CHARACTERIZATION OF A PROLINASE GENE AND ITS PRODUCT AND AN ADJACENT ABC TRANSPORTER GENE FROM LACTOBACILLUS-HELVETICUS

A prolinase (pepR) gene was cloned from an industrial Lactobacillus he lveticus strain (53/7). Three clones, hybridizing with a gene probe sp ecific for a peptidase shown to have activity against di- and tripepti des, were detected from a L. helveticus genomic library constructed in Escherichia coli. None of Science, University of the three clones, ho wever, showed enzyme activity against the di- or tripeptide substrates tested. One of the clones, carrying a vector with a 5.5 kb insert, wa s further characterized by DNA sequencing. The sequence analysis revea led the presence of two ORFs, ORF1 and ORF2 of 912 and 1602 bp, respec tively. ORF2, located upstream of and in the opposite orientation to O RF1, had a promoter region overlapping that of ORF1. ORF1 had the capa city to encode a 35083 Da protein. When amplified by PCR, ORF1 with it s control regions specified a 35 kDa protein in E. coli that was able to hydrolyse dipeptides, with highest activity against Pro-Leu, wherea s from the tripeptides tested, only Leu-Leu-Leu was slowly degraded. B y the substrate-specificity profile and protein homologies, the 35 kDa protein was identified as a prolinase. The activity of the cloned pro linase was inhibited by p-hydroxymercuribenzoate. Northern and primer- extension analyses of ORF1 revealed a 1.25 kb transcript and two adjac ent transcription start sites, respectively, thus confirming the DNA s equence data. ORF2 had encoding capacity for a 59.5 kDa protein that s howed significant homology to several members of the family of ABC tra nsporters. Determination of the mRNA levels at different growth phases revealed that the pepR gene and ORF2 are transcribed in L. helveticus at the exponential and stationary phases of growth, respectively. Fur thermore, two ORF2 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.