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Isolation of a new insertion element of Yersinia intermedia closely related to remnants of mobile genetic elements present on Yersinia plasmids harboring the Yop virulon

Eckhard Strauch , Bernd Hoffmann , Gudrun Heins , Bernd Appel
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09399.x 37-44 First published online: 1 December 2000


A new insertion element present in two alleles, designated IS1635.1 and IS1635.2, was identified on a plasmid of a Yersinia intermedia strain by hybridization with the Yersinia enterocolitica pYV virulence plasmid. IS1635.1 and IS1635.2 are 861 bp long, carry imperfect inverted terminal repeats and possess a single open reading frame encoding a putative transposase of the IS6 family. A truncated IS1635 element is present immediately downstream of element IS1635.2. The capacity of the IS1635 elements to mediate transposition in Yersinia was demonstrated with a R6K-derived suicide vector, where a kanamycin resistance gene had been inserted between IS1635.1 and IS1635.2. Hybridization and sequence alignments showed that remnants of IS1635-like insertion elements harboring large deletions and point mutations are present on the Yop virulon harboring plasmids of pathogenic Yersinia strains. In a few cases, the IS1635 element has also been found on plasmids of apathogenic Yersinia strains.

  • IS1635
  • IS6 family
  • Distribution in Yersinia
  • pUT derived suicide vector

1 Introduction

The study of bacterial pathogenicity revealed a frequent association between insertion elements and many virulence functions and might indicate that these mobile genetic elements are involved in horizontal gene transfer of virulence genes [1]. In the genus Yersinia several insertion elements have also been found in association with various pathogenicity determinants. For instance, three IS3 elements are present in the high pathogenicity island (HPI) of Yersinia enterocolitica 0:8 strains [2], whereas an IS100 element is a component of the HPI of Yersinia pestis and Yersinia pseudotuberculosis[3]. Furthermore, an IS200-like element (IS1541) is integrated in multiple locations in the chromosome and another copy was found on the virulence plasmid pFRA of Y. pestis[4]. IS1541-like elements were also found in Y. pseudotuberculosis and in Y. enterocolitica[5]. The entire sequence of the Y. pestis plasmid pCD1 as well as the pYV plasmid of Y. enterocolitica, which encode the Yop virulon, revealed the presence of several other intact and partial insertion sequences scattered throughout the plasmid [6,7].

In this paper we describe a new insertion element present in two allelic conformations, which were designated IS1635.1 and IS1635.2. The elements were located on a plasmid (p29332) of the Yersinia intermedia strain 29332 and were identified by hybridization using the entire pYV virulence plasmid of Y. enterocolitica as a probe. The hybridization experiments were carried out in continuation of previous studies, in which we investigated if virulence genes of the pYV plasmid of Y. enterocolitica might be present on plasmids of Yersinia strains that do not belong to the pathogenic strains [8,9].

2 Materials and methods

2.1 Bacterial strains

Pathogenic strains used in this study were Y. enterocolitica 13169 serogroup 0:3, Y. enterocolitica 31084 serogroup 0:9, Y. enterocolitica H162/82 serogroup 0:8 and Y. pseudotuberculosis 29827 serogroup I [8]. Y. intermedia 29932 serogroup 0:4,33 was a foodborne isolate. The strains used for hybridization studies have been described in [8,9] and other apathogenic strains were obtained from Dr. Feuerpfeil, Umweltbundesamt Bad Elster, Germany. Y. enterocolitica H162/82 serogroup 0:8 was obtained from Dr. Aleksic, Institut für Hygiene, Hamburg, Germany.

2.2 Molecular biological techniques

Plasmid isolations were performed using an alkaline lysis method [10]. Genomic DNA of Yersinia strains was prepared as previously described [11]. Restriction enzyme analysis, ligations etc. were performed with commercially available enzymes according to the manufacturers’ recommendations. Hybridizations were carried out with fluorescein-labeled DNA probes as described previously [9]. The 6.99-kb EcoRI fragment of the plasmid p29932 carrying the IS1635 elements was cloned into pBluescript SK+ yielding plasmid pYep70. Both strands of the fragments were sequenced either by subcloning or using synthetic oligonucleotide primers. Sequencing reactions were carried out by using the Prism Big Dye™ FS Terminator Cycle Sequencing Ready Reaction kit, (PE Applied Biosystems, Weitersheim, Germany) and separated on an automated DNA sequencer (ABI 377). The sequences were analyzed with the Sequencing Analysis, Sequence Navigator and Auto Assembler software packages (PE Applied Biosystems), Mac Vector software (Oxford Molecular Group) and the Blast, FastA, Malign, Clustal programs of the HUSAR/GCG Package of the German Cancer Research Center, Heidelberg, Germany.

2.3 Amplification of DNA by PCR

The kanamycin resistance gene was amplified as a 1064-bp fragment using the vector pK18 as template [11]. A 444-bp PCR fluorescein-labeled product for detecting yopM was amplified with the primers YopMYe-S (5′-ATGGTGAACAGAGGGGAATGGC-3′) and YopMYe-AS (5′-CATAAATCGCAGTCAAGAAGGGC-3′) using the pYV plasmid of Y. enterocolitica 31084 as a template [7]. A 980-bp product of the mob region of the pUT vector was PCR-amplified using the primers R6Kmob2 (5′-ATTGTCACGCTCAAGCCCGTAG-3′) and R6Kmob3 (5′-CTTCTTCACTGTCCCTTATTCG-3′) [12]. An IS1635 probe with a size of 1360 bp was amplified using primers flanking IS1635.1 element (5′-CTTAGAGCTGCTCAACCCTG-3′ and 5′-CCATGCCGGCAGGACAAGAC-3′).

2.4 Construction of pYep86 and transposition experiments

Transposition assays were performed with the suicide vector pYep86, which was constructed as follows. The vector pUTmini-Tn5luxAB[13,14] was cleaved with SalI and religated, thus removing the Tn5 transposase and the luxAB cassette and a tetracycline resistance gene downstream of the luxAB genes. The vector pYep71 was generated by cleaving pYep70 with XbaI and religating, thus deleting a 1.6-kb fragment of the 6.99-kb p29332 insert and a short sequence of the MCS of pBluescript SK+. In the next step, ORF3 was partially removed by cleavage with BamHI, filled in with Klenow fragment, and a kanamycin resistance gene (see Section 2.3) was inserted. An NotI/EcoRI fragment of this construct harboring the IS1635 elements was ligated in the derivative of the pUT vector cut with NotI/EcoRI, and the resulting plasmid pYep86 (Fig. 4) conferring ampicillin resistance as well as kanamycin resistance was introduced into Escherichia coli S17-1(λpir). The identity of the construct was verified by partial sequencing. Mating experiments were performed on solid media by mixing the E. coli S17-1(λpir) [pYep86] donor strain with the Y. enterocolitica 29807 recipient according to standard procedures as described previously [11]. Selection for transconjugants of the recipients was done on Yersinia-selective agar containing cefsulodin-irgasan-novobiocin (CIN, Oxoid) and 100 μg ml−1 kanamycin.


Construction and restriction map for BglII, NcoI and PstI of suicide vector pYep86 (see Section 2.4). One BglII site and the NcoI site were introduced by inserting a PCR fragment carrying a kanamycin resistance gene (see Section 2.3). The gray shaded parts of the plasmid are derived from pUTmini-Tn5luxAB. Fragments I–IV, which were PCR-amplified in all transconjugants, are indicated.

2.5 Assignment of IS and nucleotide sequence accession number

Assignment of the designation IS1635 was made by Dr. E. Lederberg at the Plasmid Reference Center, Medical School of Stanford University. The nucleotide sequence reported here has been submitted to the EMBL databank under accession number Y18002.

3 Results

3.1 Isolation and characterization of the insertion elements IS1635.1 and IS1635.2

The plasmid of Y. intermedia strain 29932 with a size of approximately 40 kb was digested with EcoRI and subjected to hybridizations using a probe consisting of the complete pYV plasmid of the Y. enterocolitica strain 13169 (serogroup 0:3) [9]. Three EcoRI fragments of the plasmid of Y. intermedia 29332 hybridized to the pYV plasmid probe (Fig. 1A, lane 3). Two of these fragments were shown to hybridize to a probe with homology to the replication region of the pYV plasmid [9], while an EcoRI fragment with a size of 6.99 kb hybridized to a fragment of the pYV plasmid carrying the yopM region (see below). The same hybridization pattern was obtained using the pYV plasmid of a Y. enterocolitica serogroup 0:9 (strain 31084) as a probe (data not shown).


Identification and physical map of a fragment of plasmid p29932 carrying the IS1635 elements. A: Gel electrophoresis (left panel) and Southern-blot hybridization (right panel) using the entire pYV plasmid of Y. enterocolitica 0:3 as a probe. Lane 1, BamHI digest of pYV plasmid of Y. enterocolitica 31084 (0:9); lane 2, BamHI digest of pYV plasmid of Y. enterocolitica 13169 (0:3); lane 3, EcoRI digest of plasmid p29332 of Y. intermedia 29332; lane 4, EcoRI digest of pYep70; lanes 5–8, PCR fragments from nucleotides 1–1629 (lane 5), 1511–2871 (lane 6), 2898–3825 (lane 7) and 5314–6994 (lane 8) of the 6.99-kb EcoRI-fragment of p29931; see also bottom of B. B: Physical map of the 6.99-kb EcoRI-fragment of plasmid p29932 of Y. intermedia inserted into the vector pBluescript SK+ giving recombinant plasmid pYep70. ORFs are indicated by solid arrows; IR=inverted repeat (see also Fig. 3). Horizontal bars below the restriction map represent PCR fragments used to determine the regions hybridizing to the pYV plasmids of Y. enterocolitica strains 13169 and 31084 (+=positive hybridization; −=no hybridization). Numbers indicate location in the sequenced fragment (see also A, lanes 5–8).

Sequence analysis of the 6994-bp EcoRI fragment revealed the presence of two complete putative insertion elements (IS1635.1 and IS1635.2) with a length of 861 bp and a truncated element (ΔIS1635) with a length of 425 bp (Fig. 1B). IS1635.1 spans the region between nucleotides (nt) 1688 and 2548. IS1635.2 extends from nt 4006 to 4866, followed immediately downstream by the truncated element ΔIS1635 from nt 4867 to 5291. The full size IS elements differ in 18 nucleotides, they possess two imperfect terminal repeats (see below) and carry single open reading frames (ORF2, ORF4) encoding putative proteins of 245 amino acids. The deduced protein sequences of ORF2 and ORF4 differ in eight amino acid residues. The putative gene products showed the highest similarity to the transposase of IS6100 (67% identity), an insertion element found in Mycobacterium fortuitum and Flavobacterium sp. [15,16]. Comparison of the deduced protein sequences to a consensus sequence for IS6-like transposases [1] revealed that a DDEK motif involved in transposition catalysis and several additional conserved amino acid residues are present in IS1635.1 and IS1635.2 suggesting that these elements are members of the IS6-family. In contrast, the truncated IS1635-like element (ΔIS1635), immediately downstream of IS1635.2 only comprises the 3′ region of the transposase gene (ORF5) and a single inverted repeat. An additional open reading frame, ORF3 (nt 3105–3947), is located between IS1635.1 and IS1635.2, encoding a protein with a deduced size of 331 amino acids. However, searches of protein databases did not identify protein sequences with significant homology to the predicted gene product.

The left and right sides of the fragment flanking the IS elements carry a complete and a partial open reading frame (ORF1 and ORF6) encoding putative proteins with significant similarity (48 and 81%, respectively) to the E. coli ParA protein, which is involved in the distribution of newly synthesized plasmids to daughter cells [17].

To analyze if related IS elements are also encoded by the virulence plasmid of pathogenic Yersinia species, the entire fragment was split into smaller fragments by generating PCR fragments using synthetic oligonucleotides and hybridized to the pYV virulence plasmids of Y. enterocolitica. Only the fragment carrying IS1635.1 hybridized to the pYV plasmids (Fig. 1A, lane 6; Fig. 1B, bottom). Further hybridization experiments showed that this fragment hybridized to regions of the pYV plasmid of strains Y. enterocolitica 31084 and 13169 carrying the yopM gene (data not shown).

3.2 Comparison of IS1635 to remnants of insertion sequences present on Yersinia plasmids carrying the Yop virulon

Database searches revealed that repetitive sequences flanking the yopM gene of the virulence plasmid of the three pathogenic Yersinia species (plasmid pCD1 of Y. pestis, plasmid pIB1 of Y. pseudotuberculosis and plasmid pYV of Y. enterocolitica) exhibit a strong homology to the IS1635 insertion elements. Fig. 2 shows a schematic alignment of the newly identified IS1635 elements and the truncated IS1635 element to the published Y. pestis, Y. pseudotuberculosis and Y. enterocolitica sequences. All sequences showed an overall sequence homology of more than 90%, indicating that the repetitive sequences are remnants of IS1635-like insertion elements, which underwent point mutations and extensive deletions.


Schematic alignment of IS1635 sequences of Y. intermedia and published sequences with homology to IS1635 present on plasmid pCD1 of Y. pestis[6], on plasmid pIB1 of Y. pseudotuberculosis[19,20] and on plasmid pYV of Y. enterocolitica[7]. Numbers indicate nucleotide positions in relation to IS1635.1. White rectangles indicate the region of homology to IS1635, gray rectangles inside the Y. pestis (Y.p.) elements show the repetitive sequences R1, R2 and R3 described in [18]. Y.ps. seq.1: Y. pseudotuberculosis sequence upstream of yopM[19]Y.ps. seq.2: Y. pseudotuberculosis sequence downstream of yopM; *end of published sequence [20]; Y.e. seq.1 and Y.e. seq.2: Y. enterocoltica sequences upstream of yopM. Dashed lines in Y.p. R1, Y.e. seq.1 and Y.e. seq.2 indicate deletions.

The pCD1 plasmid of Y. pestis harbors three repetitive sequences (R1, R2, R3) with a length of 189 bases (R1), 274 bases (R2) and 275 bases (R3) described by Reisner and Straley [18]. To define the length of these IS1635-like remnants more precisely a detailed sequence comparison has been performed. The longest IS1635 remnant contains the R1 region (indicated by a gray box in Fig. 2) Because of point mutations and a deletion, only an internal part of the putative transposase gene is present, unlikely to encode a functional protein. The R2 and R3 repeats downstream of the yopM gene correspond to the 3′-region of the putative transposase gene and the inverted repeat of IS1635.

Two remnants of the IS1635 element, one upstream [19] and one downstream [20] of the yopM gene, have been found in the pIB1 plasmid of Y. pseudotuberculosis. In the remnant upstream of yopM (seq. 2) of the Y. pseudotuberculosis plasmid an open reading frame was found, whose 5′-coding region has not been sequenced [19]. Compared to the putative tnp gene of IS1635.1 and IS1635.2 this partial ORF harbors point mutations in the 3′-region leading to a frameshift, indicating that this ORF does not encode a functional protein. The pYV plasmid of a Y. enterocolitica serogroup O:9 strain [7] possesses two remnants of insertion elements with high similarity to the IS1635 element upstream of the yopM gene.

To define the length of the terminal inverted repeats and identify putative target sequences of the IS1635 elements, the sequences of the IS1635 remnants of the Y. pestis pCD1 plasmid, Y. pseudotuberculosis plasmid pIB1 and Y. enterocolitica plasmid pYV were included in the sequence analysis. The alignment of the terminal repeats of all elements indicates that the inverted repeats are 26 bp long (Fig. 3). The various integration sites do not indicate a specific target sequence. In addition, no sequence duplication was found.


Terminal inverted repeats (IR) of IS1635.1, IS1635.2 and truncated IS1635 element (ΔIS1635) of Y. intermedia. Inverted repeats present in the IS remnants containing R1, R2 and R3 of Y. pestis (Y.p. R1; Y.p. R2, Y.p. R3) [18] and in the IS remnant downstream of yopM of Y. pseudotuberculosis (Y.ps. seq.2) [20]. Y.e. seq.1 and Y.e. seq.2=Inverted repeats of IS remnants of pYV plasmid of Y. enterocolitica upstream of yopM[7]. IR1=inverted repeat downstream of the tnp gene, IR2=inverted repeat upstream of the tnp gene. Complement indicates that the published sequences have been inverted for the alignment.

3.3 Transposition of IS1635 elements

The ability of IS1635 elements to transpose was tested in the Y. enterocolitica strain 29807 after mating with E. coli S17-1(λpir) harboring the suicide plasmid pYep86. Plasmid pYep86 is a R6K-derivative harboring a kanamycin resistance gene between the IS1635.1 and IS1635.2 elements (see Section 2.4, Fig. 4). By standard mating procedure the plasmid pYep86 was introduced into Y. enterocolitica 29807 at a frequency of about 10−4 to 10−5. This rate was comparable to transfer rates achieved with the vector pUTKm [14], which was used as a control in all experiments. The mechanism of transposition of IS6-like elements is described as the formation of cointegrates of donor and target replicon, which results in the integration of the entire vector plus a new copy of the IS element into the chromosome [1]. In analogy to similar experiments with IS6100[15] we analyzed 73 transconjugants for the presence of an additional copy of an IS1635 element by hybridizing an IS1635 probe to BglII digests of genomic DNA. BglII cuts twice in pYep86 generating two fragments carrying the IS1635 elements (Fig. 5A, lane 1). Of all the transconjugants analyzed, the genomic DNA of seven transconjugants showed three hybridizing fragments. The DNA of these seven transconjugants was additionally cut with NcoI that cuts once in pYep86 in the kanamycin resistance gene. In the transconjugants two hybridizing bands were observed because of the integration into the chromosome (Southern blot analysis of two transconjugants are shown; Fig. 5A, lanes 6 and 9). PstI digest of pYep86 also separates IS1635.1 and IS1635.2, an NcoI/PstI double digest of pYep86 yielded two IS1635 carrying fragments (Fig. 5A, lane 3), while an NcoI/PstI digest of the transconjugants again yielded three hybridizing bands, because of a duplication of one IS1635 element. Fig. 5B depicts a schematic drawing of the integration after transposition. Transconjugant 29807-Tra2 contains a duplication of the IS1635.1 element, while transconjugant 29807-Tra31 exhibits a duplication of IS1635.2. Furthermore by carrying out PCR reactions with primers binding within the IS1635 elements and in the flanking DNA of pYep86 (PCR I to PCR IV, see Fig. 4) and an additional hybridization using a vector specific probe containing the mob region of the R6K vector, we found that the entire vector was present in all transconjugants. The DNA of the 66 remaining transconjugants possessed only two BglII fragments hybridizing to the IS1635 probe. Analysis of some of these transconjugants suggests that they harbor the vector pYep86 integrated into the chromosome by recombinative processes, as in all three digests only two hybridizing bands were observed (data not shown).


Analysis of transposition of IS1635 elements in Y. enterocolitica 29807. A: Southern-blot hybridization of pYep86 and genomic DNA of strain Y. enterocolitica 29807, transconjugants 29807-Tra2 and 29807-Tra31 using an IS1635 probe. Lanes 1–3, hybridization pattern of pYep86 digested with BglII, NcoI and NcoI/PstI; lane 4, BglII digest of genomic DNA of strain Y. enterocolitica 29807; lanes 5–7, genomic DNA of transconjugant 29807-Tra2 digested with BglII, NcoI and NcoI/PstI, Lanes 8–10, genomic DNA of transconjugant 29807-Tra31 digested with BglII, NcoI and NcoI/PstI. B: Schematic drawing of transposition events. Genomic DNA of transconjugant 29807-Tra2 possesses three BglII-fragments hybridizing to IS1635 probe (lane 5). A 3.4-kb BglII-fragment (lane 5) and a 2.9-kb NcoI/PstI-fragment (lane 7) carrying the IS1635.2 element are conserved, indicating that IS1635.1 was duplicated. Transconjugant 29807-Tra31 harbors three BglII-fragments, while in this case a 6.4-kb BgllII-fragment (lane 8) and a 5.0-kb NcoI/PstI-fragment (lane 10) carrying IS1635.1 are conserved, indicating that IS1635.2 was duplicated. Dashed lines indicate genomic DNA of transconjugants.

3.4 Distribution of IS1635 in different Yersinia strains

A number of Yersinia strains were investigated for the presence of IS sequences with homology to IS1635. Genomic DNA from 43 non-pathogenic Yersinia strains (29 Y. enterocolitica biogroup 1A strains, four Yersinia frederiksenii strains, three Yersinia mollaretii strains, three Yersinia kristensenii strains and four Y. intermedia strains) from different sources (environmental, clinical and foodborne isolates), three pathogenic Y. enterocolitica strains of the serogroups 0:3; 0:8 and 0:9 and one pathogenic Y. pseudotuberculosis strain were hybridized to an IS1635 probe. Positive hybridization signals were only observed with three plasmid-harboring Yersinia strains (one Y. enterocolitica biogroup 1A strain, one Y. intermedia and one Y. mollaretii strain) and the pathogenic strains harboring the Yop virulon. In case of the three apathogenic Yersinia strains an additional PCR with internal primers derived from the complete IS1635 elements amplified a 545-bp region containing the 5′region of the transposase gene (data not shown).

Preparations of CsCl-purified plasmid DNA and of genomic DNA of these strains, the Y. intermedia strain 29932 and the four pathogenic strains, were HindIII-digested and hybridized to an IS1635 probe. As the observed hybridization patterns of both, the plasmids and the genomic DNA were identical, the IS1635-related sequences seemed to be present on the plasmids of all the strains investigated (data not shown).

An intriguing feature of the IS1635 remnants is their neighborhood to the yopM gene, which is an effector protein of the Yop virulon. As IS6-like elements can form composite transposons, we investigated, if the plasmid and chromosomal DNA of the four non-pathogenic strains that hybridized to the IS1635.1 probe, carry sequences related to yopM. However, PCR reactions with primers derived from the published Y. enterocolitica yopM sequence failed to produce a PCR product and a fluorescein-labeled yopM probe (see Section 2.2) did not hybridize to the DNA of the four strains (data not shown).

4 Discussion

The new insertion elements IS1635.1 and IS1635.2, which have been identified on a cryptic plasmid of Y. intermedia, belong to the family of IS6-like insertion elements. The analysis of their insertion sites in plasmid p29332 did not show a specific target site nor a duplication of a particular target sequence, as was also reported for the related IS6100 element [16]. However, as most IS6-like elements create direct target repeats [1], an analysis of insertion sites derived from recent transposition events still has to be performed. The arrangement of IS1635.1, IS1635.2 and the enclosed unknown open reading frame (ORF3) may indicate that these genetic elements form a composite transposon, which has been observed for many IS elements of the IS6 family [1]. The pUT-derived transposition system yielded few transconjugants, which harbored a new copy of an IS1635 element in the chromosome and the completely integrated vector. The hybridization patterns of these two transconjugants suggest that either IS1635.1 or ISI635.2 were duplicated indicating that both elements are functional. In our analysis, we also observed, that more than 90% of all transconjugants harbored single copies of the suicide vector integrated into the bacterial chromosome. This integration event seems to be a general feature of the R6K-based suicide system in Yersinia, as experiments with other pUT vectors (e.g., pUTmini-Tn5Km or pUTmini-Tn5luxAB) performed in parallel, also led to the integration of these plasmids into the chromosome of Y. enterocolitica 29807 and other Y. enterocolitica strains in most of the analyzed transconjugants, although in the case of Tn5 transposition occurs via a ‘cut and paste’ mechanism [21].

The origin of the truncated element downstream of IS1635.2 is unknown, the truncated element deviates in 11 positions from IS1635.1 and in seven positions from IS1635.2 (out of 425 bases). Therefore, it does not appear to be a mere duplication of the corresponding region of IS1635.2. A truncated element adjacent to a complete IS element was also observed in the case of an IS6100 element on a plasmid of Flavobacterium sp. strain K172 [16].

An intriguing feature of the IS1635 elements is their close relationship to remnants of insertion sequences flanking the yopM gene of the virulence plasmid of pathogenic Yersinia carrying the Yop virulon. Our data suggest that these remnants are derived from IS1635-like insertion elements, which underwent large deletions and mutations. Under the assumption that IS1635 elements may form a composite transposon, it is conceivable that the yopM gene was integrated into the Yersinia virulence plasmids as part of a related mobile genetic element. This was also discussed by Reisner and Straley [18], who discovered the R1, R2 and R3 sequences of the pCD1 plasmid of Y. pestis. However, they did not find an experimental support for this hypothesis, as plasmid DNA of Shigella flexneri, which harbored the yopM homolog ipaH, did not hybridize to an R2 probe. An association between IS1635 elements and yopM was not found in the non-pathogenic strains harboring IS1635-related sequences.

Hybridizations of an IS1635 probe to genomic DNA and plasmid DNA of more apathogenic Yersinia strains from different sources (environmental, clinical and foodborne strains) made it likely that IS1635-related sequences were plasmid-associated, although the R6K-derived transposition system yielded integration of the IS1635 elements into the chromosome. Reisner and Straley, who described the repeated sequences R1, R2 and R3 of the pCD1 plasmid of Y. pestis[18], also did not find a chromosomal counterpart of these sequences in Y. pestis.


We thank Gudrun Hultsch, Anne Bernhard and Christoph Schaudinn for technical assistance and Drs. Petra Dersch and Astrid Lewin for critical reading of the manuscript.


  • 1Friedrich Loeffler-Institut, Bundesforschungsanstalt für Viruskrankheiten der Tiere, D-17498 Insel Riems, Germany.


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