A prolinase gene was cloned from an industrial strain (53/7). Three clones, hybridizing with a gene probe specific for a peptidase shown to have activity against di- and tripeptides, were detected from a genomic library constructed in None of the three clones, however, showed enzyme activity against the di- or 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, ORF1 and ORF2 of 912 and 1602 bp, respectively, ORF2, located upstream of and in the opposite orientation to ORF1, had a promoter region overlapping that of ORF1. ORF1 had the capacity to encode a 35083 Da protein. When amplified by PCR, ORF1 with its control regions specified a 35 kDa protein in 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-specificity profile and protein homologies, the 35 kDa protein was identified as a prolinase. The activity of the cloned prolinase was inhibited by -hydroxymercuribenzoate. Northern and primer-extension analyses of ORF1 revealed a 1.25 kb transcript and two adjacent transcription start sites, respectively, thus confirming the DNA sequence data. ORF2 had encoding capacity for a 59.5 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 gene and ORF2 are transcribed in at the exponential and stationary phases of growth, respectively. Furthermore, two ORF2 deletion constructs, carrying the intact gene, showed that this upstream operon adversely affected PepR activity in which explains the enzymic inactivity of the original clones.


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