X-prolyl dipeptidyl aminopeptidase gene (pepX) is part of the glnRA operonin Lactobacillus rhamnosus

A peptidase gene expressing X-prolyl dipeptidyl aminopeptidase (PepX) activ ity was cloned from Lactobacillus rhamnosus 1/6 by using the chromogenic su bstrate L-glycyl-L-prolyl-beta-naphthylamide for screening of a genomic lib rary in Escherichia coli, The nucleotide sequence of a 3.5-kb HindIII fragm ent expressing the peptidase activity revealed one complete open reading fr ame (ORF) of 2,391 nucleotides. The 797-amino-acid protein encoded by this ORF was shown to be 40, 39, and 36% identical with PepXs from Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis, respectivel y. By Northern analysis with a pepX-specific probe, transcripts of 4.5 and 7.0 kb were detected, indicating that pepX is part of a polycistronic opero n in L. rhamnosus. Cloning and sequencing of the upstream region of pepX re vealed the presence of two ORFs of 360 and 1,338 bp that were shown to be a ble to encode proteins with high homology to GlnR and GlnA proteins, respec tively. By multiple primer extension analyses, the only functional promoter in the pepX region was located 25 nucleotides upstream of glnR, Northern a nalysis with glnA- and pepX-specific probes indicated that transcription fr om glnR promoter results in a 2.0-kb dicistronic glnR-glnA transcript and a lso in a longer read-through polycistronic transcript of 7.0 kb that was de tected with both probes in samples from cells in exponential growth phase, The glnA gene was disrupted by a single crossover recombinant event using a nonreplicative plasmid carrying an internal part of glnA, In the disruptio n mutant, glnRA specific transcription was derepressed 10-fold compared to the wild type, but the 7,0-kb transcript was no longer detectable with eith er the glnA- or pepX-specific probe, demonstrating that pepX is indeed part of glnRA operon in L. rhamnosus, Reverse transcription-PCR analysis furthe r supported this operon structure. An extended stem-loop structure was iden tified immediately upstream of pepX in the glnA-pepX intergenic region, a s equence that showed homology to a 23S-5S intergenic spacer and to several o ther L. rhamnosus-related entries in data banks.