Identification of Curtobacterium flaccumfaciens pv. flaccumfaciens specific genomic sequences by suppressive subtractive hybridization

Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) and C. flaccumfaciens pv betae (Cfb) are very closely related bacteria but having a different host range. In particular Cff is the causal agent of the “bacterial wilt disease” of Phaseolus spp. and a seed-borne phytopathogen of EPPO A2 quarantine list. Suppressive subtractive hybridization (SSH) was used to identify specific genomic sequences unique Cff and related to the pathogenicity/virulence and to the host range of this bacterium. Genomic DNA from Cff was used ad the tester DNA, while genomic DNA from Cfb served as the driver DNA. Progressive screening of the SSH library obtained by PCR, dotand Southern blot analyses identified 18 different clones that were specific for Cff. There of them gave interesting results according to the close matches found with the proteic sequences present in main databases and related to sugar ABC transporters and transposase-like proteins of Mycobacterium tubercolosis and Micrococcus spp., and to the catalytic moiety of the neurotoxin of Clostridium botulinum. Specific primer pairs, designed according to the nucleotide sequences of each one of these clones, were used to amplify genomic DNA of Cff and Cfb. Only one amplification fragment, of the expected length and sequence, was obtained using each one of these primer pairs and Cff DNA as template. These amplicons were then used as probes to fish, from a Cff mini-genomic library, large genomic fragments containing the whole sequences of the genes related to the SSH clones