Molecular Characterization of IS1541Insertions in the Genome of Yersinia pestis

Distribution of IS1541 in the Y. pestis6/69M genome.We have previously shown that the genome of Y. pestis 6/69M, which belongs to biotype Orientalis and ribotype B (12), contains multiple copies of the IS1541element (30). By hybridization with an IS1541probe of bacterial DNA digested by NotI or SpeI, restriction endonucleases that infrequently cut Y. pestisDNA (12, 17) and do not cut the IS element, we found that 18 of 27 NotI fragments and 17 of 29 SpeI fragments hybridized with this probe (Fig. 1A). This result demonstrates that the IS1541 elements are not clustered in the genome.

• Open in new tab
• Download powerpoint

Fig. 1.

Distribution of IS1541 elements in Y. pestis 6/69M. Bacterial DNA was digested by appropriate restriction endonucleases according to the manufacturer’s recommendations (New England Biolabs), and DNA fragments were separated by electrophoresis. Pulsed-field gel electrophoresis of macrorestricted genomic DNA was carried out as described by Guiyoule et al. (12). After separation, DNA fragments were depurinated, denaturated, and then transferred to nylon membranes (Boehringer Gmbh, Mannheim, Germany) by the Southern technique (28). The IS probe preparation, [³²P]dCTP (Amersham France, Courtaboeuf, France) labeling, and DNA-DNA hybridization were performed as previously described (20). (A) Lanes 1 and 2, pulsed-field gel electrophoresis of DNA digested byNotI (lane 1) or SpeI (lane 2); lanes 3 and 4, Southern blot hybridization of DNA digested by NotI (lane 3) or SpeI (lane 4) with the radiolabeled IS probe. (B) Lane 1, agarose gel electrophoresis of a total plasmid extract (pYV, pPst, and pFra) prepared by an alkaline lysis procedure and digested by the restriction endonuclease EcoRV; lane 2, Southern blot hybridization of EcoRV-digested DNA with the radiolabeled IS probe. (C) Southern blot hybridization of HincII-digested DNA from Pgm⁺ (lane 1) and Pgm⁻(lane 2) cells with the radiolabeled IS probe. Molecular size markers (in kilobases) are indicated on the left. A strong hybridization signal given by some bands may correspond to the presence of multiple IS.

As in almost all strains of Y. pestis (5), strain 6/69M contains one plasmid each of 110 (pFra), 70 (pYV), and 9.5 (pPst) kb. Plasmid DNAs were extracted from this strain, digested withEcoRV (which does cleave within IS1541), separated by electrophoresis, transferred onto a nylon membrane, and hybridized with a ³²P-labeled IS1541-specific DNA probe (28). A single hybridizing band of about 3.2 kb was detected, and on the basis of the known restriction maps of individual plasmids, this fragment was assigned to pFra (Fig. 1B). Thus, almost all IS1541 copies were located on the bacterial chromosome.

Finally, we looked for the presence of the IS within the pgmlocus. This was indirectly achieved by comparing the IS1541hybridization profiles of DNA from the parental strain Pgm⁺and its derivative strain Pgm⁻, digested byHincII, an enzyme which does not cleave within IS1541. As depicted in the autoradiogram presented in Fig.1C, there was no detectable difference in the IS patterns exhibited by the two strains. This result suggested that there is no IS1541 element within the pgm locus. Moreover,E. coli recombinant clones carrying DNA fragments spanning the pgm locus did not hybridize with an IS1541-specific DNA probe (4).

Cloning of IS1541 flanking regions of the Y. pestis 6/69M genome.The autoradiogram presented in Fig. 1C revealed the presence of numerous hybridizing bands, mostly ranging from 2 to 6 kb in size. To identify regions of the Y. pestisgenome in which the IS element was inserted,HincII-restricted DNA from strain 6/69M was separated by electrophoresis through a 1% agarose gel in Tris-acetate buffer, and 2- to 6-kb DNA fragments were extracted from the gel by using β-agarase (New England Biolabs, Beverly, Mass.) and ligated with T4 DNA ligase into a SmaI-digested pUC19 vector (Appligene, Illkirch, France) (28). Recombinant plasmids were introduced into E. coli JM105 by transformation (28), and transformants were selected on Luria-Bertani agar containing ampicillin (100 μg ml⁻¹) and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). White transformants resistant to ampicillin were screened for the presence of the IS1541-related sequences by colony blot hybridization (28) with the specific probe. Endonuclease restriction (EcoRI, HindIII, and SphI) followed by Southern blot hybridization with the IS1541probe of plasmids from recombinant clones allowed discrimination of 14 different inserts having a size less than 4.5 kb and containing a single copy of the IS element. Following digestion with the appropriate enzymes (EcoRI and/or SphI), these inserts were purified and put into M13mp18 and -mp19.

IS1541 insertion specificity in Y. pestis6/69M.IS200 does not possess terminal inverted repeats and preferentially inserts into T/A-rich short segments (15, 21). Consequently, the ends of this element have not been definitively assigned and a duplication event at the target site, if any, has not been precisely characterized. We previously reported the insertion of IS1541 within the inv gene ofY. pestis, and in the present work we sequenced about 350 nucleotides of both IS-chromosome junction fragments in 14 differentY. pestis loci (MO951, MO952, MO953, MO954, MO955, MO956, MO957, MO958, MO959, MO960, MO962, MO963, MO964, and MO965). In all cases, the nucleotide sequence (100 bp) of both termini of IS1541 were identical to those of the prototype sequence (30). Sequence analysis revealed that the right extremity of IS1541, as defined in Fig. 2A, is almost identical to that of IS200, whereas their left extremities display sequence divergence within their outermost 7-bp segments. However, the ends of IS1541, like those of IS200, do not contain inverted repeats. Comparison of the sequences of the 14 target sites of IS1541 revealed that in all cases insertion occurred within a T/A-rich short (≤10-bp) segment (Fig. 2B). Two T’s were invariably present in both sides of the IS element, but since the sequence of targets prior to insertion is not known, it is not possible to determine if these motifs belong to the element or whether they result from an IS-mediated duplication event. More interestingly, in 13 of the 14 targets studied, sequence analysis revealed that the region upstream of the IS could form stem-loop structures having a minimum energy formation of <−10 kcal/mol. These motifs resemble rho-independent transcription terminators and might constitute hot spots for insertion of IS1541 within theY. pestis genome. Precise characterization of the transposition behavior of IS1541 will require the development of a qualitative and quantitative transposition assay.

• Open in new tab
• Download powerpoint

Fig. 2.

Nucleotide sequences adjacent to IS1541 inY. pestis 6/69M. Sequencing of the IS flanking regions was achieved by the dideoxynucleotide chain termination method (29) with modified T7 DNA polymerase (Sequenase version 2.0; Amersham France) and primers located at the left (5′-CATTTGCAGTTGCCAG-3′) and right (5′-GTTTACGGGCCGTAA-3′) ends of the IS (30). In each case, the sequence of the complementary strand was carried out by using synthetic oligonucleotides designated from the sequence previously determined. (A) The 5′ and 3′ ends of IS200 and IS1541 are shown. Asterisks indicate identical nucleotides. (B) Nucleotide sequences (36 bp) upstream and downstream of 15 IS elements are shown. Inverted and complementary nucleotides are depicted by arrows below the sequences. The loop free energies (ΔG, in kilocalories) were calculated as reported by Zuker (33).

In none of the 14 target sequences was IS1541 inserted within large open reading frames (ORFs) (data not shown). However, sequence analysis using the BlastX program revealed that, in 9 of 14 cases, this IS inserted in the vicinity of an ORF (designatedorf₁ to orf₉, of which only orf₄ was entire) coding for a peptide highly homologous (47 to 98% identity) to a known bacterial protein from E. coli or Salmonella typhimurium (Fig.3). In eight cases, IS1541inserted downstream of genes thought to be either transcribed monocistronically (cspG, envZ, gltX, and suhB) or which constituted the last gene (crr, ilvH, rpoC, and secF) of an operon. These findings are consistent with our proposal that the hairpin structures present at the corresponding IS1541insertion sites might correspond to transcriptional terminators. Insertion of IS1541 in the 5′ region of leuA, the first gene of the leucine operon, constitutes the only case of insertion of this IS upstream from a gene. By analogy with the S. typhimurium system, it is likely that this insertion has occurred in a hairpin structure belonging to the transcriptional regulatory region of the leu operon (10).

• Open in new tab
• Download powerpoint

Fig. 3.

Insertions of IS1541 in eight coding regions of the Y. pestis 6/69M genome. Arrows represent ORFs. Asterisks and periods indicate identical and similar amino acids, respectively. Symbols: •, IS insertion site; ○∣, hairpin structure. Left and right extremities of IS1541 are indicated by L and R, respectively. LeuA is the E. coliα-isopropylmalate synthase (26); SuhB is the extragenic suppressor protein of E. coli (31) which possesses inositol monophosphatase activity (18); RpoC is the β′ subunit of the E. coli RNA polymerase (22); CspG is an E. coli cold shock protein (19); EnvZ is the osmotic sensor from the two-component system OmpR-EnvZ of S. typhimurium (16); Crr is the component III (IIIGlc) of the phosphoenolpyruvate-dependent phosphotransferase system of E. coli (27); SecF is a protein export membrane protein of E. coli(9); IlvH is an acetolactate synthase of S. typhimurium (13) involved in isoleucine and valine synthesis; GltX is the glutamyl-tRNA synthetase of E. coli(2).

Characterization of IS1541 insertions in unrelatedY. pestis strains.The next step was to consider whether, in other strains of Y. pestis, the IS1541 element flanked the same genes as in strain 6/69M. For this purpose, strains Hambourg 12 (biotype Orientalis, ribotype A), PKR XXIV (biotype Medievalis, ribotype O), Sénégal Th (biotype Orientalis, ribotype B), and Saı̈gon 55-1239 (biotype Orientalis, ribotype E) from the Centre National de Référence des Yersinia (Institut Pasteur, Paris, France) were investigated. As previously reported (30), these strains, isolated from different periods of time and geographical areas, displayed different IS1541 hybridization patterns.Y. pestis DNA was amplified by PCR with different sets of primers, each of them consisting of (i) a forward primer located within one of the previously defined ORFs (F1, 5′-CTGAGCGATAGTACG-3′ [orf₁]; F2, 5′-CTATTGGTTCGTGAG-3′ [orf₂]; F3, 5′-CGTGATGAATTGCGTGGC-3′ [orf₃]; F4, 5′-GAAGTATCTCTATGTC-3′ [orf₄]; F5, 5′-CGTGTTAAATCAGCG-3′ [orf₅]; F6, 5′-CGTATCGCTGAAGAA-3′ [orf₆]; F7, 5′-CGTCGTGGAACGCCT-3′ [orf₇]; F8, 5′-CTCACATGTTGAGCG-3′ [orf₈]; or F9, 5′-CGCCATCACCGAATG-3′[orf₉]) and (ii) a reverse primer chosen in the IS element sequence (R1, 5′-CATTTGCAGTTGCCAG-3′ [used in PCR with F2, F5, F7, F8, and F9] or R2, 5′-TGCGGTCTGGCAACT-3′ [used in PCR with F1, F3, F4, and F6]). PCRs were performed as previously described (30), and amplimers were analyzed by 1% agarose gel electrophoresis. For the five unrelated strains, the IS element was always found, on the basis of product size, in the vicinity ofleuA, suhB, rpoC, crr,secF, ilvH, envZ, or gltXbut was not associated with suhB in strain PKR XXIV (data not shown). We previously reported that an identical copy of IS1541 was inserted at the same nucleotide position within the inv gene of strains 6/69M, Hambourg 12, PKR XXIV, Sénégal Th, and Saı̈gon 55-1239 (30). This indicates that acquisition of IS1541 by these strains was prior to their divergence. Data also suggest that insertions of IS1541 at a given loci constitute a stable event, which, in turn, raises the question of the efficiency of the intracellular mobility of these IS elements in Y. pestis. This last point is currently being investigated in the laboratory.

Nucleotide sequence accession numbers.The sequences oforf₁, orf₂,orf₃, orf₄,orf₅, orf₆,orf₇, orf₈, andorf₉ have been submitted to the EMBL nucleotide sequence database under accession numbers Z97975 , Z97976 , Z97977 ,Z97978 , Z97979 , Z97980 , Z97981 , Z97982 , and Z97983 , respectively.