pUB6060: A Broad-Host-Range, DNA Polymerase-I-Independent ColE2-like Plasmid

Plasmid 45, 88–100 (2001) doi:10.1006/plas.2000.1511, available online at http://www.academicpress.com on

pUB6060: A Broad-Host-Range, DNA Polymerase-I-Independent ColE2-like Plasmid Matthew B. Avison,1 Timothy R. Walsh, and Peter M. Bennett Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom Received September 12, 2000; revised November 27, 2000 A ColE2-like, cryptic plasmid, pUB6060, of 5.8 kb has been found in a clinical isolate of Plesiomonas shigelloides. The complete sequence of pUB6060 has been determined and reveals a number of interesting features about the plasmid. The ColE2-like replication locus is linked to a functional ColE1-like mobilization locus. Replication is, unusually for ColE2 replicons, DNA polymerase-I-independent and may involve two, rather than the usual one, plasmid-encoded functions. Additionally, it carries two ORFs encoding products of unknown function. The pUB6060 replicon maintains a moderate plasmid copy number (10 per chromosome copy) and permits replication in diverse gram-negative bacteria. © 2001 Academic Press

Plesiomonas shigelloides is a gram-negative, oxidase-positive rod, assigned to the family Vibrionaceae (Schubert, 1984). The organism causes diarrheal disease in humans (Soweid and Clarkston, 1995) and increasing numbers of other types of infection are being attributed to the bacterium, including bacteremias, meningitis, and peritonitis (Billiet et al., 1989; Alcaniz et al., 1995). Immunosuppressed individuals show increased susceptibility to P. shigelloides infection and many patients infected extraintestinally die (Herrington et al., 1987; Abbott et al., 1991). Antibiotic-resistant strains of P. shigelloides have been reported, with resistance to aminoglycosides and penicillins being most common (Clark et al., 1990). We have recently demonstrated that several clinical and environmental isolates of P. shigelloides each produce a single constitutively expressed β-lactamase (Avison et al., 2000). Three enzyme types were identified, penicillinases, carbenicillinases and oxacillinases. However, only 50% of isolates tested produced a β-lactamase. Because of this, we speculated that some, if not all of the β-lac-

1 To whom correspondence and reprint requests should be addressed. Fax: (44) 117 9288274. E-mail: Matthewb. [email protected].

tamase genes had been acquired on transmissible elements. Accordingly, the presence of plasmids in β-lactamase-producing strains was investigated. A single small plasmid, designated pUB6060, was found in P. shigelloides isolate 11184. This isolate is of particular interest because it produces a class 2d oxacillinase (Bush et al., 1995; Avison et al., 2000), but was isolated in the Czech Republic in 1966, several years before oxacillin was first used there (Avison et al., 2000). Although plasmid pUB6060 does not encode the β-lactamase, it is still of interest because of its architecture and its replication, which are reported in this paper. MATERIALS AND METHODS Materials, Bacterial Strains, and DNA Manipulation Bacterial strains and plasmids used in this study are described in Table 1. Bacterial growth media were from Oxoid plc. (Basingstoke, UK). All general chemicals were obtained from Sigma Chemical Company or BDH (both of Poole, UK). Enzymes for DNA manipulation were from Life Technologies, Inc.; DNA manipulation protocols were as described previously (Sambrook et al., 1989). All oligonucleotide primers were obtained from Sigma-Genosys Ltd. (Pampisford, UK). 88

0147-619X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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pUB6060, AN UNUSUAL ColE2 PLASMID TABLE 1. Bacterial Strainsa and Plasmids Used in This Study Strain/plasmid name E. coli DH5α E. coli JC3272 E. coli UB2272 Ps. aeruginosa 55 A. hydrophila T429125 P. shigelloides 11184 P. shigelloides 8085 S. maltophilia J675Ib R388 pK18 pUC18 pSU18 pUB6060 pUB6061 pUB6062 pUB6063 pUB6064 pUB6065

Relevant genotype/features

Source/reference

supE44, ∆lacU169 (φ80 lacZ ∆M15), hsdR17, Hanahan (1983) recA1, endA1, gyrA96, relA1, thi Jenkins and Bennett (1976) his, trp, lys, lac, rpsL, rpoB, StrR JC3272 polA Jenkins and Bennett (1976) Clinical isolate Bristol Royal Infirmary, Bristol, UK Clinical isolate Royal Hobart Hospital, Tasmania, Australia Clinical isolate Avison et al., (2000) Clinical isolate Avison et al., (2000) Clinical isolate Bristol Royal Infirmary, Bristol, UK IncW, tra+, TpR SulR Albiger et al., (2000) KmR, lacZ′, pMB1 origin Pridmore (1987) AmpR, lacZ′, pMB1 origin Lin-Chao et al., (1992) ChlR, lacZ′, p15A origin Martinez et al., (1988) From P. shigelloides 11184 This study 2.8-kb EcoRI fragment of pUB6060 in pK18 This study 2.2-kb EcoRI fragment of pUB6060 in pK18 This study 0.8-kb EcoRI fragment of pUB6060 in pK18 This study 2.2-kb EcoRI fragment of pUB6060 in pUC18 This study 2.2-kb EcoRI fragment of pUB6060 in pSU18 This study

a

Genus abbreviations: E., Escherichia; Ps., Pseudomonas; A., Aeromonas; P., Plesiomonas; S., Stenotrophomonas.

Isolation and Sequencing of Plasmid pUB6060 P. shigelloides isolate 11184 was cultured in Nutrient Broth at 37°C with shaking for 18 h. Plasmid pUB6060 was isolated from 1.5 ml culture using a Hybaid Plasmid Recovery kit (Hybaid, Teddington, UK) according to the manufacturer’s instructions. Plasmid DNA was digested with EcoRI to yield three fragments (2.8, 2.2, and 0.8 kb), which were cloned into dephosphorylated EcoRI-digested plasmid vector pK18 (Pridmore, 1987). Escherichia coli strain DH5α (Hanahan, 1983) was transformed to kanamycin resistance with the ligation mix; transformants were selected on Nutrient Agar containing kanamycin (30 mg L−1), IPTG (1 mM), and X-GAL (40 mg L−1). A set of white colonies was chosen and the plasmid in each transformant was analyzed by colony PCR (Jones et al., 1994) using primers based on the multiple cloning site of pK18 (MCS+, 5′CAGCTGGCACGACAGGTT, and MCS−, 5′GTTGGCAGTCACGACGTT), to determine the size of the insert carried by each plasmid recombinant. Three distinct plasmid recombinant types were identified (pUB6061–6063 are typi-

cal), each of which carried one of the three EcoRI fragments of pUB6060 (2.8, 2.2, and 0.8 kb, respectively). The inserts were sequenced on both strands with an ABI PRISM 377 automated sequencer using dye termination chemistry, following the manufacturer’s protocol, and a primer-walking approach with a custom primer designed every 500 bp walked. With each recombinant, the first round of sequencing used MCS+ or MCS− primers. Finally, purified plasmid pUB6060 was used directly as template DNA for sequencing across each of the three EcoRI sites, with primers based on sequences at the ends of the three cloned fragments. This allowed the three fragments to be correctly oriented on pUB6060 and confirmed that no small EcoRI fragments had been missed. The sequence obtained for pUB6060 has been deposited with the EMBL database under Accession No. AJ249644. Bacterial Mating Experiments An overnight culture of each bacterium to be mated was prepared using Nutrient Broth. One hundred microliters of each culture was dis-

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cretely mixed on Nutrient Agar containing no antibiotics and incubated overnight at 37°C. A loopful of the mixed growth was then streaked on Nutrient Agar containing the appropriate selective agents and incubated overnight at 37°C. RESULTS AND DISCUSSION General Features of pUB6060 Plasmid pUB6060 is a cryptic element of 5804 bp (Fig. 1) with an overall G + C content of 46%, which is somewhat lower than the average 51% G + C content of the P. shigelloides genome (Schubert, 1984). The zero coordinate for the plasmid was placed at the start of the sole

BglII site (Fig. 1). Eight putative open reading frames (ORFs) have been located; each follows a correctly positioned, credible ribosome recognition sequence. pUB6060 Encodes a ColE1-like Mobilization System Encoded between plasmid coordinates 1472 and 3521 is a sequence, mob (Figs. 1 and 2A), which shows significant similarity to the mobilization locus of the E. coli plasmid ColE1 (Boyd et al., 1989). The region encodes four putative peptides, MobA, 528 amino acids, MobB, 161 amino acids, MobC, 108 amino acids, and

FIG. 1. Circular map of pUB6060. The zero co-ordinate of the plasmid was located at the beginning of the sole BglII site. Cut sites for a variety of common restriction enzymes are labeled. The AT-rich region of the plasmid is marked. The reading frame orientations of the mobCABD, repAB, and orf1,2 operons are defined with arrows.

pUB6060, AN UNUSUAL ColE2 PLASMID

FIG. 2.

Continued

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FIG. 2. Sequence of the insert of pUB6061 containing the mob region of pUB6060. In (A), the sequence is of the 2.8-kb EcoRI fragment of pUB6060. Open reading frames are marked with arrows (dark gray, mobC; white, mobA; light gray, mobB; black, mobD). Other regions of interest are underlined and labeled. The coordinates of pUB6060 are used. In (B), the putative pUB6060 oriT locus is aligned with that from plasmid ColE1 (Boyd et al., 1989). Mismatches are shaded.

MobD, 77 amino acids (Fig. 2A). Percentages of amino acid similarities of the proteins with their appropriate ColE1 equivalents are, for MobA, 44%, for MobB, 33%, for MobC, 47% and for MobD, 39%.

The mob genes form an operon, mobCABD, and, as at the ColE1 mobilization locus (Boyd et al., 1989), the genes overlap one another (Fig. 2A). No convincing promoter, based on the E. coli σ70 consensus (Record et al., 1996), could

pUB6060, AN UNUSUAL ColE2 PLASMID

be identified from visual analysis of the sequence upstream of mobC. A possible −10 box, TAGCAT, is located at nucleotide positions 1563–1568, but this is not associated with a consensus −35 sequence. The putative nick site, oriT, of pUB6060 is between nucleotides G and C at plasmid coordinates 1487 and 1488, 130 nucleotides upstream from the translation start of mobC (Fig. 2A). The 25nucleotide pUB6060 oriT locus (coordinates 1474–1498) shares 68% identity with the equivalent region of ColE1 (Boyd et al., 1989) (Fig. 2B). Cloning vector pK18, encoding resistance to kanamycin, is a nonmobilizable plasmid lacking mob genes and an oriT (Pridmore, 1987). The pK18 derivative pUB6061 contains the 2.8-kb EcoRI fragment of pUB6060, defined by the sites at pUB6060 coordinates 1298–1303 and 4113–4118, which encodes all four mob genes and contains oriT (Figs. 1 and 2A). Hence, to determine if the mob locus of pUB6060 is functional, mobilization of pUB6061 by the conjugative IncW plasmid R388, which encodes resistance to trimethoprim and mobilizes other plasmids with ColE1-like Mob functions (Albiger et al., 2000), was investigated. R388 was introduced into the nalidixic acid-resistant E. coli strain, DH5α carrying pUB6061, by conjugation. Transconjugants were resistant to naladixic acid, kanamycin (each at 30 mg L−1), and trimethoprim (50 mg L−1). Several were purified and crossed with the streptomycin-resistant E. coli strain JC3272 (Jenkins and Bennett, 1976; Tavakoli et al., 2000). Transconjugants resistant to streptomycin and kanamycin and carrying pUB6061 were readily obtained in each case. In contrast, when pK18 was substituted for pUB6061, no kanamycin-resistant transconjugants were obtained. In both crosses, R388 was transferred independently at high frequency. These results confirm that the fragment of pUB6060 cloned in pUB6061 encodes a functional mobilization system. The Replication System of pUB6060 Is ColE2like Located between plasmid coordinates 4231 and 5664 are two ORFs that overlap by 1 nucleotide, not including the stop codon, which are predicted to form a two-gene operon, repAB

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(Figs. 1 and 3A). The first of these is located over coordinates 4231–5124 and its putative product, RepA, is 297 amino acids long (Fig. 3A) and shows greatest similarity (60%) to the Rep protein of the E. coli plasmid ColE7-K317 (Hiraga et al., 1994). High levels of similarity to other Rep proteins of ColE2-related plasmids are also seen. Approximately 200 nucleotides upstream of repA is what we would predict to be a good promoter, with −35 and −10 boxes of GTGACA and TAGTAT, respectively, separated by the canonical 17 nucleotides (plasmid coordinates 3984–4012) (Fig. 2A) (Record et al., 1996). Transcription from this point would give a transcript leader of about 210 nucleotides. Within this region, there are candidates for promoters that would direct transcription of an antisense RNA, but none are wholly convincing. The best of these is at coordinates 4144–4174, which would generate a transcript with a sequence of approximately 150 nucleotides complementary to leader sequences on the repAB transcript (Fig. 3A). At coordinates 4113 to 4152 there is a potential stem–loop structure containing the sequence TTGGCGGAA at coordinates 4130–4138. This sequence is often found in the loop regions of antisense RNA molecules (del Solar et al., 1998). It could be that this region represents an antisense RNA system for regulating repAB gene expression. The second gene, repB, encompasses coordinates 5111–5669, encoding a protein of 182 amino acids (Fig. 3A) that shows 39% similarity to Soj from Bacillus cereus (Ogasawra and Yoshikawa, 1992; Ireton et al., 1994; Gal-Mor et al., 1998). Soj is believed to be involved in chromosome segregation during sporulation (Ireton et al., 1994; Sharpe and Errington, 1996). RepB is also similar to other members of the ParA family of proteins that are often responsible for active partitioning of diverse bacterial plasmids (Motallebi-Veshareh et al., 1990). These observed similarities to known partition/segregation proteins, together with the gene array, suggest that RepB may be involved in pUB6060 segregation. Replication of ColE2-like plasmids is reportedly DNA polymerase-I-dependent (Espinosa et al., 2000). This aspect of pUB6060 replication was tested as follows. Plasmid recombinant

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FIG. 3.

Continued

pUB6060, AN UNUSUAL ColE2 PLASMID

95

FIG. 3. Sequence of the insert of pUB6062 containing the rep region of pUB6060. In (A), the sequence is of the 2.2-kb EcoRI fragment of pUB6060. Open reading frames are marked with arrows (dark gray, repA; white, repB; light gray, orf1 (fragment)). Other regions of interest are underlined and labeled. The coordinates of pUB6060 are used. Note the BglII site at the zero coordinate. In (B), the putative oriV1 and oriV2 of pUB6060 are aligned with a series of oriV sequences from other ColE2-like plasmids (Espinosa et al., 2000). Mismatches compared to ColE2–P9 (Hiraga et al., 1994; Espinosa et al., 2000) are shaded, and gaps (−) are inserted as appropriate to facilitate the alignment. The AGA priming site is highlighted in bold.

pUB6062 comprises pK18 and the 2.2-kb EcoRI fragment of pUB6060 that encodes both RepA and RepB (Figs. 1 and 3A). The cloning vector, pK18, has a ColE1-like replication system (Pridmore, 1987) that is DNA polymeraseI-dependent and cannot establish in the E. coli polA derivative UB2272 (Jenkins and Bennett, 1976; Tavakoli et al., 2000). Therefore, we investigated if pUB6062 could transform UB2272 to kanamycin resistance. As control, both pK18 and pUB6062 were used to transform JC3272, the polA+ parent strain (Jenkins and Bennett, 1976). When JC3272 was the recipient, kanamycin-resistant clones were obtained with both pK18 and pUB6062 at similar frequencies, as expected; however, when the recipient was UB2272, kanamycin-resistant transformants were obtained only with pUB6062. Furthermore, pUB6062 was recovered from kanamycin-resistant colonies, confirming that resistance was due to maintenance of the plasmid. This result tells us two things: (a) the pUB6060 sequence on pUB6062 encodes a

replication system, since the ColE1-like replication system of pK18 cannot support plasmid replication in UB2272. This point suggests that RepA is a functional primase of the ColE2 family of primases (Takechi et al., 1995). (b) The pUB6060 replication system is DNA polymerase-I-independent. The ability of pUB6062 to establish and be maintained in UB2272 must reflect the function of repA and/or repB, since these are the only intact genes acquired from pUB6060 (Figs. 1 and 3A). The independence of pUB6060 replication from DNA polymerase I might reflect the activity of RepB, since DNA polymerase-I-dependent ColE2-like plasmids appear only to have RepA homologues (Hiraga and Itoh, 1994; del Solar et al., 1998; Espinosa et al., 2000). If this were proved to be the case, the involvement of two plasmid-encoded functions in plasmid replication would be a departure for a ColE2-like replicon, among which a single Rep protein is the norm (Hiraga et al., 1994; del Solar et al., 1998). More generally, however, involvement of two plasmid-encoded

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functions in plasmid replication is not uncommon (del Solar et al., 1998; Espinosa et al., 2000). The origins of replication, oriV, of ColE2like plasmids show a marked degree of sequence conservation (Fig. 3B) (del Solar et al., 1998; Espinosa et al., 2000). A sequence similar to these origins of replication, oriV1, is located immediately distal to repB, covering coordinates 5666–5685, and includes the invariant AGA priming site (Espinosa et al., 2000) at coordinates 5674–5676 (Figs. 3A and 3B). No other sequence strongly resembling a ColE2-like replication origin was identified on the pUB6062 insert. Neither is there any evidence for an iteron-based replication origin (Espinosa et al., 2000). A second ori-like sequence, oriV2, is found on pUB6060 at plasmid coordinates 1403–1422 (Fig. 2A), but this is not present on the insert of pUB6062, which is clearly able to replicate in UB2272. The evidence suggests that pUB6060 has a ColE2-like replicon that has evolved to be DNA polymerase-I-independent. How this has been achieved is unknown, but involvement of RepB is a possibility. Targeted disruption of these loci will enable a more complete analysis of the replication of pUB6060. Copy Number of pUB6060 The replication region of pUB6060 incorporating repAB and oriV1 was subcloned from pUB6062 into pUC18 on the 2.2-kb EcoRI fragment. In the new construct, pUB6064, the pUB6060 replication region was linked to a TEM β-lactamase gene, expression of which is constitutive and directly proportional to gene copy number. pUB6064 was introduced into E. coli JC3272 (polA+) and UB2272 (polA) (Jenkins and Bennett, 1976) by transformation, with selection for ampicillin resistance. The β-lactamase activity in extracts of JC3272:pUB6064 was fivefold greater than that in extracts of UB2272:pUB6064 and equal to that in extracts of JC3272:pUC18. These data confirm that in the DNA polymerase-I-proficient strain, pUB6064 replicates from its ColE1-like origin, while in the DNA polymerase-I-deficient strain replication is switched to the ColE2 mode, producing a fivefold decrease in plasmid copy number.

Given that the copy number of pUC18 has been determined as approximately 50 copies per chromosome during growth at 37°C (Lin-Chao et al., 1992), then the copy number of the pUB6060 replicon is approximately 10 per chromosome copy. Host Range of the pUB6060 Replicon The 2.2-kb EcoRI fragment from pUB6062, encoding the pUB6060 replicon, was introduced into the EcoRI site of pSU18, a derivative of the p15A-based cloning vector pACYC184 (Martinez et al., 1988), to give construct pUB6065. This rearrangement linked the replication region of pUB6060 to a chloramphenicol resistance determinant originating on Tn9 (Martinez et al., 1988). Next, recombinant plasmids pUB6062 (Kmr), pUB6064 (Apr), or pUB6065 (Cmr) were used to transform a variety of gram-negative bacteria to kanamycin, ampicillin, or chloramphenicol resistance, depending on the drug susceptibility status of the potential recipient. All bacterial strains tested were transformed by at least one of the plasmid recombinants (Table 2). In parallel, pK18, pUC18, or pSU18 was used to transform the same set of bacteria, to determine which, if any, of the cloning vectors could establish in the new hosts. The results indicate that the pUB6060 replicon can function in pylogenetically diverse bacteria and, therefore, pUB6060 can be considered a broad-host-range plasmid. Its suitability for cloning vector construction is obvious. Since the modes of replication of p15A and ColE1 are very similar, though the plasmids are compatible, in that both use E. coli RNA polymerase, RNase H, and DNA polymerase I for replication (Selzer et al., 1983), it is possible that the reason for more efficient maintenance of pUB6062/pUB6065 compared to pK18/pSU18 (Table 2) is due to stimulation of ColE1/p15A-type replication by a additional feature encoded on the 2.2-kb EcoRI fragment of pUB6060. This needs to be tested in more detail by removing the native origins from pUB6062/pUB6065. pUB6060 Encodes Two Novel Functions Beyond the putative oriV1 downstream of repB are two ORFs (Fig. 1) that show no simi-

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pUB6060, AN UNUSUAL ColE2 PLASMID TABLE 2. The Ability of the pUB6060 Replicon to be Maintained in Diverse Gram-Negative Bacteria Vectora Organism

Ps. aeruginosa 55 A. hydrophila T429125 S. maltophilia J675Ib P. shigelloides 8085

pUC18

pSU18

pK18

pUB6064

pUB6065

pUB6062

RESb RES RES 258

10c 0 0 237

64 18 RES 274

RES RES RES 284

228 185 129 218

287 162 RES 269

a Selection was with ampicillin (50 mg/L), chloramphenicol (30 mg/L), or kanamycin (30 mg/L), respectively, for pUC18/pUB6064, pSU18/pUB6065, and pK18/pUB6062. b RES, The untransformed organism was resistant to this antibiotic at the concentration used. c The figures represent the numbers of resistant colonies formed per 106 cells, following their transformation with 10 ng of DNA using a Gene pulsar (Bio-Rad, München, Germany) with a single pulse at 2.5 V, 25 µF, 200 Ω.

larity to any gene in the databases. The first, orf1, is located at plasmid coordinates 147–689 and encodes a peptide of 180 amino acids. The termination codon of orf1 overlaps the initiation codon of the second, orf2, by one nucleotide, the second putative gene, orf2, being located over coordinates 689–1387 and potentially encoding a peptide of 232 amino acids. The overlapping nature of these putative genes suggests that they form an operon. Possible promoter sequences are located upstream of orf1, between it and oriV1 (Figs. 1 and 3A), but none shows such a high degree of similarity to the E. coli σ70 consensus (Record et al., 1996) that it can be assigned promoter status with confidence. Standard amino acid sequence analysis of the predicted products of orf1 and orf2 give no clues as to their functions. At this time, therefore, pUB6060 is cryptic, i.e., it confers no recognizable phenotype on its host. What use it is to its host, if any, is not known. Nonetheless, it is a remarkably stable plasmid. P. shigelloides 11184 was isolated in 1966 in Czechoslovakia (Avison et al., 2000). No obvious plasmid stability functions (Gerdes et al., 2000) have been identified, although the products of orf1 and orf2 may fulfil this function. Many ColE2-like plasmids encode colicins (Gerdes et al., 2000). Accordingly, bacteriocin production/immunity encoded by orf1 and orf2 was considered a distinct possibility. No such activity connected with pUB6060 was identified, however. P. shigelloides 11184 does not

produce a compound that is toxic to laboratory strains of E. coli or to other strains of P. shigelloides from various geographical locations (Avison et al., 2000) (data not shown). It is still possible, of course, that orf1 and orf2 encode a bacteriocin system toxic for bacterial species other than those investigated. Construction of pUB6060 The base composition of pUB6060 indicates that it is probably a hybrid structure assembled from DNA from at least two sources. The region between plasmid coordinates 1255 and 5003 (Fig. 1), encompassing mob and repA, has an average GC base composition of 54% (range 46–61%), consistent with coevolution of these functions, probably within the Enterobacteriaceae, where ColE2-like plasmids are common (Hiraga et al., 1994). In contrast, the remainder of the plasmid (approximately 2 kb), responsible for repB, orf1, and orf2 (Fig. 1), has an average GC composition of just 35.5% (range 25–46%), indicating a different heritage from that of the mob/repA genes. A bacterial sequence with such a low GC content is more typical of a gram-positive organism than a gramnegative one, although Proteus spp. have genomes with average GC contents of 38–40% (Marmur et al., 1963). One curious feature is that the putative DNA polymerase-I-independent replication origin, oriV1, is located at the end of repB, centered on coordinate 5675, within the region of low GC

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composition (Fig. 3A), while the putative primase gene has an average GC content of about 55%. This is suggestive of different evolutionary origins for what are now fundamentally integrated functions. It is interesting to find, however, that the replication origin found immediately downstream of repA in many ColE2-like plasmids (Fig. 4) (Hiraga et al., 1994; Espinosa et al., 2000) appears to have been retained in pUB6060 (oriV2, centred at coordinate 1414) (Fig. 2A). This arrangement implies that the AT-rich region containing repB, oriV1, orf1, and orf2 has been inserted into a standard ColE2-type plasmid between repA and oriV2 (Fig. 4). It is not known if oriV2 is functional in pUB6060. The region of pUB6060 encoding mob and repA is also found in another plasmid, pMUT2 (EMBL Accession No. A95448). The level of DNA sequence identity is approximately 90% across the 4.0-kb region, which contains the same oriT, mob, repA array.

There has also been an insertion of an AT-rich sequence after repA in pMUT2 (Fig. 4). In this case, however, the AT-rich region shares no significant homology with that of pUB6060; in particular, there is no repB-like gene and no additional oriV. It is not known whether pMUT2 requires DNA polymerase I for replication. The base compositions of the two sections of pUB6060, 54% GC and 35.5% GC, compare with 51% GC for the chromosome of P. shigelloides (Schubert, 1984), suggesting that the organism in which it was found does not represent the genus/genera where it evolved. Rather, the evidence would suggest a heritage more typical of some of the Enterobacteriaceae, with subsequent mobilization into P. shigelloides. The plasmid pMUT2, which has a similar architecture to pUB6060, was found in E. coli strain DSM 6601 (U. Sonnenborn and H. Proppert, unpublished data). It is highly probable, therefore, that P. shigelloides is able to share DNA

FIG. 4. Arrangement of the repA/oriV regions of pUB6060 and pMUT2. The usual arrangement of repA–oriV found in ColE2-like plasmids (Hiraga et al., 1994) has been disrupted by the insertion of an AT-rich region in pUB6060 and pMUT2, though the insertion in each case is different. In pUB6060, the insertion introduces repB and an additional oriV, so the original ColE2 oriV (white) has been renamed oriV2.

pUB6060, AN UNUSUAL ColE2 PLASMID

with members of the Enterobacteriaceae. From this aspect, it is interesting to note that, on the basis of rRNA analysis, it has been suggested that P. shigelloides be moved from the Vibrionaceae into the Enterobacteriaceae (East, et al., 1992; Martinez-Murcia et al., 1992 Ruimy et al., 1994). ACKNOWLEDGMENTS This work was supported by grants from the Wellcome Trust and the British Society for Antimicrobial Chemotherapy. We thank Dr. Eva Aldová, National Institute of Public Health, ˇ Srobárova, Czech Republic, for donating the P. shigelloides isolate and Rhiannon Murry, Department of Biochemistry, University of Bristol, for DNA sequencing. REFERENCES Abbott, S. L., Kokka, R. P., and Janda, J. M. (1991). Laboratory investigation on the low pathogenic potential of Plesiomonas shigelloides. J. Clin. Microbiol. 29, 148–153. Albiger, B., Comanducci, A., Dodd, H. M., Tavakoli, N., Lett, M.-C., and Bennett, P. M. (2000). pUB2380: Characterization of a ColD-like resistance plasmid. Plasmid 44, 54–65. Alcaniz, J. P., de Cuenca, M. B., Gomez, R. M., Martinez, A. J. L., and Garcia, J. (1995). Spontaneous bacterial peritonitis due to Plesiomonas shigelloides. Am. J. Gastroenterol. 90, 1529–1530. Avison, M. B., Bennett, P. M., and Walsh, T. R. (2000). βLactamase expression in Plesiomonas shigelloides. J. Antimicrob. Chemother. 45, 877–880. Billiet, J., Kuypers, S., Van Lierde, S., and Verhaegen, J. (1989). Plesiomonas shigelloides meningitis and septicaemia in a neonate: Report of a case and review of the literature. J. Infect. 19, 267–271. Boyd, A. C., Archer, J. A. K., and Sherratt, D. J. (1989). Characterization of the ColE1 mobilization region and its protein products. Mol. Gen. Genet. 217, 488–498. Bush, K., Jacoby, G. A., and Medeiros, A. A. (1995). A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39, 1211–1233. Clark, R. B., Lister, P. D., Arneson-Rotert, L., and Janda, J. M. (1990). In vitro susceptibilities of Plesiomonas shigelloides to 24 antibiotics and antibiotic-β-lactamase-inhibitor combinations. Antimicrob. Agents Chemother. 34, 159–160. del Solar, G., Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M., and Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microbiol. Mol. Biol. Rev. 62, 434–464. East, A. K., Allaway, D., and Collins, M. D. (1992). Analysis of the DNA encoding 23S rRNA and 16S-23S

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