Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR

Authors

  • Abdelmounaaïm Allaoui,

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    • Present address: Laboratoire de Recherche sur les Trypanosomatidae. Institut Pasteur, 1 rue du Professeur Calmette, BP 245, 59019 Lille, France.

  • Ralf Schulte,

    1. Microbial Pathogenesis Unit, International Institute of Cellular and Molecular Pathology and Faculté de Médecine, Université Catholique de Louvain, UCL 74-49, B-1200 Brussels, Belgium.
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  • Guy R. Cornelis

    Corresponding author
    • *For correspondence. E-mail Cornelis@mipa.ucl.ac.be: Tel. (2) 764 74 49; Fax (2) 764 74 98.

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Abstract

Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC. In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca2+ (Cl) for their growth at 37°C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a Cl phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD50 for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multicopy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH—LacZ′ protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.

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