Cloning, sequence and expression of the gene (aprV5) encoding extracellular serine acidic protease V5 from Dichelobacter nodosus.

The acidic protease V5-encoding gene (aprV5) from Gram- Dichelobacter nodosus virulent strain 198 was isolated from a cosmid bank by activity screening and sequenced. The 2371-bp nucleotide (nt) sequence contained an open reading frame coding for a protein precursor of 595 amino acid (aa) residues composed of a signal peptide, a pro-region, a mature active protease of 347 aa and a C-terminal extension region of 120 aa. The deduced aa sequence of the pre-pro-mature protease regions showed about 65% similarity to that of D. nodosus basic protease while the C-terminal extension region showed only about 26% similarity. The aprV5 gene, without its C-terminal extension region, was cloned and expressed in Escherichia coli. The acidic protease B5-encoding gene (aprB5) from non-virulent strain 305 was also cloned and sequenced. The aprB5 nt sequence showed 99% homology to that of aprV5 with two single-aa changes occurring in the precursor.

Amino acid sequence of extracellular acidic protease V5 of Dichelobacter nodosus, the causative organism of ovine footrot.

Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative organism of ovine footrot, secretes a family of extracellular serine proteases with pI's in the range of 5.2 to 5.6 and a serine basic protease with a pI of approximately 9.5. The primary structure of acidic protease V5 (pI approximately 5.2) from D. nodosus virulent strain 198 was determined by direct amino acid sequencing. This protease consists of a single polypeptide chain of 347 amino acids, contains two disulfide bonds and has a M(r) of 35960. Comparison of the D. nodosus acidic protease V5 sequence with that of other serine proteases showed that it is a member of the subtilisin family of proteases with strong conservation of identity around the catalytic residues. The sequence of protease V5 showed 64% identity to D. nodosus basic protease (pI approximately 9.5) and 53% identity to the extracellular serine protease of Xanthomonas campestris, a plant pathogen but only 25-35% identity to other proteases of the subtilisin family. The D. nodosus proteases are similar in length to X. campestris protease (but some 70 residues shorter than the subtilisins) and they share two conserved disulfide bonds with the X. campestris protease, a feature not observed for other members of the subtilisin family.