Lactobacilli carry cryptic genes encoding peptidase-related proteins: characterization of a prolidase gene (pepQ) and a related cryptic gene (orfZ) from Lactobacillus delbrueckii subsp. bulgarkus

Abstract

Two genes, pepQ and orfZ, encoding a prolidase and a prolidase-like protein, 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 preceded by a putative promoter region. Northern analysis of pepQ mRNA revealed a 1.1 kb transcript indicating that pepQ forms a monocistronic transcriptional unit. Under the growth conditions used, no evidence was obtained that orfZ was expressed, either by mRNA size determination in Northern analysis or by primer extension analysis. With reverse transcription-PCR, however, the presence of monocistronic orfZ transcripts was established. The orfZ gene could also be overexpressed in E. coli using the vector pKK223-3. The size 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 specificity characteristic of prolidase enzymes, no enzymic activity for the 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 noteworthy that homologues of the Lb. delbrueckii subsp. bulgaricus orfZ and pepQ genes appeared to be present in both Lb. delbrueckii subsp. lactis and Lactobacillus helveticus.