A multipurpose broad host range cloning vector and its use to characterise an extracellular protease gene of Xanthomonas campestris pathovar campestris

Abstract

A multipurpose broad host range plasmid, pIJ3200, was constructed by inserting the polylinker-containing 445 bp PvuII fragment of Bluescript M13 into the EcoRI site of the cosmid pLAFR1. Using this vector a protease gene of Xanthomonas campestris pathovar campestris, previously cloned in the recombinant plasmid pIJ3070, was located by deletion to a 2.2 kb DNA region. Sequencing of the protease gene revealed an open reading frame encoding a 580 amino acid polypeptide with molecular weight of 57,000. The deduced amino acid sequence showed strong homology with the subtilisin family of serine proteases. This, together with its sensitivity to inhibition by phenylmethylsulphonyl fluoride, suggests that the enzyme belongs to this family of proteases.