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 enco ding a siderophore uptake system, These genes are contained in a 102-kb chr omosomal pgm locus that is lost in a high-frequency deletion event, resulti ng 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 resu lt from a RecA-mediated deletion of the IS100-flanked pgm locus. Two Pgm-as sociated phenotypes (Crb+ and pesticin sensitivity [Pst(s)]) were used as m arkers 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 Cr b(+) 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 resista nce mutation frequencies (2 x 10(-7)), These RecA-independent Crb(-) mutant s were caused by mutations in both the hmsHFRS locus and in a newly identif ied gene, hmsT. Nonpigmented Yersinia pseudotuberculosis and Escherichia co li strains transformed with both hmsT and hmsHFRS became Crb(+). This study demonstrates that in a laboratory culture, the Crb(+) phenotype is unstabl e, independent of the pgm locus deletion. We propose that a lack of selecti on for the CR-binding ability of Y, pestis in vitro mag contribute to the m utation frequencies observed at the hmsHFRS and hmsT loci.