High-frequency RecA-dependent and -independent mechanisms of Congo red binding mutations in Yersinia pestis

Yersinia pestis, which causes bubonic and pneumonic plague, forms pigmented red colonies on Congo red (CR) dye agar, The hmsHFRS genes required for CR binding (Crb(+)) are genetically linked to virulence-associated genes encoding a siderophore uptake system, These genes are contained in a 102-kb chromosomal pgm locus that is lost in a high-frequency deletion event, resulting in loss of the Crb(+) phenotype, We constructed a recA mutant strain of Y. pestis KIM10+ (YPRA) to test whether the high frequency Crb mutants result from a RecA-mediated deletion of the IS100-flanked pgm locus. Two Pgm-associated phenotypes (Crb+ and pesticin sensitivity [Pst(s)]) were used as markers for the presence of the pgm locus in the RecA(+) KIM10+ and RecA(-) YPRA strains. In KIM10+, both phenotypes were lost at a very high (2 x 10(-3)) frequency, due to the deletion of the entire pgm locus. In YPRA, the Crb(+) phenotype was still lost at a high frequency (4.5 x 10(-5)), although the loss of the Pst(s) phenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 x 10(-7)), These RecA-independent Crb(-) mutants were caused by mutations in both the hmsHFRS locus and in a newly identified gene, hmsT. Nonpigmented Yersinia pseudotuberculosis and Escherichia coli strains transformed with both hmsT and hmsHFRS became Crb(+). This study demonstrates that in a laboratory culture, the Crb(+) phenotype is unstable, independent of the pgm locus deletion. We propose that a lack of selection for the CR-binding ability of Y, pestis in vitro mag contribute to the mutation frequencies observed at the hmsHFRS and hmsT loci.