rorn Lactobacillus helveticus

2 Department of Food Science, University of Wisconsi nMad ison, WI 53706, USA A prolinase (pepR) gene was cloned from an industrial Lactobacillus helveticus strain (53/7). Three clones, hybridizing with a gene probe specific for a peptidase shown to have activity against diand tripeptides, were detected from a L. helveticus genomic library constructed in Escherichia coli. None of the three clones, however, showed enzyme activity against the dior tripeptide substrates tested. One of the clones, carrying a vector with a 5.5 kb insert, was further characterized by DNA sequencing. The sequence analysis revealed the presence of two ORFs, ORFI and ORF2 of 912 and 1602 bp, respectively. ORF2, located upstream of and in the opposite orientation to ORFI, had a promoter region overlapping that of ORFI. ORFI had the capacity to encode a 35083 Da protein. When amplified by PCR, ORFI with its control regions specified a 35 kDa protein in E. coli that was able to hydrolyse dipeptides, with highest activity against Pro-Leu, whereas from the tripeptides tested, only Leu-Leu-Leu was slowly degraded. By the substrate-specif icity profile and protein homologies, the 35 kDa protein was identified as a prolinase. The activity of the cloned prolinase was inhibited by phydroxymercuribenzoate. Northern and primer-extension analyses of ORFI revealed a 1.25 kb transcript and two adjacent transcription start sites, respectively, thus confirming the DNA sequence data. ORFZ had encoding capacity for a 595 kDa protein that showed significant homology to several members of the family of ABC transporters. Determination of the mRNA levels at different growth phases revealed that the pepR gene and ORFZ are transcribed in L. helveticus at the exponential and stationary phases of growth, respectively. Furthermore, two ORFZ deletion constructs, carrying the intact pepR gene, showed that this upstream operon adversely affected PepR activity in E. coli, which explains the enzymic inactivity of the original clones.