Salmonella enterica serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make S. enterica serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of S. enterica serovar Pullorum RKS5078, using I-CeuI, XbaI, AvrII, and SpeI and Tn10 insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas. However, the genome of S. enterica serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most S. enterica serovar Pullorum strains examined, with different structures in a minority of S. enterica serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with S. enterica serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among S. enterica serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the S. enterica serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.

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.

Background: Chromosomal DNA replication in bacteria starts at the origin (ori) and the two replicores propagate in opposite directions up to the terminus (ter) region. We hypothesize that the two replicores need to reach ter at the same time to maintain a physical balance; DNA insertion would disrupt such a balance, requiring chromosomal rearrangements to restore the balance. To test this hypothesis, we needed to demonstrate that ori and ter are in a physical balance in bacterial chromosomes. Using wavelet analysis, we documented GC skew, AT skew, purine excess and keto excess on the published bacterial genomic sequences to locate the turning (minimum and maximum) points on the curves. Previously, the minimum point had been supposed to correlate with ori and the maximum to correlate with ter. Results: We observed a strong tendency of the bacterial chromosomes towards a physical balance, with the minima and maxima corresponding to the known or putative ori and ter and being about half chromosome separated in most of the bacteria studied. A nonparametric method based on wavelet transformation was employed to perform significance tests for the predicted loci. Conclusions: The wavelet approach can reliably predict the ori and ter regions and the bacterial chromosomes have a strong tendency towards a physical balance between ori and ter.

The genomic cleavage map of Salmonella typhi Ty2, 4,780 kb in size, was determined through digestion of the genomic DNA with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 33, 26, 7, and 35 sites for the enzymes XbaI, BlnI, I-CeuI, and SpeI, respectively. The fragments were arranged around the chromosome through excision of fragments from the gel, redigestion with a second enzyme, end labelling with 32P, and reelectrophoresis and named in alphabetical order. Tn10 transposons inserted in 82 different genes of Salmonella typhimurium were transduced by phage P22 into S. typhi, and the location of Tn10, and thus of the gene, was mapped through the XbaI and BlnI sites of Tn10. All seven I-CeuI sites (in rrl genes for 23S rRNA) were conserved, and the gene order within the I-CeuI fragments resembles that of S. typhimurium LT2, but the order of I-CeuI fragments is rearranged from ABCDEFG in S. typhimurium LT2 to AGCEFDB in S. typhi. In addition, there is a 500-kb inversion which covers the terminus region. Comparisons of lengths of segments between genes showed that S. typhi has segments which differ in size from those in S. typhimurium. The viaB locus, for synthesis of the Vi antigen of S. typhi, was shown to be within a 118-kb loop (a segment of DNA with no homolog in most other Salmonella species) between mel and poxA on the chromosome.

Salmonella typhi is the only species of Salmonella which grows exclusively in humans, in whom it causes enteric typhoid fever. Strains of S. typhi show very little variation in electrophoretic types, restriction fragment length polymorphisms, cell envelope proteins, and intervening sequences, but the same strains are very heterogeneous for ribotypes which are detected with the restriction endonuclease PstI. In addition, the genome of S. typhi has been proven to undergo genomic rearrangement due to homologous recombination between the seven copies of rrn genes. The relationship between ribotype heterogeneity and genomic rearrangement was investigated. Strains of S. typhi which belong to 23 different genome types were analyzed by ribotyping. A limited number of ribotypes were found within the same genome type group; e.g., most strains of genome type 3 belonged to only two different ribotypes, which result from recombination between rrnH and rrnG operons. Different genome type groups normally have different ribotypes. The size and identity of the PstI fragment containing each of the seven different rrn operons from S. typhi Ty2 were determined, and from these data, one can infer how genomic rearrangement forms new ribotypes. It is postulated that genomic rearrangement, rather than mutation, is largely responsible for producing the ribotype heterogeneity in S. typhi.