Identification of homologues to the pathogenicity factor Pat-1, a putative serine protease of Clavibacter michiganensis subsp. michiganensis

Hybridization of Clavibacter michiganensis subsp. michiganensis total DNA against the pathogenicity gene pat-1 indicated the presence of pat-1 homologous nucleotide sequences on the chromosome and on plasmid pCM2. Isolation of the corresponding DNA fragments and nucleotide sequence determination showed that there are three pat-1 homologous genes: chpA (chromosome) and phpA and phpB (plasmid pCM2). The gene products share common characteristics, i.e. a signal sequence for Sec-dependent secretion, a serine protease motif, and six cysteine residues at conserved positions. Gene chpA located on the chromosome is a pseudogene since it contains a translational stop codon after 97 of 280 amino acids. In contrast to pat-1, cloning of the plasmid encoded homologs phpA and phpB into the avirulent plasmid free Cmm strain CMM100 did not result in a virulent phenotype. So far no proteolytic activity could be demonstrated for Pat-1, however, site specific mutagenesis of pat-1 showed that the serine residue in the motif GDSGG is required for the virulent phenotype of pat-1 and thus Pat-1 could be a functional protease.