JOURNAL OF BACTERIOLOGY, June 2002, p. 3194–3202 Vol. 184, No. 120021-9193/02/$04.00�0 DOI: 10.1128/JB.184.12.3194–3202.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Complete Nucleotide Sequence of Plasmid Rts1: Implications forEvolution of Large Plasmid Genomes

Takahiro Murata,1 Makoto Ohnishi,2 Takeshi Ara,3 Jun Kaneko,4 Chang-Gyun Han,5 Yong Fang Li,1†Kayoko Takashima,6‡ Hideaki Nojima,3§ Keisuke Nakayama,2 Akira Kaji,7 Yoshiyuki Kamio,4

Takeyoshi Miki,8 Hirotada Mori,3,6 Eiichi Ohtsubo,5 Yoshiro Terawaki,1 and Tetsuya Hayashi2*Department of Bacteriology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621,1 Department of

Microbiology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692,2 Institute of Advanced Biosciences, KeioUniversity, 14-1 Baba, Tsuruoka, Yamagata 997-0035,3 Laboratory of Applied Microbiology, Department of Molecular and

Cell Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumi-dori, Amamiya-machi, Aoba-ku,Sendai, Miyagi 981-8555,4 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi,

Bunkyo-ku, Tokyo 113,5 Research and Education Center for Genetic Information, Nara Institute ofScience and Technology, 8916-5 Takayama, Ikoma, Nara 630-01,6 and Department of MolecularMicrobiology, Kyushu University Graduate School of Pharmaceutical Sciences, 3-1-1 Maidashi,

Higashi-ku, Fukuoka 812-8582,8 Japan, and Department of Microbiology, University ofPennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-60767

Received 23 October 2001/Accepted 26 February 2002

Rts1, a large conjugative plasmid originally isolated from Proteus vulgaris, is a prototype for the IncTplasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide se-quence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs).Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequencesimilarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has beenidentified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Ofparticular interest was the presence of tus-like genes that could be involved in replication termination.Inspection of the overall genome organization revealed that the Rts1 genome is composed of four largemodules, providing an example of modular evolution of plasmid genomes.

Rts1 is a low-copy-number kanamycin resistance plasmidoriginally isolated from a clinical strain of Proteus vulgaris (56).Its molecular mass was originally estimated to be about 140MDa (29). This large plasmid is the prototype for the T in-compatibility group (9) and expresses pleiotropic thermosen-sitive phenotypes in autonomous replication (13, 58), conjuga-tive transfer (56), host cell growth (12, 57), and restriction ofT-even phages (28, 32, 66).

As in many large plasmids of gram-negative bacteria, Rts1requires two elements for its autonomous replication, a repli-cation initiation protein encoded by the plasmid (repA) and ashort segment containing the replication origin, ori (30, 34, 59).Because the replication of this mini-Rts1 plasmid is stable at37°C but is inhibited at 42°C in Escherichia coli (30), as ob-served for Rts1 (56, 58), the replication machinery of Rts1

itself is thermosensitive. A locus termed tdi (temperature-de-pendent instability) has also been identified as another locusresponsible for the temperature sensitivity (48, 55), but its realphysiological role in the replication (or maintenance) of Rts1is unknown.

As for the temperature-sensitive effect on host cell growth,Terawaki et al. (57) first reported that Rts1 inhibits its host cellgrowth at 42°C but not at 37°C. Since then, two loci responsiblefor this phenotype have been identified, tsg (43, 47) and hig(60–62). Since the tsg locus contains no open reading frame(ORF), the AT-rich DNA segment itself is thought to be re-sponsible for the thermosensitivity of host cell growth. On theother hand, the hig locus encodes a system belonging to theproteic killer family, where the HigB and HigA proteins func-tion as a toxin and an antitoxin, respectively. The temperature-sensitive host cell growth conferred by the hig system is medi-ated by selectively killing the host that has lost all copies ofRts1 at a nonpermissive temperature (postsegregation killing).

Conjugation of Rts1 is also thermosensitive, but the temper-ature range for conjugation differs from that for replication,efficient at 25°C but not at 37°C (56). Thus, the thermosensi-tivity of conjugation is a phenomenon unrelated to that ofreplication. The molecular mechanism underlying the thermo-sensitive conjugation is unknown except that the lengths ofRts1 sex pili were reported to be shorter at 37°C than at 30°C(8). No Rts1 gene for conjugation has been identified.

Another temperature-sensitive phenotype shown by Rts1 isthe restriction of T-even phages: Rts1 inhibits the multiplica-

* Corresponding author. Mailing address: Department of Microbi-ology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki889-1692, Japan. Phone: 81-985-85-0871. Fax: 81-985-85-6475. E-mail:thayash@post.miyazaki-med.ac.jp.

† Present address: Gene Regulation and Chromosome Biology Lab-oratory, Division of Basic Science, National Cancer Institute–Fred-erick Cancer Research and Development Center, Frederick, MD21702.

‡ Present address: Specific Toxicology Research R&D, KISSEIPharmaceutical Co., Ltd., 2320-1 Maki, Hotaka, Minamiazumi, Na-gano 399-8305, Japan.

§ Present address: Division of Molecular Oncology, Osaka Univer-sity Graduate School of Medicine (C7), 2-2, Yamada-oka, Suita, Osaka565-0871, Japan.

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tion of T-even phages at 30°C but not at 43°C (66). A locusencoding a restriction enzyme, PvuRts1I, is responsible for thisphenomenon (28, 32).

Except for above-mentioned thermosensitive phenotypes,only a few Rts1-related phenomena have been documented sofar, and only nine genes have actually been cloned and ana-lyzed. They indeed represent a very small portion of the largegene repertoire encoded on the Rts1 genome. In this study, wedetermined the entire nucleotide sequence of Rts1, comprising217,182 nucleotides, and identified all the potential genes en-coded by the plasmid. They include the set of genes for theconjugation function and a large number of genes involved invarious processes of DNA metabolism. Furthermore, by in-specting the overall genome organization, we identified fourmodules constituting the Rts1 genome. Among them, two weregenerated by a large segment duplication and subsequently bystructural and sequence diversification. These findings providenew insights into the mechanisms of the evolution and generepertoire expansion of large plasmids in general.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. Plasmid Rts1, which was originallyisolated from P. vulgaris strain UR-75, was conjugatively transferred to andmaintained in E. coli ER1648 [F� fhuA2 �(lacZ)r1 supE44 trp31 mcrA1272::Tn10(Tetr) his1 rpsL104(Strr) xyl-7 mtl-2 metB1�(mcrBC-hsdSMR-mrr)102::Tn10(Tetr)] (32). Plasmid pUC18 (65) and E. coli strains DH5� MCR and DH5�(both from Gibco-BRL) were used to construct random shotgun libraries of theRts1 genome. E. coli HB101 (leuB6 supE44 thi-1 hsdS20 recA13 ara-14 proABlacY1 galK2 rpsL20 xyl-5 ntl-1) (7) was used as a recipient strain in the mobili-zation assay. All strains were cultivated with Luria-Bertani (LB) or 2xYT me-dium supplemented with appropriate concentrations of antibiotics (ampicillin,100 to 200 �g ml�1; kanamycin, 30 �g ml�1; and streptomycin, 500 �g ml�1).

DNA manipulation and sequencing determination. Routine DNA manipula-tions were carried out by the standard methods (53). To purify the intact Rts1plasmid, E. coli strain ER1648 containing Rts1 was cultivated overnight in 2xYTmedium containing kanamycin, and the plasmid DNA was extracted with aplasmid kit (Qiagen). The DNA was further purified by cesium chloride densitygradient centrifugation. The whole genome sequence was determined by therandom shotgun method as described previously (45, 46). Collected sequenceswere assembled by the Sequencher sequencing software (version 3.0, genecodes). After the initial assembling of 4,750 sequences obtained by the forwardprimer (�500 bases in average), a short region remained to be determined. Tofill the sequence gap, 15 clones that covered the gap were selected and sequencedby the reverse primer and custom primers. In addition, 178 clones, the oppositeends of whose inserts were expected to cover the regions with any sequenceambiguity, were selected and sequenced by the reverse primer. The final redun-dancy of sequencing was about 11-fold, and both strands were read at least oncethroughout the entire genome.

ORF prediction and computer analysis. ORFs were predicted with the soft-ware GeneMark (6) trained with the matrix generated from a set of genes fromvarious phages and plasmids. In principle, ORFs larger than 150 bp preceded byrecognizable ribosome-binding sequences were searched. By this search, 223ORFs were identified. In the regions where no ORF was identified by Gene-Mark, ORFs larger than 150 bp with typical ribosome-binding sequences weresearched for manually. DNA and protein sequences were compared to the publicsequence databases with the Blast program (4) through DDBJ. Multiple align-ments of protein sequences were made with ClustalW through DDBJ.

Construction of the physical map. To construct the physical map of Rts1, thepurified plasmid DNA was digested with XbaI, SpeI, SalI, or various combina-tions of these enzymes. Digested DNAs were analyzed by conventional agarosegel electrophoresis with 0.35 to 0.8% agarose gels prepared by AgaroseH (Nip-ponGene). Pulsed-field gel electrophoresis (PFGE) was also employed to pre-cisely determine the sizes of large DNA fragments. PFGE was performed withthe CHEF Mapper apparatus (Bio-Rad) on 1% agarose gels (pulsed-field cer-tified agarose, Bio-Rad) in 0.5� TBE (Tris-borate-EDTA) and regularly run at6 V/cm at 14°C with a linear increase in pulse intervals. To resolve fragments of20 to 120 kb in size, pulse times were ramped from 2.98 to 10.29 s for 26.56 h.

Identification of the oriT locus. To identify the oriT locus of Rts1, we screenedthe random shotgun library of Rts1 by the mobilization assay. To construct apUC18-based library, the purified plasmid DNA was fragmented by sonication,and fragments of 0.5 to 5.0 kb in length were cloned into the SmaI site of pUC18.Resulting plasmids were introduced into E. coli DH5� containing the intact Rts1that served as a helper plasmid. After precultivation at 28°C, each transformantwas incubated with HB101 overnight at 28°C in 96-well microtiter plates. Eachcoculture was then transferred to fresh medium containing streptomycin andincubated overnight to reduce the background in the screening of transconju-gants. Selection of transconjugants was done on LB agar plates containing strep-tomycin and ampicillin. Sizes of pUC18 derivatives in each transconjugant wereanalyzed and compared with those in the donor clones to confirm the absence ofrearrangement during the mobilization. Frequency of kanamycin-sensitivetransconjugants was also examined for each clone to determine whether eachclone was transferred by mobilization or cointegration into the helper Rts1plasmid. Finally, both ends of the inserts were sequenced to map their positionson the Rts1 genome. To further localize the oriT locus, various lengths of DNAfragments covering the oriT-containing region identified by the random screen-ing were generated by PCR, cloned into pUC18, and subjected to the mobiliza-tion assay. Nucleotide sequences of all the clones generated by PCR wereconfirmed by sequencing.

Nucleotide sequence accession number. The annotated sequence of Rts1 hasbeen deposited in DDBJ/GenBank/EMBL under accession number AP004237.

RESULTS AND DISCUSSION

General overview. The Rts1 genome is 217,182 bp in length.The nucleotide position 1 was defined as the guanine thatcorresponds to position 1441 of the former mini-Rts1 coordi-nate (34). The physical map deduced from the sequence wasthe same as that determined experimentally except for an XbaIsite at position 127284, but the site contained a GATC se-quence which is methylated by Dam methylase (data notshown). The calculated G�C content is 45.7%, close to thevalue determined by chemical analysis (45%) (19). This valueis, however, significantly higher than that of the chromosomeof P. vulgaris, from which Rts1 was isolated (39.3% � 1.2%)(15). This suggests that the original host of Rts1 is not P.vulgaris. There was no substantial region showing atypical basecomposition (data not shown).

Rts1 encodes 300 ORFs (001 to 165 and 167 to 301, Fig. 1).They indeed include the nine Rts1 genes that were identifiedpreviously. Among the 300 ORFs, 253 are oriented in the samedirection, left to right in Fig. 1. This remarkable bias mayindicate that Rts1 genome replication proceeds mostly in thisorientation. In the homology search, the products of 141 ORFsshowed significant sequence similarity to known proteins, andamong these, 99 were homologous to proteins whose functionsare known or predicted. No ORFs directly related to bacterialpathogenesis were identified.

In the following sections, we will describe the features ofRts1 that have been revealed by analyzing the genome se-quence.

Transposon and IS elements. Rts1 contains six kinds ofinsertion sequence (IS) elements (nine copies in total) andthree transposons (Table 1, Fig. 1). All together, they comprise28,644 bp, 13% of the plasmid genome. IS610 and IS611 arethe ones newly identified in this study, and both belong to theIS3 family. IS611B and IS611C are located very closely (444 bpapart), but they do not appear to form a composite transposonbecause no target sequence duplication was detected in theregions flanking the two IS elements.

Tn2680 is a previously identified composite transposon thatcarries a kanamycin resistance gene [apH(3)] and is composed

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of two copies of IS26 and one copy of IS903.B (27, 44), but thecomplete sequence has been determined in this study. Tn2680contains additional three ORFs (ORF167, ORF168, andORF169). Although the functions of ORF168 and ORF169 areunknown, ORF167 is highly homologous to the C-terminalpart of a putative transposase (R0148) of Salmonella entericaserovar Typhi plasmid R27 (54). The deduced amino acidsequence in the upstream region of ORF167 is also similar tothe N-terminal part of R0148 but contains several frameshiftmutations.

Tn9199 and Tn6901 are newly identified class II trans-posons. Besides the genes for transposases and resolvases,each of these transposons carries four ORFs. ORF088 andORF089 of Tn9199 are similar to a putative ATPase and aputative sulfate permease encoded by Tn2501 on Yersinia en-terocolitica plasmid pGC1, respectively (25, 68). The N-termi-nally truncated arsC gene (ORF090) is present in the upstreamregion of the two ORFs. ORF087 is similar to BetT, a trans-porter of choline, which is a precursor of glycine betaine,serving as an osmoprotectant (38). ORF009, ORF010, andORF012 on Tn6901 are highly homologous to YaiN (90%amino acid sequence identity over the entire length), alcoholdehydrogenase AdhC (94%), and YaiM (78%), respectively, ofE. coli K-12. The gene organization is also similar to the yaiN-adhC-yaiM locus on the K-12 chromosome, though an ORF ofunknown function is inserted between ORF010 and ORF012in Rts1.

Conjugation system. Genes for the conjugation functionsare clustered in two regions separated by a 16-kb segment.Most of the gene products show the highest similarities tothose of the conjugation systems of plasmid F or plasmid R27,the prototype IncFI and IncHI1 plasmids, respectively (16, 54).

Genes required for the conjugal transfer of plasmid F havebeen most intensively analyzed, and they are categorized intofive groups according to their functions: pilus biosynthesis,aggregate stabilization, conjugal DNA metabolism, surface ex-clusion, and regulation of gene expression. The genes for sexpilus formation are further divided into two subgroups, thosefor pilin synthesis and for pilus assembly (reference 16 andreferences therein). Homologues of all the F genes for pilusassembly except trbI were identified in Rts1. ORF216 showshomology to two pilus assembly proteins of F plasmid, theN-terminal half to TrbC and the C-terminal half to TraW.Although little is known about the exact functions of these Fproteins, both are known to be located in the periplasmic space(16). Fusion of the two genes probably indicates that theyparticipate in the same or closely related operational steps inpilus assembly. The same type of gene fusion is observed inplasmid R27, and most of the pilus assembly genes of Rts1 arealso similar to the R27 genes (54).

Making a sharp contrast to the pilus assembly genes, nohomologues of the F genes for pilin synthesis, traA, traQ, andtraX, were identified in Rts1. Instead, Rts1 contains a gene(ORF215) homologous to traF of RP4, which encodes a pro-

pilin-specific peptidase (14, 20). R27 also contains a traF ho-mologue, TrhF (54). It should be also noted that ORF185encodes a type I leader peptidase, which could participate inthe first processing step of pilin maturation, removal of thesignal peptide.

The aggregate stabilization system of Rts1 is also similar tothat of plasmid F, since ORF242 and ORF244 showed simi-larity to TraG and TraN of plasmid F, respectively. However,no genes homologous to the F genes related to DNA metab-olism, surface exclusion, and regulation of gene expressionwere identified. Instead, ORF201 and ORF202 exhibit signif-icant similarities to a putative nickase (TraI) and an anchoring/coupling protein (TraG) of plasmid R27, respectively (54).These proteins are required for DNA transfer in R27, and thusthe DNA transfer system of Rts1 is similar to that of R27.

Among the F genes that are not essential for conjugation butare located in the transfer region, only trbB exhibits a weak andlocal homology to an Rts1 gene, ORF212. ORF212 is a homo-logue of DsbC, which catalyzes disulfide bond formation in theperiplasmic space (50). A predicted active-site motif, Phe-(X)4-Cys-Pro-Tyr-Cys (42), is conserved in ORF212, and thehomology between ORF212 and TrbB of F is limited to asegment containing the motif. DsbC homologues are alsopresent in the transfer regions of R27 (54) and pNL1 (52),suggesting that they play some role as redox proteins in theconjugation process.

ORF197 encodes a protein similar to Nuc, the EDTA resis-tance endonuclease of pKM101 (49). Nuc homologues areencoded in close proximity to transfer regions in many plas-mids belonging to several incompatibility groups (36, 49),though their roles in conjugation are unknown. ORF197 is alsolocated just upstream of the tra gene clusters in Rts1. Anextracellular nuclease activity was previously detected in E. coliharboring Rts1 (41), but the relationship between the nucleaseactivity and ORF197 remains to be elucidated.

TABLE 1. Transposon and IS elements on the Rts1 genome

Element TSDa

(bp)TIRb

(bp)Location

(length in bp)% Homology

(accession no.)

Tn6901 5� 52 5877–12777 (6,901) Newly identifiedIS610A 3� 38 15663–16892 (1,230) Newly identifiedIS5A 4 16 25205–26399 (1,195) 100 (J01734)IS611A 3 38 31503–32764 (1,262) Newly identifiedTn9199 — 78 60227–69425 (9,199) Newly identifiedIS611B 3 38 106039–107300 (1,262) Newly identifiedIS611C 3 38 107745–109006 (1,262) Newly identifiedTn2680 8 820 114687–119688 (5,002) Newly identifiedIS26A — 14 114687–115506 (820) 100 (X00011)IS903.B 9 18 116718–117774 (1,057) 99.9 (X02527)IS26B — 14 118869–119688 (820) 100 (X00011)IS2A 5 42 169698–171028 (1,331) 100 (V00279)

a Elements, in which one base of the target site duplication (TSD) sequencesbetween the right and left ends are different are indicated by asterisks. Dashesindicate that no such sequences were found.

b TIR, terminal inverted repeat.

FIG. 1. Genome organization of Rts1. Boxes indicate ORFs identified on Rts1, and numbers above and below the boxes are ORF numbers.ORFs shown above and below the line are transcribed left to right and right to left, respectively. Predicted functions of ORFs were categorizedinto eight groups, and ORFs in the same group are indicated by the same color. Locations of ori, oriT, IS elements, and transposons are alsoindicated.

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Identification of the oriT region. In the conjugation process,transfer of DNA initiates at a specific site, termed oriT. oriT isusually located at or close to the end of the tra gene cluster,and the tra genes are organized so that they are transmittedlate in the conjugation process. Since our first attempt to iden-tify the oriT site of Rts1 by sequence similarity to known oriTsequences of other conjugative plasmids (39) was unsuccessful,we tried to identify the oriT region of Rts1 by screening thegenomic DNA library by the mobilization assay as described inMaterials and Methods. The principle of this approach is thatwhen the oriT region is cloned into a nonmobilizable plasmid,pUC18, the recombinant plasmid can be mobilized by cohab-itation of the parent plasmid (64).

By this screening, we obtained 20 ampicillin-resistant clones.Except for one clone that contained the entire IS611(B), 19clones were mapped to one of four regions (Fig. 2): positions2145 to 6073 (region I), 56019 to 57984 (region II), 110435 to111997 (region III), and 186537 to 193835 (region IV). In thecases of clones derived from regions I, II, and III, kanamycinresistance, a marker of the helper Rts1 plasmid, was alwayscotransferred with ampicillin resistance (Fig. 2A), implyingthat they were probably transferred to the recipient cells bycointegration into the helper Rts1 plasmid. In contrast, trans-fer of the clones derived from region IV was accomplishedindependently with the helper plasmid, indicating that regionIV contains the true oriT site of Rts1. The region was in factlocated in close proximity to the transfer region.

The minimum region common to all the nine clones mappedto region IV, named region T2 (Fig. 2B), is 455 bp in size andlocated at positions 188398 to 188852. When the DNA frag-ment corresponding to region T2 was cloned into pUC18, therecombinant plasmid was mobilized, but plasmids containingfragments outside of T2 were not (Fig. 2A and 2B). This resultconfirmed that the oriT site of Rts1 is located in region T2.Region T2 roughly corresponds to the intergenic region be-tween ORF251 and ORF252 and contains three inverted re-peats. This somewhat resembles the common structure for oriTsequences (64).

Replication and maintenance systems. Rts1 contains onereplicon, consisting of the repA gene (ORF001), encoding areplication initiator protein, and the ori segment, containingseveral sequence elements characteristic of plasmid repliconsthat employ the iteron-based replication initiation and controlmechanism (10, 31, 34, 67). The minimum segment for repli-cation initiation defined by Itoh et al. (31), which containsduplicated dnaA boxes, four GATC sequences, and five 19-bpiterons, spans from position 51 to 238.

In Rts1, no partitioning system for stable maintenance wasidentified before, but a system highly similar to the parABSpartitioning system of plasmid P1 was identified. ORF293 andORF294 are highly homologous to ParA and ParB of P1,respectively, and are arranged in the same order as in P1 (1).Furthermore, a parS-like structure containing three copies ofrepeated sequence of 7 bp and an integration host factor bind-

FIG. 2. Identification of the oriT locus of Rts1. (A) Summary of oriT screening. Clones obtained in the oriT screening of the random shotgunlibrary of the Rts1 genome are listed, and their map positions and kanamycin sensitivities are presented. All the clones were derived from one ofthe four regions, I to IV, but the clones derived from regions I, II, and III were always cotransferred with kanamycin resistance, a marker of helperplasmid Rts1. (B) Map positions of the clones derived from region IV and those prepared by PCR. The T2 segment represents the minimumconsensus of the eight random clones obtained in the oriT screening. The T2 segment was able to mobilized by Rts1, but the segments outside ofT2 were not (O1, O2, and O5), indicating that the oriT locus of Rts1 is located in the T2 segment.

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ing motif were identified just downstream of ORF294 (11, 17).Although two additional ORFs, ORF190 and ORF273, alsoexhibited weak similarities to ParB of P1, they are not accom-panied by parA-like genes. Rts1 contains a proteic killer systemencoded by higBA (ORF261 and ORF262), which functions asa plasmid maintenance system (61, 62). The tdi locus could alsobe involved in the maintenance of Rts1 (48, 55). Thus, thestable maintenance of Rts1 is achieved by multiple systems.

Large segment duplication on the Rts1 genome. By examin-ing the sequence similarity among all the gene products ofRts1, we identified 33 pairs of homologous genes which werelocated on the first 100-kb region of the Rts1 genome (Fig. 3).Genes comprising each pair are separately located in the firstand the second halves of the 100-kb region, and the gene orderis completely conserved. This finding indicates that the 100-kbregion was created by a large segment duplication. Homologyobserved in each pair of genes is, however, not so remarkable(up to 65.6% amino acid sequence identity), implying that eachsubregion has undergone significant sequence diversificationafter duplication, and each pair of genes may have functionallydiverged. In addition, the two subregions contain different setsof IS or transposon elements, and several genes or gene clus-ters seem to have been deleted or replaced after the duplica-tion.

Genes on the duplicated segments. It is noteworthy that theduplicated segments encode many genes related to variouskinds of DNA metabolisms, such as repair, recombination,restriction/modification, and replication (Fig. 1). Among these,the two copies of tus homologues (ORF015 and ORF091) areof particular interest, because no plasmid-borne tus gene hasbeen reported so far, except that Koch et al. (35) recentlyidentified a tus-like gene from R394, a close relative of Rts1.Tus dictates the arrest of replication progression by binding toa set of ter sequences (21, 22, 26) and plays a crucial role inreplication termination of chromosomes (5, 23). Tus is alsonecessary for efficient replication termination in some plas-mids, such as R6K and R1 (26, 37). These plasmid genomescontain ter consensus sequences but not the tus genes. Instead,the host-encoded Tus protein participates in the replicationtermination of these plasmids.

ORF015 and ORF091 of Rts1 both exhibit significant se-quence similarity to the enterobacterial Tus proteins through-out the entire molecules. The sequences known to be essentialfor the Tus functions are well conserved (33), implying thatboth or either ORF015 and ORF091 encode Rts1-specific Tusproteins involved in the replication termination of Rts1. Be-cause the 11-bp core sequence of the ter consensus (TGTTGTAACTA) is absent on the Rts1 genome, Rts1 Tus homo-logues must recognize different sequences.

ORF031 and ORF116 are highly homologous to the E. coliSSB protein (70% amino acid sequence identity over 147 res-idues and 84% over 113 residues, respectively). The ability ofRts1 to rescue the replication defect of an E. coli ssb mutant(18) could be explained by the presence of these ssb homo-logues. Rts1 contains another ssb homologue (ORF222) in theregion between the two transfer gene clusters, but it is verydistantly related to E. coli SSB (32% identity over 95 residues)and has an extremely long C-terminal tail, suggesting that ithas some distinct function. ORF007/ORF008 and ORF079/ORF080 both encode UmuDC homologues, which mediate

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error-prone repair in the SOS repair system (51, 63). AlthoughORF008 is C-terminally truncated by the insertion of Tn6901,the other set of genes (ORF079/ORF080) appears to be intactand is probably responsible for the recently identified umu-complementing activity of Rts1 (35).

ORF053 and ORF131 are homologous to Dam methylase,which plays important roles in the initiation of replication andthe sequestration of newly replicated oriC segments to the cellmembrane (40). Both ORFs have been confirmed to possessDam methylase activity by the SeqA focus formation experi-ment with the dam null mutant KA468 (24; T. Onogi and S.Hiraga, personal communication). Homologues of the betaand theta subunits of DNA polymerase III holoenzyme arealso encoded (ORF050 and ORF081, respectively).

The gene for PvuRts1I, a T-even phage restriction enzyme,also resided in this region (corresponding to ORF063). In aprevious study, an ORF encoding 290 amino acids was identi-fied upstream of the PvuRts1I gene, but its protection activityrelative to PvuRts1I was not detected (32). In this study, how-ever, this ORF (ORF062) was found to actually encode alarger protein of 593 amino acids homologous to TraG1, aputative restriction methylase of plasmid R478. The functionof this ORF needs to be reexamined with the possibility that itmay have protection activity against PvuRts1I.

Genome organization and evolution of Rts1. When the ge-nome organization of Rts1 is overviewed, the genome can beconsidered to be composed of four modules (Fig. 4), two mod-ules created by duplication (M1a and M1b), a transfer region-containing module (M2), and a module containing the genes

required for replication and stable maintenance of Rts1 (M3).As for the large segment duplication, some site-specific recom-bination system might be involved in the event, because all theclones that formed cointegrates with Rts1 in the oriT screeningwere derived from three regions (regions I, II, and III), eachcorresponding to the predicted boundary of the duplicatedsegments. Since the oriT screening was performed in the recAmutant background, some site-specific recombination mustmediate cointegrate formation. Rts1 encodes several genesrelated to the recombination function, and some of them mightparticipate in cointegrate formation and segment duplication.

The boundary between the M2 and M3 modules is not pre-cisely defined but seems to lie somewhere between oriT andhigAB. It is noteworthy that Rts1 contains many homologues ofthe S. enterica serovar Typhi plasmid R27 genes. Among these,26 exhibited highest similarities to the R27 genes in the ho-mology search. Most of them resided on the M2 module (19out of 26 genes). Rts1 and R27 in fact share a temperature-sensitive phenotype in conjugation transfer, but the high se-quence similarity observed between the two plasmids is notlimited to the genes for conjugation. This suggests that the M2module has evolved from an ancestor common to that of R27.The similarity between the replication initiation systems ofRts1 and P1 had been noticed as well (2, 3, 34). It is now clearthat their partitioning systems are also highly homologous.This finding further supports the evolutionary relationship ofthe two plasmids.

Concluding remarks. Determination of the complete se-quence of the large conjugative plasmid Rts1 provides severallines of important information on plasmid biology. All thegenes potentially related to the conjugation function have beenidentified, which will facilitate comparative and experimentalapproaches to the full understanding of bacterial conjugationsystems. An unexpected finding is the presence of multiplegenes related to various types of DNA metabolism. They in-clude plasmid-encoded tus homologues. The analysis of thegenome organization of Rts1 provided a prominent example ofthe modular evolution of a large plasmid genome. The ex-change or acquisition of modules from other replicons and thecapture of IS elements and transposons are deeply involved inthe generation of Rts1, as has been repeatedly discussed forplasmid genome evolution. In Rts1, however, another mecha-nism, the duplication of a large module and subsequent struc-tural and functional diversification of the duplicated modules,has also played an important role in genome evolution andgene repertoire expansion.

ACKNOWLEDGMENTS

We thank Y. Itoh, A. Tabuchi, K. Tanimoto, M. Tsuda, and T.Komano for useful suggestions; K. Sato, M. Takahashi, and S. Setsufor technical assistance; and Y. Hayashi for editorial assistance.

This work was supported by a Grant-in-aid for Scientific Researchfrom the Ministry of Education, Science, and Culture of Japan andgrants from the Yakult Foundation.

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