Francisella tularensis is the etiological agent of tularemia, a serious disease in several Northern hemisphere countries. The organism has fastidious growth requirements and is very poorly understood at the genetic and molecular levels. Given the lack of data on this organism, we undertook the sample sequencing of its genome. A random library of DNA fragments from a highly virulent strain (Schu 4) of F. tularensis was constructed and the nucleotide sequences of 13,904 cloned fragments were determined and assembled into 353 contigs. A total of 1.83 Mb of nucleotide sequence was obtained that had a G+C content of 33.2%. Genes located on plasmids pOM1 and pNFL10, which had been previously isolated from low virulence strains of F. tularensis, were absent but all of the other known F. tularensis genes were represented in the assembled data. F. tularensis Schu4 was able to grow in the absence of aromatic amino acids and orthologues of genes which could encode enzymes in the shikimate pathway in other bacteria were identified in the assembled data. Genes that could encode all of the enzymes in the purine biosynthetic and most of the en- zymes in the purine salvage pathways were also identified. This data will be used to develop defined rationally attenuated mutants of F. tularensis, which could be used as replacements for the existing genetically undefined live vaccine strain.

The complete nucleotide sequence and organization of the Yersinia enterocolitica serotype 0:8 low-calciumresponse (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for ylpA. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in Yersinia pestis plasmid pCD1 or in Y. enterocolitica serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, ISYen1, was a new IS belonging to the IS256 family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by Y. enterocolitica serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the spy/sopABC loci of pCD1 and pYVe227. Sequence analysis suggests that Yersinia LCR plasmids are from a common ancestor but that Y. enterocolitica serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of Y. enterocolitica serotype 0:8 plasmids from those of Y. enterocolitica serotype 0:9 and Y. pestis LCR plasmids.Pathogenic Yersinia enterocolitica is a well-established foodborne pathogen (29). Infection usually results in a self-limiting gastroenteritis, but in immunocompromised individuals septicemia and hepatic abscesses may occur. Postinfection complications include arthritis and erythema nodosum (8). Y. enterocolitica is a serologically diverse species that includes saprophytes as well as pathogens. Certain serotypes are consistently associated with human infection (30). Serotypes 0:3 and 0:9 are most frequently isolated in Europe, Japan, and Canada, while serotype 0:8 causes most infections in the United States. Serotype 0:8 is also associated with more severe invasive disease (3, 5). So far, only one phenotypic trait has been identified in Y. enterocolitica 0:8 to account for these observed differences in pathogenicity (37).Essential virulence genes are carried on a ca.-70-kb plasmid in Y. enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis (42, 43). The virulence plasmid encodes virulence proteins called Yops (Yersinia outer proteins), a type III secretion system, the V antigen, and regulatory proteins. The virulence plasmid encodes the low-calcium response (LCR) (53), which refers to a complex response to in vitro growth conditions of temperature (37°C) and extracellular calcium concentration (less than 2.5 mM Ca 2ϩ ). Under these conditions, pathogenic Yersinia shifts from vegetative growth to the production a...

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