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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 IS 100 -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 × 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 × 10 −5 ), although the loss of the Pst s phenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 × 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 may contribute to the mutation frequencies observed at the  hmsHFRS  and hmsT  loci.

High-Frequency RecA-Dependent and -Independent Mechanisms of Congo Red Binding Mutations in Yersinia pestis