Endonuclease digestion of the 4,800-kb chromosome of SabloneUa lyphimurium LT2 yielded 24 XbaI fragments, 12 BlnI fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis. The 90-kb plasmid pSLT has one XbaI site and one BinI site. The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography. Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome. One hundred nine independent Tn 10 insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and Bin1 sites in Tn 10. The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn 10 insertions were located by centisome around the map. There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes. All seven rRNA genes were located on the map; all have the CeuI digestion site, all four with the tRNA gene for glutamate have the XbaI and the BinI sites, but only four of the seven have the BinI site in the 16S rRNA (rrs) gene. Their inferred orientation of transcription is the same as in Escherichia coli. A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E. coli, observed earlier by others, has been confirmed. The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E. coli, but the XbaI and BinI sites outside the mrn genes show very little conservation.

The enzyme I-CeuI, encoded by a class I mobile intron inserted in the gene for 23S rRNA in Chlamydomonas eugamatos, cleaves a specific 19-bp sequence in this gene. This sequence is present only in the seven genes for rRNA in Salmonella typhimurium and Escherichia coli. Partial digestion with I-CeuI of DNA from 17 wild-type strains of S. typhimurium indicates that the chromosome of these strains is strongly conserved, for the digestion products closely resemble those of strain LT2. The lengths and order of chromosomal segments are conserved in 15 of the strains; 2 show some rearrangements. XbaI digestion indicated heterogeneity without revealing the genomic structure. Because of conservation of I-CeuI sites in genes for rRNA and conservation of the number and locations of these genes, I-CeuI provides an excellent tool for the rapid examination of the chromosomes of related species of bacteria; differences in the fingerprints indicate the occurrence of chromosomal rearrangements such as insertions or inversions.

Genome plasticity resulting from frequent rearrangement of the bacterial genome is a fascinating but poorly understood phenomenon. First reported in Salmonella typhi, it has been observed only in a small number of Salmonella serovars, although the over 2,500 known Salmonella serovars are all very closely related. To gain insights into this phenomenon and elucidate its roles in bacterial evolution, especially those involved in the formation of particular pathogens, we systematically analyzed the genomes of 127 wild-type S. typhi strains isolated from many places of the world and compared them with the two sequenced strains, Ty2 and CT18, attempting to find possible associations between genome rearrangement and other significant genomic features. Like other host-adapted Salmonella serovars, S. typhi contained large genome insertions, including the 134 kb Salmonella pathogenicity island, SPI7. Our analyses showed that SPI7 disrupted the physical balance of the bacterial genome between the replication origin (ori) and terminus (ter) when this DNA segment was inserted into the genome, and rearrangement in individual strains further changed the genome balance status, with a general tendency toward a better balanced genome structure. In a given S. typhi strain, genome diversification occurred and resulted in different structures among cells in the culture. Under a stressed condition, bacterial cells with better balanced genome structures were selected to greatly increase in proportion; in such cases, bacteria with better balanced genomes formed larger colonies and grew with shorter generation times. Our results support the hypothesis that genome plasticity as a result of frequent rearrangement provides the opportunity for the bacterial genome to adopt a better balanced structure and thus eventually stabilizes the genome during evolution.

Salmonella enterica serovar Gallinarum is a fowl-adapted pathogen, causing typhoid fever in chickens. It has the same antigenic formula (1,9,12:-:-) as S. enterica serovar Pullorum, which is also adapted to fowl but causes pullorum disease (diarrhea). The close relatedness but distinct pathogeneses make this pair of fowl pathogens good models for studies of bacterial genomic evolution and the way these organisms acquired pathogenicity. To locate and characterize the genomic differences between serovar Gallinarum and other salmonellae, we constructed a physical map of serovar Gallinarum strain SARB21 by using I-CeuI, XbaI, and AvrII with pulsed-field gel electrophoresis techniques. In the 4,740-kb genome, we located two insertions and six deletions relative to the genome of S. enterica serovar Typhimurium LT2, which we used as a reference Salmonella genome. Four of the genomic regions with reduced lengths corresponded to the four prophages in the genome of serovar Typhimurium LT2, and the others contained several smaller deletions relative to serovar Typhimurium LT2, including regions containing srfJ, std, and stj and gene clusters encoding a type I restriction system in serovar Typhimurium LT2. The map also revealed some rare rearrangements, including two inversions and several translocations. Further characterization of these insertions, deletions, and rearrangements will provide new insights into the molecular basis for the specific host-pathogen interactions and mechanisms of genomic evolution to create a new pathogen.

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.