The cellulase encoded by the native plasmid of Clavibacter michiganensis ssp sepedonicus plays a role in virulence and contains an expansin-like domain

We are examining the molecular basis of pathogenicity for Clavibacter michi ganensis ssp. sepedonicus, a gram positive coryneform bacterium that causes the economically important potato ring rot disease. We present here a comp lete restriction map of the native plasmid pCS1, on which we localize the c ellulase gene that it contains. A mutant produced by chemical mutagenesis t hat does not produce cellulase as well as a naturally occurring strain that does not contain the pCS1 plasmid were both shown to be markedly reduced i n virulence on eggplant. Both strains became significantly more virulent af ter the cellulase gene was introduced into the cells by transformation. The complete nucleotide sequence of the cellulase gene was determined and show n to encode a protein of 727 amino acids which would have a predicted molec ular weight of 71.5 kDa. The sequence shows a leader sequence for secretion and two typical cellulase domains (a catalytic domain and a cellulose bind ing domain). Additionally, we identify an unexpected third domain that show s similarity to a plant protein called expansin; which is believed to inter act with cellulose microfibrils during plant cell expansion. (C) 2000 Acade mic Press.