Molecular characterization of IS1541 insertions in the genome of Yersinia pestis

Abstract

The genome of Yersinia pestis, the causative agent of plague, contains at least 30 copies of an element, designated IS1541, which is structurally related to IS200 (85% identity). One such element is inserted within the chromosomal inv gene (M. Simonet, B. Riot, N. Fortineau, and P. Berche, Infect. Immun. 64:375-379, 1996). We characterized other IS1541 insertions by cloning 14 different Y. pestis 6/69M loci carrying a single copy of this insertion sequence (IS) into Escherichia coli and, for each element, sequencing 250 bp of both flanking regions. In no case was this IS element inserted into large open reading frames; however, in eight cases, it was detected downstream (17 to 139 bp) of genes thought to be transcribed monocistronically or which constituted the last gene of an operon, and in only one case was it detected upstream (37 bp) of the first gene of an operon. Sequence analysis revealed stem-loop structures (deltaG, < -10 kcal) resembling rho-independent transcription terminators in 8 of the 14 insertion sites. These motifs might constitute hot spots for insertion of this IS1541 element within the Y. pestis genome.

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 by NotI (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.

FIG. 2

Nucleotide sequences adjacent to IS1541 in Y. 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).

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. coli α-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).