Genetic Analysis of Transfer-Related Regions of the Vancomycin Resistance Enterococcus Conjugative Plasmid pHT{beta}: Identification of oriT and a Putative Relaxase Gene

The pHT plasmids pHT ${alpha}$ (65.9 kbp), pHTß (63.7 kbp),and pHT ${gamma}$ (66.5 kbp) are highly conjugative pheromone-independentpMG1-like plasmids that carry Tn1546-like transposons encodingvancomycin resistance. pHTß is the prototype plasmid,and the pHT ${alpha}$ and pHT ${gamma}$ plasmids are derivatives of the insertioninto pHTß of an IS232-like (2.2 kbp) element and agroup II intron (2.8 kbp), respectively. The complete nucleotidesequence of the pHTß plasmid was determined and, withthe exception of the Tn1546-like insertion (10,851 bp), wasfound to be 52,890 bp. Sixty-one open reading frames (ORFs)having the same transcript orientation were identified. A homologysearch revealed that 22 of the pHTß (pHT) plasmidORFs showed similarities to the ORFs identified on the pXO2plasmid (96.2 kbp), which is the virulence plasmid essentialfor capsule formation by Bacillus anthracis; however, the functionsof most of the ORFs remain unknown. Most other ORFs did notshow any significant homology to reported genes for which functionshave been analyzed. To investigate the highly efficient transfermechanism of the pHT plasmid, mutations with 174 unique insertionsof transposon Tn917-lac insertion mutants of pHTßwere obtained. Of the 174 derivatives, 92 showed decrease orloss in transfer frequency, and 74 showed normal transfer frequencyand LacZ expression. Eight derivatives showed normal transferand no LacZ expression. Inserts within the 174 derivatives weremapped to 124 different sites on pHTß. The Tn917-lacinsertions which resulted in altered transfer frequency mappedto three separate regions designated I, II, and III, which wereseparated by segments in which insertions of Tn917-lac did notaffect transfer. There was no region homologous to the previouslyreported oriT sequences in the pHT plasmid. The oriT was clonedby selection for the ability to mobilize the vector plasmidpAM401. The oriT region resided in a noncoding region (192 bp)between ORF31 and ORF32 and contained three direct repeat sequencesand two inverted repeat sequences. ORF34, encoding a 506-amino-acidprotein which was located downstream of the oriT region, containsthe three conserved motifs (I to III) of the DNA relaxase/nickaseof mobile plasmids. The transfer abilities of the Tn917-lac-insertionmutants of ORF34 or a mutant of ORF34 with an in-frame motifIII deletion were completely abolished. The sequence of theoriT region and the deduced relaxase/nickase protein of ORF34showed no significant similarity to the oriT and relaxase/nickaseof other conjugative plasmids, respectively. The putative relaxase/nickaseprotein of ORF34 could be classified as a new member of theMOB_MG family.

INTRODUCTION

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Introduction

The isolation of vancomycin-resistant enterococci (VRE) wasfirst reported in 1988 in the United Kingdom (43) and France(25), and shortly thereafter, VRE were detected in hospitalsin the United States (34). Since then, VRE have emerged withunanticipated rapidity and are now encountered in most hospitals,especially in the United States (28).

Increased drug resistance is linked to direct selective pressureby the use of antibiotics and often the presence of a genetictransfer system for spread of resistance (5). Most VRE clinicalisolates are Enterococcus faecium strains (28). Little is knownabout systems of efficient plasmid transfer in E. faecium. Previously,we described the isolation of the pheromone-independent gentamicinresistance conjugative plasmid pMG1 (65.1 kbp) from an E. faeciumclinical strain in Japan, which was the first report describingefficient plasmid transfer in E. faecium (21). pMG1 transfersamong enterococcus strains during broth mating at a frequencyof about 10^–4 per donor strain. Southern hybridizationanalysis revealed no similarity to other gram-positive conjugativeplasmids, and pMG1 was categorized as a new type of conjugativeplasmid. Our epidemiological study revealed that pMG1-like plasmidsare widely disseminated in vancomycin-resistant E. faecium clinicalisolates from a hospital in the United States, suggesting thatpMG1-like plasmids may contribute to the efficient disseminationof vancomycin resistance in enterococcus strains (41).

Recently, we reported the isolation of pMG1-like vancomycinresistance pHT plasmids from clinical Enterococcus faecium andEnterococcus avium strains in Japan (42). pHT plasmids, includingpHT ${alpha}$ (65.9 kbp), pHTß (63.7 kbp), and pHT ${gamma}$ (66.5 kbp),are highly conjugative plasmids carrying Tn1546-like transposons(3) that encode vancomycin resistance (VanA). The pHT plasmidsare related to the gentamicin resistance conjugative plasmidpMG1 with respect to DNA hybridization, and they are thoughtto contain the same efficient conjugation system. The transfergene traA, which is involved in the formation of mating aggregates,is conserved in all pMG1-like plasmids (36, 42).

In this report, sequence comparisons and genetic analysis ofpHTß obtained by generating Tn917-lac insertion mutantsled to identification of a novel type of oriT region and a putativerelaxase/nickase gene designated traI.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains, plasmids, oligonucleotides, media, and reagents. The bacterial strains, plasmids, and oligonucleotides used inthis study are listed in Table 1 (and see Table S6 in the supplementalmaterial). E. faecalis strains were grown in Todd-Hewitt broth(THB) (Difco Laboratories) at 37°C. The following antibioticconcentrations were used for selection of E. faecalis: erythromycin,12.5 µg ml^–1; streptomycin, 250 µg ml^–1;kanamycin, 250 µg ml^–1; spectinomycin, 250 µgml^–1; chloramphenical, 20 µg ml^–1; rifampin,25 µg ml^–1; and fusidic acid, 25 µg ml^–1.Antibiotic concentrations for selection of Escherichia coliwere as follows: ampicillin, 100 µg ml^–1; kanamycin,40 µg ml^–1; chloramphenicol, 50 µg ml^–1;and spectinomycin, 50 µg ml^–1. All antibiotics wereobtained from Sigma Chemical Co. X-Gal (5-bromo-4-chloro-3 indolyl-ß-D-galactopyranoside)was used at 40 µg ml^–1.

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TABLE 1. Bacterial strains and plasmids used in this study

Plasmid/DNA methodology. Recombinant DNA methodology, analyses of plasmid DNA with restrictionenzymes, and agarose gel electrophoresis were carried out bystandard methods (35). Introduction of plasmid DNA into bacterialcells was by electrotransformation, as described previously(13). Plasmid DNA was purified from E. faecalis as previouslydescribed (44). Restriction enzymes were purchased from NewEngland Biolabs and Roche Co. The PCR was performed with a Perkin-ElmerCetus apparatus. Specific primers were purchased from Invitrogen,and Taq DNA polymerase was obtained from Takara.

Construction of the clone sets of the pHTß plasmid. For determination of the physical map of the pHTßand for DNA sequence analysis, the clone sets of the pHTßwere constructed as previously described (14, 39, 40). The partiallyHindIII-digested fragments of pHTß plasmid were clonedinto vector plasmid pMW119. Plasmid pACYC184 was also used forcloning of the partially BclI-digested pHTß plasmidDNA.

DNA sequence analysis. Sequence analysis was performed using the Dye primer and Dyeterminator cycle sequencing kit (Applied Biosystems) and a 377DNA Sequencer and 310 Gene Analyzer (ABI PRISM). To determinethe DNA sequence of the pHTß plasmid, a GPS in vitrotransposition system kit (New England BioLabs) and shotgun cloningmethod were used (35). To determine the DNA sequences in thegap regions, PCR amplification was performed (LA-Taq; Takara,Japan) to obtain PCR products covering the gaps. The PCR productswere sequenced directly using custom primers. Open reading frames(ORFs) were identified and initially analyzed using Genetyxversion 5.1 computer software and the BLAST (1) database tosearch for putative genes.

Conjugation experiments. Filter matings and solid surface matings were performed as previouslydescribed (7, 20). Broth matings (in THB) were for 4 h. Transferfrequencies are expressed as the number of transconjugants perdonor cell (at the end of mating).

Identification and genetic analyses of the oriT region of the pHT plasmid. Various segments of pHTß containing sequences thatwere related to the consensus oriT sequence of IncP or IncQplasmids were amplified by PCR (2, 22). Segments containinginverted repeat (IR) or direct repeat (DR) sequences were alsoamplified. The amplified DNAs were cloned into the pAM401 vectorplasmid. Two clones, pAM401::119/113 and pAM401::119/96, wereconstructed as chimeric plasmids between pAM401 and the clonespMW119::113 and pMW119::96, respectively (see Fig. 1 and seeTable S6 in the supplemental material). Each of the pAM401 derivativescarrying pHTß segments to be tested for oriT activitywas introduced by electrotransformation into Enterococcus faecalisUV202, which is defective in homologous recombination (46).Then, the conjugative plasmid pHTß was introducedinto each of the UV202 transformants carrying the pAM401 derivative(Cm^r) by conjugation. Both broth matings and filter matingswere performed using the transconjugants carrying the two plasmidsas donor strains and JH2SS as the recipient strain.

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FIG. 1. Genetic map and ORFs deduced from the complete plasmid sequence of pHTß plasmid. Boxes indicate ORFs identified on the pHT plasmid. The locations of the Tn917-lac insertions of pHTß (vertical bar with circular head) are shown on the map. Each head color shows the transfer frequency of the derivatives (white; transfer frequency the same as parent plasmid, black; transfer frequency less than one-fifth of the frequency of the parent plasmid or no transfer by broth mating). The number on the insert (1 through 29) indicates the representative plasmids of the derivatives, which are also shown in Fig. 2 and Table 3. Horizontal bars designated as Tra regions I, II, and III on the map indicate the transfer-related regions. DR and IR indicate the direct repeat sequences and inverted repeat sequences. The asterisks under the map indicate the sequence resembling the consensus oriT sites of IncP, IncQ, or oriT of the F plasmid. Black horizontal bars designated a through q under the map show the cloned DNA segments used for the identification of the oriT region of the pHT plasmid. The clones are as follows: a, pAM401::ORF1; b, pAM401::IR4; c, pAM401::IR3; d, pAM401::119/113; e, pAM401::ORF19/21; f, pAM401::119/96; g, pAM401::IR5-1; h, pAM401::ORF39/40; i, pAM401::IR1; j, pAM401::DR1; k, pAM401::ORF51/52; l, pAM401::ORF53/56; m, pAM401::traA; n, pAM401::IR2; o, pAM401::ORF58; p, pAM401::rep5'; and q, pAM401::ORF60/61.

Transposon mutagenesis and isolation of pHTß derivatives with Tn917-lac insertions. The transposon delivery vector pTV32Ts (31) was used for mutagenesis(19, 44). Strain OG1X/pTV32Ts was originally constructed byprotoplast formation (45), and plasmid pHTß was introducedinto this strain by conjugation. The isolation of pHTß::Tn917-lacinsertion mutants was performed as previously described (44).The locations of transposon inserts in the pHTß plasmidwere determined by PCR amplification and DNA sequencing, usingprimers that amplify the segment containing the junction ofthe inserts.

Construction of the ORF34 in-frame mutant. The overlapping PCR technique was used to construct the ORF34deletion mutant. The internal region of ORF34, which containedthe 30-bp deletion corresponding to 10 amino acid residues (HRNTEHIHIH)of motif III, was amplified using the specific primer set (seeFig. 5 and see Table S6 in the supplemental material). The amplifiedDNA was cloned into the pBluescript vector plasmid, resultingin pBS::ORF34del. The 1.1-kbp DNA fragment amplified by PCR,which carries a spectinomycin resistance gene, aad(9), was clonedinto pBS::ORF34del, to give pBS::ORF34del-Spc (24). pBS::ORF34del-Spcwas introduced into E. faecalis FA2-2/pHTß by electrotransformation,and recombinants occurring via double homologous recombinationwere selected as previously described (38). A representativerecombinant carrying the 30-bp deletion of motif III of ORF34,the deduced relaxase gene, was designated as pHTß/ORF34del.

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FIG. 5. Comparison of the N-terminal region of the deduced ORF34 protein (TraI) of pHTß with the hypothetical proteins found in sequence databases. The bold characters indicate the conserved amino acid residues in each protein. The asterisks on the sequences show the key residues, Tyr, Ser, and His₃ (3His) in motifs I, II, and III, respectively. There are two motif I candidates (Ia and Ib) in most of the proteins. The gray box in motif III indicates the deleted 10-amino-acid residues of ORF34 resulting in pHTß/ORF34del. The lowest sequence shows the putative consensus His₃ motif (motif III) of these proteins (designated as MOB_MG family), H(x₂)T(x₃)HxH(x₄)E(x₄)R. The accession numbers for the proteins are as follows: NP_569166 for Listeria innocua pLI100 (81,905 bp), NP_734852 for Streptococcus agalactiae MEN316, NP_765038 for Staphylococcus epidermidis ATCC 12228, NP_053238 for B. anthracis pXO2-84, and NP_150032 for Clostridium perfringens strain 13 plasmid pCP13 (54,310 bp), respectively.

Nucleotide sequence accession number. The complete nucleotide sequence of the pHTß plasmidhas been deposited in DDBJ/EMBL/GenBank under accession no.AB183714.

RESULTS AND DISCUSSION

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Results and Discussion

DNA sequence and gene organization of the pHT plasmid. The total size of the pHTß plasmid, excluding theTn1546-like insertion, was 52,890 bp. The nucleotide sequenceof the Tn1546-like element inserted in the pHTß plasmidwas almost identical to that of the prototype Tn1546 (10,851bp). There was no amino acid substitution of the VanS proteinfound in Japanese VRE isolates that show low-level teicoplaninresistance (17). The G-C content of the core region of the pHTplasmid excluding the Tn1546-like transposon was 31.35, andthat of the Tn1546-like region was 38.66. The Tn1546-like transposonis inserted at the same location in the three pHT plasmids ${alpha}$ ,ß, and ${gamma}$ . The hexa-oligonucleotide sequence 5'-GATTAT-3'was duplicated at the junctions. These results suggested thatthe pHT plasmid resulted from the transposition of the Tn1546-likeelement into an original plasmid without a drug resistance determinant.Sixty-one open reading frames were identified in the 52,884-bpcore region of the pHT plasmid, excluding the Tn1546-like elementand the 6-bp repeat (i.e., 5'-GATTAT-3'), as shown in Fig. 1and as listed in Table 2. All the ORFs were transcribed in thesame direction on the plasmid, with the exception of ORF1 ofTn1546-like transposon (Fig. 1). The nucleotide next to theright end of the Tn1546-like transposon was designated as thefirst base pair of the plasmid. Transcription of the genes encodedon the pHTß plasmid was in a counterclockwise directionfrom the physical map described in our previous report (42).

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TABLE 2. Open reading frames identified in pHT plasmids^a

A homology search of 61 ORFs encoded on the pHT plasmid wasperformed by BLAST against the protein databases, and the resultsare shown in Table 2. Only six ORFs (ORF1, -14, -22, -27, -57,and -60) showed significant similarity to transfer-related genesof other plasmids (Fig. 1) (16). Twenty-two ORFs that are locatedbetween ORF1 and -42 and ORF59 (gray-colored ORFs shown in Fig.1) had significant degrees of similarity to the ORFs encodedon the Bacillus anthracis virulence plasmid pXO2 (96,231 bp;accession no. NC_002146; 94,829 bp, accession no. NC_003981)(15, 32). The pXO2 plasmid carries capsule genes and is necessaryto cause the disease anthrax (27). The degree of identity betweenthe deduced amino acid residues of the homologous ORFs rangedfrom about 10 to 50% (Table 2).

Although most of the functions of the homologues are not known,the putative functions of several proteins were assigned basedon their similarity to other well-characterized proteins (Table2). ORF1 encoded 787 amino acid residues, but the Tn1546-likeinsert at the 142nd amino acid residue (Trp) separated ORF1into an N-terminal 142-amino-acid residue portion and a C-terminal645-amino-acid residue portion. The intact protein was related(about 40% identity) to the trsI gene carried on the Lactococcuslactis bacteriocin plasmid pMRC01 (60.2 kbp) (6). TrsI belongsto the DNA topoisomerase family, which is frequently found inthe transfer-related region of conjugative plasmids (26). ThreepHT plasmids, ${alpha}$ , ß, and ${gamma}$ , that carried Tn1546-likeinsertions within ORF1 could transfer at high frequencies. Thissuggested that the topoisomerase homologue (ORF1) was not essentialfor plasmid transfer.

The sequence comparisons of the ORFs on the pHT plasmid suggestedthat a relatively large portion of the plasmid from ORF57 throughORF61 and from ORF1 through ORF28 (a region spanning about 33kb) could be associated with the transfer region of the pHTplasmid (Fig. 1). However, many pHT ORFs did not have significanthomology with any reported proteins, and the plasmid was thereforecategorized as a new type of conjugative plasmid, as shown byour previous genetic analysis (21). There are reports describinghighly efficient self-transferable large plasmids in Bacillusspecies and the mobilization of the anthrax toxin plasmid pXO1(181.7 kbp) and pXO2 (4, 29, 33). The pMG1-like plasmids, includingthe pHT plasmids, could be closely related to these efficientconjugative plasmids.

Analysis of Tn917-lac transposon insertion mutants of pHTß plasmid. For genetic analysis of the transfer system of pHT plasmids,we isolated mutants which altered the transferability of thepHTß plasmid. Transposon-insertional mutagenesis ofpHTß plasmid using Tn917-lac (Em^r) was performed,and 1,000 independent insertion derivatives were obtained. Eachderivative was examined for transferability in broth matingand LacZ expression in OG1X harboring the plasmid on THB platescontaining X-Gal reagent. A total of 352 derivatives which showedaltered transferability or expressed LacZ activity were chosenfor further analysis. Agarose gel electrophoresis analysis ofNdeI-digested plasmid DNA showed that 289 of 352 derivativeshad a single insertion of Tn917-lac with no deletion or recombination.Of the 289 derivatives carrying a single insertion, the locationof each insertion in 174 representative derivatives was determinedby DNA sequencing and mapped on pHTß, excluding theTn1546-like region (Fig. 1). Of 174 derivatives, 92 showed alteredtransfer frequency and 74 showed normal transfer and positiveLacZ expression. Eight derivatives showed normal transfer andno LacZ expression after repeated examinations. There were hotspots for Tn917-lac insertion on pHTß plasmid, andthe inserts were mapped to 124 different sites within the pHTßplasmid. The locations of the Tn917-lac insertions into theORFs within the 124 derivatives are shown in Fig. 1. The transferfrequency of each of the 124 representative derivatives wasexamined in broth mating. The mating experiment was repeatedusing different hosts: E. faecalis FA2-2 and JH2SS, E. faecalisOG1RF and OG1SS, or E. faecium BM4105RF and BM4105SS, respectively.Ninety-two derivatives showed an altered transfer frequency,which was either a reduced transfer frequency or an inabilityto transfer at a frequency of greater than 10^–7 per donorcell. Some representative plasmids and the results of the matingexperiments are shown in Table 3.

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TABLE 3. Transfer of pHTß::Tn917-lac derivatives

Inserts that decreased in the transfer frequency of pHTßwere mapped within ORFs in three separate regions designatedI, II, and III (Fig. 1). Region I could span a relatively largeportion of the plasmid totaling 39.3 kb lying between 2.8 kbpand 42.1 kbp and contained 46 ORFs from ORF3 to ORF48. The preciseborders of region I were not defined, since the insertions withinthe ORFs between ORF3 and ORF7 or between ORF35 and ORF48 couldnot be obtained (Fig. 1). Region II spanned a small portionof 1.7 kb between 47.0 kb and 48.7 kb and contained ORF56 andORF57, and region III consisted of ORF61 located between 52.0kb and 52.4 kb. Of the 22 ORFs on pHTß which showedhomology with the ORFs on the pXO2 plasmid of B. anthracis,20 were in region I. Inserts in 11 of these 20 ORFs and insertionwithin the noncoding region upstream of ORF19 were obtained,and all of these inserts resulted in decreased transfer frequency.

Inserts in ORF56 of region II resulted in the inability to transferin broth mating. ORF57 downstream of region II was almost identicalto traA of pMG1, which is involved in the formation or stabilityof mating aggregate and is expressed in the early stage of mating(21, 36, 42). Although an insert in pHTß ORF57 hasnot been isolated, insertion into traA of pMG1 resulted in theinability to transfer in broth mating (36). Insertion into ORF61of region III resulted in a reduced transfer frequency. Thepredicted amino acid sequence of ORF60, which lies upstreamof ORF61, was homologous with PrgN of the pheromone responsiveplasmid pCF10, which is the protein involved in the negativeregulation of expression of the mating aggregation substance,and insertion into ORF60 did not affect the transfer frequency(18).

We cannot exclude any potential polar effects on an adjacentgene or genes by transposon insertions in region I and regionII. Research is now under way to determine the function of eachORF. Analysis of transferability and mapping of the insertionmutants implied that many ORFs in region I occupied a relativelylarge portion of pHTß plasmid and could be relatedto transfer of the plasmid. The transfer-related ORFs in regionI showed significant homology to ORFs on the pXO2 plasmid ofB. anthracis. Regions I, II, and III were separated by Tn1546or several ORFs where inserts did not affect the transfer frequency.The ORFs in region II and region III might be necessary fortrans-regulating expression of the ORF(s) of region I.

Identification of the fragment containing the oriT region of pHTß. The transfer origin (oriT) is thought to be characteristic ofthe conjugative plasmid and essential for the transfer of thetransferable or mobile element (11, 47). The oriT functionsin cis to generate the single-stranded plasmid intermediate,after DNA relaxase cleaves a specific phosphodiester bond ofthe nic site. The known oriTs are classified into several groupsbased on sequence similarities (47). To identify the oriT region,which involves identification of the relaxase recognition sequence(i.e., direct repeat sequences) and oriT (site), the degreeof homology between the DNA sequences determined for the pHTplasmid and the reported consensus sequences of the oriT regionwas analyzed. No sequence that was identical or similar to knownoriT regions of gram-positive conjugative plasmids was foundin the pHTß sequences. It is characteristic of theoriT region that direct repeat sequences flank the oriT siteand that the oriT sites are present within inverted repeat sequences.Thus, segments containing direct repeats (DR) and inverted repeats(IR) were selected as candidates for the oriT region. Thesecandidates for the oriT region in the plasmid are indicatedby DR1 and DR2 and from IR1 through IR5 in Fig. 1. Sequenceswhich had similarities (more than 80% identity) to sequencesnear the oriT site (nic site) of the IncP, IncQ, or F plasmids(see Fig. 4) were also screened as candidates for the oriT site.

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FIG. 4. Comparison of the oriT consensus nucleotide sequences (nic region) of the representative mobile plasmids and the possible nick site of the pHTß plasmid. The oriT region containing the possible nick site of the pHT plasmid found in the inverted repeat sequences is shown. The consensus oriT region found in the pheromone-responsive conjugative plasmids (PRP) and the previously reported consensus oriT sites found in mobile plasmids are shown. The consensus oriT sequences of IncP (MOB_P), the pMV158 superfamily, the MOB_Q family (R1162, etc.), and the F plasmid are indicated. The consensus double-stranded replication origin (dso) of RCR plasmids is also shown. The circular marks indicate the conserved nucleotides, centrally located TG site, and an A located four residues away. The black arrowheads indicate the nick sites determined in the oriT region.

DNA segments containing potential candidates for the oriT region,which were indicated by black horizontal bars marked from ato q in Fig. 1, were cloned into pAM401. Each plasmid clone(Cm^r) was tested for its ability to be mobilized by the pHTßplasmid (Vm^r). Two of the clones, pAM401::IR5-1 and pAM401::IR2,were mobilized by the pHTß plasmid (Table 4). Theissue of the mobilization of pAM401::IR2 clone will be discussedlater as a possible oriV candidate. The segment containing theIR5 region between ORF31 and ORF32 (g in Fig. 1), conferredthe ability to transfer the pAM401::IR5-1 chimeric plasmid ata high frequency comparable to that of the pHT plasmid (Table4). Of the transconjugants selected on the agar plates containingvancomycin, about 40% of the transconjugants were resistantto vancomycin, and 60% were resistant to both vancomycin andchloramphenicol. The vancomycin-resistant transconjugants containedonly the pHTß plasmid, and the vancomycin- and chloramphenicol-resistanttransconjugants contained both the pHTß plasmid andthe pAM401::IR5-1 chimeric plasmid. Of the transconjugants selectedon agar plates containing chloramphenicol, about 10% were resistantto chloramphenicol and 90% were resistant to chloramphenicoland vancomycin. The chloramphenicol-resistant transconjugantscontained only the pAM401::IR5-1 chimeric plasmid; the chloramphenicol-and vancomycin-resistant transconjugants contained both pAM401::IR5-1and the pHTß plasmid. These results indicated thatthe chimeric plasmid pAM401::IR5-1 was mobilized in trans bythe coresident pHTß plasmid. None of the other fragmentsof pHTß except fragment n containing IR2 and ORF58could mobilize transfer, and transfer of pAM401::IR5-1 requiredthe presence of pHTß.

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TABLE 4. Mobilization frequencies of pAM401 derivatives (Cm^r) carrying the various segments of pHTß by coresident pHTß plasmid (Vm^r)

Cloning and genetic analysis of oriT region of pHTß plasmid. IR5 contained direct repeats of 13 bp and two inverted repeats(Fig. 2 and 3). The direct repeats were composed of two copiesof a 13-bp sequence and 7 bp that were part of the 13-bp sequencein the same orientation (i.e., ACTATGACCAAAA [DR-a], ACTATGACCAAAA[DR-b], and TATGACC [DR-c]). The inverted repeats were composedof short and long inverted repeats, AGTTGGC/GCCAACT (IR5S) andTAGCcACCTTCCT/AGGAAGGTGCTA (IR5L), respectively. Detailed analysisof this IR5 region was performed. Deletion mutants of the IR5-1clone were constructed (Fig. 2). Deletion mutant IR5-9, whichpossessed a 192-bp fragment lying between 30,409 bp and 30,600bp of the pHTß map, was mobilized in trans by thecoresident pHTß plasmid and was the smallest fragmentretaining the ability to transfer with a frequency equivalentto that of the pHTß plasmid. This region containedone (DR-b) of the two copies of the 13-bp direct repeat sequencesand the 7 bp (DR-c) of the 13-bp sequence, as well as two invertedrepeat sequences (i.e., IR5S and IR5L).

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FIG. 2. Genetic analysis and determination of oriT region of pHT plasmids. The horizontal bars indicate the cloned PCR fragments of the pAM401 derivatives, and the bar numbers show the end positions of the segments. The transfer frequency of each of the pAM401 derivatives is shown in Table 5.

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FIG. 3. Nucleotide sequences of the oriT region of the pHT plasmid. The 420-bp noncoding DNA sequence region between ORF31 and ORF32 is shown. The horizontal arrows under the sequences indicate the direct repeats (DR-a, DR-b, and DR-c) and inverted repeats (IR5S and IR5L) in the oriT region. The names and locations of oligonucleotide primers used for the analysis of the oriT region are shown on the sequence with the angled arrows. Two downward arrowheads on the complementary sequence show the possible nick sites in the oriT region based on the sequence comparisons with other defined nick sites (see the text and Fig. 5). The vertical arrows that are numbered as 323 and 327 indicate the locations of the two Tn917-lac insertion mutants of pHTß that abolished transfer ability. The putative promoter region (–35 and –10) and ribosome binding site (Shine-Dalgarno sequence [S.D.]) for ORF32 are shown upstream of the start codon. The asterisk marks indicate the dyad symmetric sequences overlapping the promoter region.

Deletion mutants containing only two inverted repeats, suchas pAM401::IR5-3 and pAM401::IR5-10, were still mobilized atlow frequencies by filter mating (Table 5). The pAM401::IR5-8deletion mutant, which had a deletion of two inverted repeatsequences, was completely incapable of transfer, and the transferfrequency was less than 10^–8 in filter mating. Two Tn917-lacmutants (numbered as 16 in Fig. 1 and 2) near the oriT regionof pHTß were obtained, and the insertion was mappedto 10 bp downstream of the long inverted repeats (IR5L) (Fig.3). The mutants, pHTß::Tn917-lac/323 and -327, couldnot transfer at all, even on a solid surface. These resultsindicated that the IR5 region could be the oriT region and thattwo inverted repeat sequences were essential for plasmid transfer.The sequence of the putative oriT region (IR5) showed no significantsimilarity to the reported oriT sequences.

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TABLE 5. Mobilization of pAM401 derivative plasmids by pHTß^a

Five families of oriT core sequences have been defined throughcomparison of a wide range of transfer origins (Fig. 4) (9,11, 47). There are conserved sequences within the core sequencesof an oriT family. A closer inspection revealed a consensussequence that is common even among the apparently phylogeneticallyremote oriT families (47). The most conserved sequence foundamong the families contains a centrally located TG or CG site,which is the nic site for the relaxase, and an A residue 4 bpaway, which represents the highest level of conservation amongthe nucleotides in the nic sites of oriTs and dsos. IR5L ofpHT contained a centrally located TG and an A residue 4 bp away(Fig. 4). It was possible that the nic site might be locatedwithin this region in the inverted repeat region in the pHTßplasmid (Fig. 4).

A clone, pAM401::IR2, carrying the IR2 segment (segment n inFig. 1) was also mobilized by the pHTß plasmid atlow frequencies, of around 10^–6 per donor cell, whichwas about 10% of the transfer frequency of a plasmid containingthe oriT region, by broth mating (Table 4). The donor strainUV202 was shown to carry the pHTß plasmid and thecloned pAM401::IR2 plasmid by agarose gel electrophoresis ofplasmid DNAs from the donor strains (data not shown). The transconjugantsof JH2SS were resistant to both chloramphenicol and vancomycinand harbored one chimeric plasmid formed between pHTßand the pAM401 derivative (data not shown), which could resultfrom cointegration between pHTß and pAM401::IR2. Thesedata indicated that the mode of mobilization of pAM401::IR2was different from that of pAM401::IR5-1. Analysis of the chimericplasmid formed between a fragment of the F plasmid and pSC101shows that chimeric plasmids that lack oriT but contain oriV1of the F plasmid were mobilized in cis via cointegration withthe coresident F plasmid at oriV1 in a RecA-independent recombination(23). The oriV region is essential for plasmid replication andis the start site for replication. The IR2 region of the pHTßplasmid is located just upstream of ORF59, which is highly relatedto the rep genes of rolling circle replication (RCR)-type gram-positiveplasmids. It was probable that IR2 was not a second oriT regionbut was the oriV region of the pHTß plasmid (2, 10).Site-specific recombination could occur between the predictedoriVs of the pAM401::IR2 and pHTß plasmids.

The putative DNA relaxase/nickase gene, ORF34. In addition to the oriT sequence, the relaxase/nickase is animportant feature of conjugative plasmids (11, 47) needed forthe initiation of DNA transfer. None of the ORFs encoded onthe pHT plasmid showed significant similarity to reported relaxase/nickasegenes. Similarities between amino acid residues in relaxasesencoded by different conjugative systems have been reported(30), and three common motifs are seen (11, 47). These suggesta shared DNA relaxation mechanism. Motif I contains the catalyticTyr residue involved in DNA cleavage-joining activity. MotifII was reported to be involved in DNA-protein contacts throughthe 3' end of the nick region, and a Ser residue is usuallypresent. Motif III contains three conserved His residues andis known as the His₃ motif. It has been suggested that the Hisresidues aid the nucleophilic activity of the Tyr residue inmotif I coordinate the required Mg²⁺ ions and direct activationof the active Tyr. These three motifs are thought to form partof the catalytic center of the relaxase. We examined the pHTplasmid for the presence of a relaxase by searching for theconserved motifs (residues) of the relaxase. ORF34 encoding506 amino acid residues was found to contain the three conservedmotifs I, II, and III, which contained Tyr, Ser, and three Hisresidues, respectively, and could be the relaxase for the pHTplasmid (Fig. 5).

Three Tn917-lac insertion mutants of the pHTß plasmid,pHTß::Tn917-lac/95, -197, and -223, were obtainedin the ORF34 gene, and each of the insertions was mapped. Thetransposons were inserted into residues Leu³⁶⁶, Gln⁴⁴⁰, andVal⁴⁶⁹ of the ORF34 protein, respectively. All the insertionmutants had completely lost the ability to transfer. Each phenotypeof the mutant might result from the polar effects of insertion.To confirm whether ORF34 is essential for plasmid transfer,an in-frame deletion mutant of 10 amino acids (i.e., HRNTEHIHIH)from amino acid residues 200 to 209, which is located withinmotif III of ORF34, was constructed in the pHTß plasmidas described in Materials and Methods (Fig. 5). The pHTßplasmid derivative mutant with the defective ORF34 could nottransfer in broth or on a filter (data not shown). These resultsindicated that ORF34 was the essential transfer-related gene.

A FASTA/BLAST homology search showed that sequences homologousto the motifs of the putative relaxase ORF34 were found in ORFsof other bacterial species for which function has not been determined(Fig. 5). Most of the ORFs homologous to ORF34 were found tocontain two putative motifs I, designated as motifs Ia and Ib,in the N-terminal region, and a Tyr residue was conserved ineach of the motifs (Fig. 5). Although until now there has beenno genetic information or characterization of the genes homologousto ORF34, the new pHT family relaxase represented by the putativerelaxase ORF34 could be widespread throughout a variety of bacteria.The conserved His₃ sequence in motif III of the pHT family wasdesignated as MOB_MG: i.e., H(x₂)T(x₃)HxH(x₄)E(x₄)R.

Concluding remarks. Sequence data for the pHT plasmid revealed that the pMG1-likeplasmids had little similarity to well-characterized plasmids.The ORFs encoded on pXO2, a pathogenic capsule plasmid foundin B. anthracis, shared sequence homologies with the pHT plasmids.Little is known about the transfer of pXO2 and about the functionof each ORF, although there are reports about the mobilizationof the plasmid by other highly conjugative plasmids found inBacillus species (4, 33). Based on the genetic analysis by transposoninsertional mutagenesis, a region containing ORFs from ORF3to ORF48 that was designated as region I could be related tothe transfer of the pHT plasmid: the other two regions, regionII containing ORF56 and -57 and region III consisting of ORF61,were also necessary for efficient transfer.

Both oriT and nickase/relaxase are thought to be essential andimportant characteristic features of the conjugative plasmid(11, 47). The oriT region of the pHT plasmid was geneticallydetermined, and the traI gene encoding the putative DNA relaxase/nickaseresided in the transfer-related region I. The biochemical activityof the product has not yet been elucidated.

ACKNOWLEDGMENTS

This work was supported by the grants from the Japanese Ministryof Education, Culture, Sport, Science and Technology [Tokuteiryoiki(C), Kiban (B), Kiban (C)], and the Japanese Ministry of Health,Labor and Welfare (H15-Shinko-9).

We thank Koichi Tanimoto and Maria V. Francia Gil for helpfuladvice. We also thank Elizabeth Kamei for revising the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Bacteriology and Bacterial Infection Control, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan. Phone: (81) 27 220 7992. Fax: (81) 27 220 7996. E-mail: tomitaha{at}med.gunma-u.ac.jp.

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

REFERENCES

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