Mu dI1(Ap lac) mutagenesis of Yersinia pestis plasmid pFra and identification of temperature-regulated loci associated with virulence.

The F1 capsule of Yersinia pestis, encoded by the 100 kb plasmid pFra, is often assumed to be essential for full virulence of Y. pestis. However, virulent strains of Y. pestis that are F1- and either pFra+ or pFra- have been reported. To assess the role of pFra-encoded factors in virulence, mutants in pFra with insertions of the defective transposing bacteriophage Mu dl(Ap lac) were obtained, by using the wild type (wt) and the pLcr-cured derivative of strain C092. Mutants that exhibited temperature regulation of lactose fermentation and retarded electrophoretic mobility of pFra were selected. A total of 15 insertion mutants were isolated in the wt strain (12 of which had a single insertion in the genome, in pFra); and 24 mutants in the isogenic pLcr- derivative. Four of the pLcr+ mutants, and none of the pLcr- mutants, were F1-. All F1- mutants were decreased in virulence for mice compared to the wt parent; and five of the F1+ mutants also were significantly attenuated in mice. Fusion end-joints of insert DNA were cloned into Escherichia coli by using pMLB524, a vector for rescuing operon fusions of lacZ. Recombinants were obtained which contained pFra inserts ranging from < 2kb to approximately 36 kb, and the insertions occurred at several sites on pFra. All of the four F1- mutants tested mapped within the F1 capsule operon (caf1). The remaining five attenuated mutants sequenced were F1+ and mapped outside of but near the operon. Sequencing and complete analysis of the pFra insertions mutants could facilitate identification of new potential virulence factors.