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

Abstract

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.

The Genome Sequence of the Tomato-Pathogenic ActinomyceteClavibacter michiganensissubsp.michiganensisNCPPB382 Reveals a Large Island Involved in Pathogenicity

“…michiganensis is the occurrence of numerous genes encoding extracellular proteases, although when C. michiganensis subsp. michiganensis is grown on agar plates containing casein or skim milk, it shows no proteolytic activity (9). At least 28 serine proteases belonging to three different families were identified.…”

“…The plasmid-encoded Pat-1 was shown to be necessary for symptom development (wilting), but not for colonization of the tomato plant (13). Analysis of a mutant in which the serine in the catalytic triad was replaced by a threonine via site-specific mutagenesis suggested that pat-1 probably encodes a functional serine protease (9). The other genes in pCM2, phpA and phpB, are not essential for pathogenicity because the plasmid-free strain CMM100 colonizes normally and cloning of these genes into CMM100 did not affect the nonvirulent phenotype of the strain (9,40).…”

“…Analysis of a mutant in which the serine in the catalytic triad was replaced by a threonine via site-specific mutagenesis suggested that pat-1 probably encodes a functional serine protease (9). The other genes in pCM2, phpA and phpB, are not essential for pathogenicity because the plasmid-free strain CMM100 colonizes normally and cloning of these genes into CMM100 did not affect the nonvirulent phenotype of the strain (9,40). The seven chromosomal chp members include three pseudogenes (chpA, chpB, and chpD) containing frameshifts and/or in-frame stop codons.…”

“…The second known pathogenicity factor, Pat-1, is a putative serine protease encoded on the 70-kb plasmid pCM2 (13). Further genes homologous to pat-1 have been identified both in plasmid pCM2 and in the chromosome (9). All the gene functions required for infection, successful colonization, and evasion or suppression of plant defenses are carried on the chromosome.…”

Genome of the Actinomycete Plant Pathogen Clavibacter michiganensis subsp. sepedonicus Suggests Recent Niche Adaptation

“…Importantly, these advances allowed breakthrough identification of a novel set of pathogenicity-related genes in the tomato pathogen Clavibacter michiganensis subsp. michiganensis and identification of homologues in other coryneform plant pathogens (15,27,32; see the accompanying paper). Because methodologies created for functional analysis of C. michiganensis subsp.…”