Under iron-starvation, the highly pathogenic Yersinia synthesize several iron-regulated proteins including two high-molecular-weight polypeptides (HMWP1 and HMWP2). From the chromosome of Yersinia enterocolitica serovar O:8 (strain Ye 8081), the genes coding for the HMWP2 (irp2) and its promoter were cloned into plasmid pUC18 (pIR2) and used as a probe. We show here that the irp2 gene is present only in the highly pathogenic strains and that its promoter is iron-regulated in Escherichia coli. After introduction of the pIR2 plasmid into a fur mutant of E. coli, both the iron-starved and the iron-replete bacteria expressed the HMWP2. Repressibility of irp2 by iron was restored by introduction of a plasmid carrying the fur gene. These results demonstrate that the irp2 promoter is controlled by the Fur repressor in E. coli. Mutagenesis of the chromosomal irp2 gene of Yersinia pseudotuberculosis was obtained by homologous recombination with a 1 kb fragment of this gene cloned on the suicide plasmid pJM703.1. Inactivation of irp2 resulted in the non-expression of both HMWPs, while introduction of plasmid pIR2 into the mutant strain led to the synthesis of the HMWP2 only. Therefore, it is probable that the genes coding for the HMWPs constitute an operon where irp2 is upstream of irp1. When comparing the virulence of the wild-type strain and of its irp2 mutant derivative, we found that the 50% lethality (LD50) for mice of the mutant strain was increased, whatever the route of infection, but more markedly when injected parenterally. Accordingly, these data demonstrate that a mutation in the irp2 gene alters the pathogenicity of Y. pseudotuberculosis.(ABSTRACT TRUNCATED AT 250 WORDS)