did not bind to the His-Tag binding resin following theaddition of crude extract; lane 3, BtgA protein eluted from the His-Tag columnfollowing multistep dialysis (see Materials and Methods).

FIG. 3. Binding of purified BtgA protein to the pBFTM10 oriT. Ten nano-grams of 304-bp BamHI pBFTM10 oriT fragment labeled at the 59 terminus with[g-32P]ATP was incubated with purified BtgA (see Materials and Methods).Lane 1, labeled oriT fragment without addition of protein; lanes 2 to 5, labeledoriT fragment with addition of 10, 50, 100, and 300 ng of purified BtgA protein;lanes 6 and 7, oriT fragment incubated with 300 ng of BtgA in the presence of 1and 3 mg of poly(dI-dC), respectively; lanes 8 to 10, labeled oriT fragmentincubated with 300 ng of BtgA protein plus 50-, 100-, and 300-fold excess coldcompetitor oriT-containing plasmid pGEM-7ZT, respectively.



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To further our understanding of the processes required formobilization of plasmid DNA in Bacteroides spp., we first de-termined the location of the oriT region by testing for mobili-zation in E. coli. A 304-bp fragment that contained the threesets of inverted repeats identified previously as likely targetsfor protein binding was chosen. The putative promoter of btgAis immediately downstream of IRIII and was also included inthe cloned fragment. Mobilization of the oriT region in E. coliwas demonstrated only when both btgA and btgB were providedin trans, although R751 must also be coresident. It is presumedthat the role of R751 is to provide a transfer apparatus formobilization of Bacteroides transfer factors, including pGAT400,pB8-51, pLV22a, and the conjugal transposon Tn4399, in E. coli(12, 13, 21, 25, 32). However, it was previously unknown ifcotransfer of R751 with these transfer factors was required.Normally R751 transfers at levels $1.4 orders of magnitudegreater than those for the coresident transfer factor and hasthus been found in transconjugants that received the mobilizedtransfer factor. Indeed, in these studies, R751 was efficientlyself-transferred at levels 1.4 orders of magnitude greater thanthose for the mobilized pGAT400 or pJAYC1 (12, 30). As ex-pected, we observed that 100% of pGAT400 transconjugantsalso contained R751. However, only 85% of pJAYC1 transcon-

jugants also contained R751. Thus, it appears that cotransfer ofR751 is not necessary for pJAYC1 mobilization, although itis not known why a difference between R751 cotransfer withpGAT400 and pJAYC1 was seen. This finding implies thatR751 indeed may simply provide a transfer apparatus utilizedby pBFTM10 and other transfer factors.

The function of one of the mobilization proteins, BtgA, wastested after cloning, expression, and purification in differentassays with the pBFTM10 oriT. The presence of a helix-turn-helix motif suggested that BtgA may be a DNA binding pro-tein, and we tested whether it could bind to the pBFTM10 oriT.Electrophoretic mobility shift assays demonstrated significantspecific binding of BtgA to the 304-bp oriT-containing frag-ment. Poly(dI-dC) in concentrations of up to 3 mg did not af-fect the mobility shift results. The addition of unlabeled oriTfragment contained in plasmid pJAYC1 was an effective com-petition for this binding reaction, confirming its specificity (Fig.3). However, intermediate mobility shifts were seen at proteinconcentrations ranging from 10 to 100 ng (Fig. 3). These find-ings could represent the oligomerization of BtgA either beforeor during binding. Alternatively, this could represent a concen-tration-dependent increase in binding to additional regionswithin the oriT fragment, as seen for TraK from RP4 (46), and

FIG. 4. DNase I footprint analysis of the BtgA-pBFTM10 oriT protein complex. The specifically labeled upper and lower strands of oriT were incubated withincreasing amounts of purified BtgA protein in binding reactions. The BtgA-oriT complexes were subjected to limited cleavage by DNase I and electrophoresed on 8%polyacrylamide sequencing gels. (A) Cleavage pattern of the lower strand of oriT. Quantities of purified BtgA in reaction mixtures: lane C, none; lanes 1 to 5, 100 ng,300 ng, 500 ng, 1 mg, and 3 mg, respectively. Locations of the inverted repeats are shown by arrows. L and R designate the left and right arms, respectively, of the repeats.For IRI, only the right arm of the repeat is shown since the region corresponding to the left arm is undetectable under the conditions of the footprint. The locationsof DNase I protection regions are indicated at the right by brackets. Asterisks represent the region of weak protection (see Results and Discussion). Nucleotidenumbering at the left is from the labeled end and corresponds to the published sequence (12). The inset shows the DNase I digestion pattern up to the top of the gel,indicating that no regions of protection were seen, and also demonstrates that the extents of DNase I digestion were similar for all reactions. (B) Cleavage pattern ofthe upper strand of oriT. Quantities of BtgA in reaction mixtures: lanes 1 to 6, 100 ng, 300 ng, 500 ng, 1 mg, 2 mg, and 3 mg, respectively; lane C, none.



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may be supported by the results of DNase I footprinting ex-periments (see below). Assays to determine if oligomersare involved in binding should resolve this observation.

DNase I protection assays localized BtgA binding to threeregions on the lower strand of the pBFTM10 oriT (Fig. 5). Themost prominent zones of protection were visualized at nucle-otides 462 to 497 and 501 to 535. Taken together, these tworegions span 71 nucleotides that cover parts (or all) of thethree inverted repeats within the oriT region. The first regionof protection toward the 59 terminus of the oriT region includesthe right half of IRI, which also contains the consensus nicksite sequence -TTCCTCTTG/C- (39). Results of preliminaryexperiments using labeled double-stranded linear oriT DNAcombined with BtgA support this observation (unpublisheddata). A weaker zone of protection was visualized on nucleo-tides 536 to 558, which was visualized only with higher concen-trations of protein. No large regions of protection were visu-alized on the upper strand of the pBFTM10 oriT, althoughindividual bases could be protected in more compressed areasbut not seen. This apparent asymmetric binding of BtgA pro-tein to oriT could be due to a conformational change in theDNA as a result of wrapping or bending during BtgA-oriTinteraction. This is not unusual, having been previously ob-served for single-stranded DNA binding proteins involved inreplication (protection over 70 nucleotides on one strand only[5]) and the E. coli Fpg zinc finger protein (tested by usinghigh-resolution hydroxyl radical footprinting analysis [35]), aswell as several other bacterial and eukaryotic DNA bindingproteins (7, 14, 18, 36, 41, 42, 47).

The location of BtgA binding strongly supports its role inDNA processing functions necessary for transfer initiation.Binding to recognition sequences within the oriT region is nec-essary for efficient transfer in RP4 and other plasmids. ForRP4, TraJ binds to the right arm of a 19-nucleotide inverted re-peat recognizing a 10-bp sequence within 8 nucleotides of thedownstream nick site (45). TraJ binding to supercoiled DNA isrequired for binding and nicking activity of the relaxase TraI,presumably by conformational change of the nick site. TraK isnot required for RP4 transfer but increases the efficiency ofrelaxosome formation. It binds downstream of the nick siteover an approximate 180-nucleotide region, which requires asmany as 15 to 20 monomers of TraK to form the complex re-

sulting in bending of DNA (46). The precise role of BtgA inDNA processing has not been defined, but it is required fortransfer of pBFTM10 (12). The relatively large region of pro-tection overlapping the inverted repeats, and possible nick site,raises the possibility that BtgA is multifunctional.

ACKNOWLEDGMENTS

This work was supported by Veterans Administration Medical Re-search Service Merit Review grant 001.

We thank J. Nawrocki, A. Wolfe, S. Baker, C. Hofmann, and V.Bublys for providing various materials or equipment necessary forcompletion of this project. We thank B. Wakim for synthesis and se-quencing the amino terminus of BtgA. We thank M. Malamy andC. Murphy for valuable discussions regarding R751 and pDG5 mobi-lization in E. coli.

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