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.

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.

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.

To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at 70℃ or in serovar Typhimurium LT2 stocked either at 70℃ or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at 70 ℃ and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsedfield gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.