Short-Term Signatures of Evolutionary Change in the Salmonella enterica Serovar Typhimurium 14028 Genome

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis in humans and a typhoid-like disease in mice and is often used as a model for the disease promoted by the human-adapted S. enterica serovar Typhi. Despite its health importance, the only S. Typhimurium strain for which the complete genomic sequence has been determined is the avirulent LT2 strain, which is extensively used in genetic and physiologic studies. Here, we report the complete genomic sequence of the S. Typhimurium strain 14028s, as well as those of its progenitor and two additional derivatives. Comparison of these S. Typhimurium genomes revealed differences in the patterns of sequence evolution and the complete inventory of genetic alterations incurred in virulent and avirulent strains, as well as the sequence changes accumulated during laboratory passage of pathogenic organisms.The genomes of related bacteria can differ in three ways: (i) gene content, where one bacterial species or strain harbors genes absent from the other organism; (ii) nucleotide substitutions within largely conserved DNA sequences, which can result in amino acid changes in orthologous proteins, form pseudogenes, and promote distinct expression patterns of genes present in the two organisms; and (iii) changes in gene arrangement, caused by inversions and translocations. These differences have been observed not only across bacterial species but also among strains belonging to the same species. Recent genomic analyses have revealed that many bacterial pathogens of humans are virtually monomorphic (1) and exhibit very limited sequence diversity, raising questions about the nature of the genetic changes governing distinct behaviors. Furthermore, several bacterial pathogens that have been subjected to extensive passage in the laboratory display altered virulence characteristics, but the genetic basis for these alterations remains largely unknown. Here, we address both of these questions by determining and analyzing the genome sequences of closely related isolates of Salmonella enterica serovar Typhimurium, a Gram-negative pathogen that has been used as a preeminent model to investigate basic genetic mechanisms (2, 8, 46, 59), as well as the interaction between bacterial pathogens and mammalian hosts (11, 41).The genus Salmonella is divided into two species: Salmonella bongori and Salmonella enterica, which together comprise over 2,300 serovars differing in host specificity and the disease conditions they promote in various hosts. For example, S. enterica serovar Typhi is human restricted and causes typhoid fever, whereas serovar Typhimurium is a broad-host-range organism that causes gastroenteritis in humans and a typhoid-like disease in mice. Although the complete genome sequences of 15 Salmonella enterica strains are available, there is only a single representative of S. Typhimurium—strain LT2 (31). Despite its wide application in genetic analysis, strain LT2 is highly attenuated for virulence in both in vitro and in vivo assays (52, 56), leading many investigators to use other S. Typhimurium isolates to examine the genetic basis for bacterial pathogenesis (11, 14, 16).Over 300 virulence genes (3, 5, 47) have already been identified in Salmonella enterica serovar Typhimurium 14028 (now termed S. enterica subsp. enterica serovar Typhimurium ATCC 14028), which is a descendant of CDC 60-6516, a strain isolated in 1960 from pools of hearts and livers of 4-week-old chickens (P. Fields, personal communication). Whereas strain 14028 has been typed as LT2, a designation based on phage sensitivity (27), the two strains were isolated from distinct sources decades apart, which makes their genealogy and exact relationship obscure. A derivative of the original 14028 strain with a rough colony morphology (due to changes in O-antigen expression) was designated 14028r to distinguish it from the original smooth strain, renamed 14028s, and was used in a genetic screen for Salmonella virulence genes because it retained lethality for mice and the ability to survive within murine macrophages. Strain 14028 was also used for the identification of Salmonella genes that were specifically expressed during infection of a mammalian host (30). Both 14028 and LT2 possess a 90-kb virulence plasmid promoting intracellular replication and systemic disease (14), but they differ in their prophage contents, as is often the case among S. Typhimurium strains (12, 13).To identify the individual changes that differentiate S. Typhimurium strains and to assess the nature of variation that arises during laboratory storage and passage, we determined the genome sequence of strain 14028s. This genome was then used as a reference for sequencing its progenitors, including the original source strain CDC 60-6516 and the earliest smooth and rough variants. Our analysis uncovered the genomic differences that arose during the past decades of laboratory cultivation and showed that derivatives with different virulence potentials can follow distinct patterns of sequence evolution.