CLONING, SEQUENCING AND EXPRESSION OF THE ACYLNEURAMINATE LYASE GENE FROM CLOSTRIDIUM-PERFRINGENS A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cl oned on a 3.3 kb HindIII DNA fragment identified by screening the chro mosomal DNA of this species by hybridization with an oligonucleotide p robe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli an d in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of th e cloned fragment contains the complete acylneuraminate lyase gene (OR F2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding reg ion with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected . The 3'-terminus of the lyase structural gene is followed by a furthe r ORF (ORF3). A high homology was found between the amino acid sequenc es of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% i dentical amino acids), respectively, whereas the similarity to the gen e from E. coli is low (38% identical amino acids). Based on our new se quence data, the `large' sialidase gene and the lyase gene of C. perfr ingens are not arranged next to each other on the chromosome of this s pecies.