LACTOBACILLI CARRY CRYPTIC GENES ENCODING PEPTIDASE-RELATED PROTEINS - CHARACTERIZATION OF A PROLIDASE GENE (PEPQ) AND A RELATED CRYPTIC GENE (ORFZ) FROM LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS

Two genes, pepQ and orfZ, encoding a prolidase and a prolidase-like pr otein, respectively, were cloned and characterized from Lactobacillus delbrueckii subsp. bulgaricus. The identity of the pepQ and orfZ genes with the Lactobacillus delbrueckii subsp, lactis prolidase gene (pepQ ) was shown to be 98% and 60%, respectively. Both pepQ and orfZ were p receded by a putative promoter region. Northern analysis of pepQ mRNA revealed a 1.1 kb transcript indicating that pepQ farms a monocistroni c transcriptional unit, Under the growth conditions used, no evidence was obtained that orfZ was expressed, either by mRNA size determinatio n in Northern analysis or by primer extension analysis. With reverse t ranscription-PCR, however, the presence of monocistronic orfZ transcri pts was established. The orfZ gene could also be overexpressed in E. c oil using the vector pKKZ23-3. The sire of the protein synthesized, 41 kDa, confirmed the molecular mass of OrfZ calculated according to DNA sequence analysis. In contrast to PepQ, which showed a substrate spec ificity characteristic of prolidase enzymes, no enzymic activity for t he orfZ-encoded protein was found with the peptide substrates tested. These results indicate that orfZ is a cryptic gene, which is expressed at a very low level under the growth conditions used, it is noteworth y that homologues of the Lb, delbrueckii subsp. bulgaricus orfZ and pe pQ genes appeared to be present in both Lb. delbrueckii subsp, lactis and Lactobacillus helveticus.