IDENTIFICATION AND CLONING OF A HEMIN STORAGE LOCUS INVOLVED IN THE PIGMENTATION PHENOTYPE OF YERSINIA-PESTIS

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26°C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an ~ 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing ~ 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mtuation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y. pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.