Transcriptional Pattern of Genes Coding for the Proteolytic System of Lactococcus lactis and Evidence for Coordinated Regulation of Key Enzymes by Peptide Supply

Abstract

The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), P I and P III proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) of Lactococcus lactis MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37°C, and peptide supply on the transcription of these genes. Only transcription of the pepP gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes. prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5-to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription of prtP1, prtP3, pepC, pepN, and the opp-pepO1 operon is repressed two-to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon. The significance of these results with respect to the functions of different components of the proteolytic system in L. lactis are discussed.

The Lactococcus lactis CodY Regulon

“…As anticipated, the levels of transcription of the genes that constitute the opp operon were elevated in L. lactis MG1363⌬codY, as this operon has been identified previously as being repressed by CodY (13,17). Although purified histidine-tagged CodY has been shown to directly interact with the upstream regions of the genes encoding the peptidases pepN and pepC, their expression levels were not significantly changed or only marginally elevated (below 1.5-fold), respectively, in the codY mutant (17).…”

“…The bcaT gene is repressed by CodY in a chemically defined medium supplemented with casitone, a complex source of nitrogen, and in that respect comparable with the GM17 used in this work (13,33). Although expression of bcaT was significantly altered (p Ͻ 0.001) in our experiments as well, the extent of derepression measured on the DNA microarrays was somewhat low compared with that observed by Chambellon and Yvon (54).…”

“…In fact, it has been shown recently that both transport systems have similar substrate specificities (10). As is the case for the known CodY targets, expression of dpp/opt is repressed in peptiderich media and especially in media containing peptides with BCAA residues (11,13). These observations demonstrate that dpp/opt is another member of the CodY regulon.…”

“…When L. lactis encounters limiting amounts of nitrogen, repression is relieved (13,14). From recent studies it has become apparent that the transcriptional regulator CodY is responsible for repression of the genes prtP/prtM, opp, pepN, pepC, araT, and bcaT in response to nitrogen availability (14 -17).…”

Catalytic Activity of Tripeptidase fromLactococcus lactisto Which Amino Acid Substitution Was Introduced According to Natural Mutation

“…1,2) All PepTs from various microorganisms have been shown to hydrolyze only tripeptides as substrates. 3) Tripeptidases from Lactococcus lactis were purified and characterized.…”