Diversity of genome structure in Salmonella enterica serovar Typhi populations

Abstract

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136 wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonuclease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangements due to homologous recombination between the rrn operons leading to inversions and translocations. Recombination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S. enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. enterica serovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need for genome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types 1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectional replication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb off balance), indicating that the survival of the best-balanced strains was greater. In addition, the need for appropriate gene dosage apparently selected against rearrangements which moved genes from their accustomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S. enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved in rearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmonella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a prophage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appeared to be independent of the origins of the genome types.

FIG. 1.

Location and order of the seven I-CeuI fragments on the chromosome (the rrn skeleton). oriC is the site of initiation of bidirectional replication; Ter is the termination site. The numbers with arrows represent the different primer combinations used to amplify the seven rrn operons (indicated by arrows outside the circles). The numbers outside the circles indicate the sizes of the I-CeuI fragments (in kilobases) based on the previously published sequences (45, 50). (A) S. enterica serovar Typhimurium LT2. (B) S. enterica serovar Typhi CT18.

FIG. 2.

Partial digestion of DNA of strains of S. enterica serovar Typhi with endonuclease I-CeuI, separation by PFGE, and staining with ethidium bromide. The gel is shown on the left. The fragments are shown on the right, and the inferred composition and sizes (in kilobases) are indicated. Lane 1, strain SARB63 (fragment order, CBEFDG; genome type 25); lane 2, SARB64 (fragment order, BDCEFG; genome type 19); lane 3, 27566 (fragment order, ECBFDG; genome type 26); lane 4, SA4864 (fragment order, BCFDEG; genome type 6); lane 5, SA4665 (fragment order, GFCEDB; genome type 16); lane 6, Ty2 (fragment order, GCEFDB; genome type 9).

FIG. 3.

Proposed model of genomic rearrangements due to homologous recombination between rrn operons resulting in inversions or translocations. oriC is indicated by a shaded circle in fragment C (which corresponds to I-CeuI-C), and Ter is indicated by a shaded square in fragment A (I-CeuI-A). pro (proline requirement) and his (histidine requirement) indicate the positions of standard genes. (A) Both the A and C fragments are in the normal, uninverted orientation (A+C+), and the fragment order is I-CeuI-ABCDEFG. (B and C) Inversion. Recombination between rrnH and rrnG results in inversion of fragment A. (D to F) Translocation. Recombination between rrnC and rrnA deletes fragment D, which is reinserted by recombination with rrnE; this results in translocation to produce the fragment order ABCEFDG.

FIG. 4.

Order and orientation of I-CeuI fragments in 136 independent wild-type strains of S. enterica serovar Typhi. The sizes (in kilobases) of the fragments based on the sizes in CT18 (50) are indicated at the top, shown approximately to scale. The order of I-CeuI fragments B to G was determined by PFGE (Fig. 2) and was confirmed by PCR (Fig. 5). The I-CeuI-A fragment (2,422 kb) is inferred to join the left end to the right end of a fragment to form a circle. The orientation of I-CeuI fragments B, D, E, F, and G was inferred from the polarity of the rrn genes and was confirmed by PCR. The order and sizes of fragments for E. coli K-12 and S. enterica serovar Typhimurium LT2 (STM LT2) and the orientation of rrn operons are indicated at the bottom. The chromosomes of the different genome types are shown in the A+C+ orientation (with both the A and C fragments uninverted); the open square in fragment A indicates pro (proline utilization), and the open triangle indicates his (histidine requirement). Since both I-CeuI-C and I-CeuI-A are flanked by inverted rrn operons, these fragments could be inverted. The number of strains of each genome type that fall into each of the four sets of orientation of A and C fragments was determined from the PCR data (see Fig. 5). The dot in the I-CeuI-C fragment indicates the location of oriC; T indicates the terminus. The sizes of the fragments (in kilobases) were calculated from previously published sequences of S. enterica serovar Typhimurium LT2 (45) (GenBank accession no. NC_003197) and E. coli K-12 (5) (GenBank accession no. NC_000913); these fragments are shown at the bottom.

FIG. 5.

PCR analysis of the rrn skeleton of S. enterica serovar Typhi genome type 3 strains. The primer pairs are indicated above the gel showing the PCR products. Lane M contained the marker (HindIII-digested lambda). The inferred rrn skeleton is shown on the left. (Upper set) Template DNA of strain 26.047 (genome type 3, A+C+). (Lower set) Template DNA of strain 425Ty (genome type 3, A−C+).

FIG. 6.

Analysis of genome balance. The chromosome structure of genome type 3 A+C+ illustrates the genome balance. Since the chromosome is bidirectionally replicated from oriC, two replichores are shown. The dot in fragment C represents oriC. (A) Linear form. The cross-hatched line represents calculation of the total fragment sizes to determine the length of replichore 1 and replichore 2. (B) Circular form. Rep1, replichore 1; Rep2, replichore 2.