Probing Direct Interactions between CodY and theoppDPromoter ofLactococcus lactis

Abstract

CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes include pepN, pepC, opp-pepO1, and probably prtPM, pepX, and pepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate that L. lactis CodY interacts directly with a region upstream of the promoter of its major target known so far, the opp system. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region of oppD yielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.Genetic and biochemical research over the past decades has led to a clear picture of the proteolytic system of lactic acid bacteria. To ensure a proper nitrogen balance, several regulators are present that respond to changes in intracellular concentrations of nitrogen-containing compounds (16,24,28,30). The lactic acid bacterium Lactococcus lactis is auxotrophic for several amino acids (6). For optimal growth in milk, it has to degrade milk proteins (e.g., ␣ S1 and ␤-and -casein), because only limited amounts of free amino acids are present in this environment. An elaborate proteolytic system to release amino acids from casein, involving a number of enzymatic activities that are subject to medium-dependent regulation, has evolved in L. lactis. Casein degradation by L. lactis is a process that can be divided into three successive steps (25,51). First, the extracellular cell wall-bound serine proteinase (PrtP) liberates peptides of various sizes from casein. In the second step, the casein-derived peptides are transported into the cell by the oligopeptide transport system (Opp) or by the di-and tripeptide transport systems (DtpP and DtpT, respectively) (45). In the last step, the internalized peptides are degraded into smaller peptides and free amino acids by a large number of cytoplasmic peptidases. Two major groups of peptidases can be discerned: the endopeptidases (e.g., PepO and PepF), which perform endolytic hydrolysis of their substrates, and the aminopeptidases (e.g., PepN, PepX, and PepC), which cleave off one or two amino acids from the free N termini of their substrates (51).Transcription of a number of lactococcal genes encoding the proteins that constitute the proteolytic system is regulated similarly in response to peptide availability in the medium (17).Transcriptional luxAB fusions with the promoters of a number of peptidase, protease, and transporter genes were used t...

Complete Genome Sequence of the Prototype Lactic Acid Bacterium Lactococcus lactis subsp. cremoris MG1363

“…From the genome comparison with L. lactis IL1403, it can be concluded that opp-2 is the original copy of this locus. In L. lactis MG1363, the oppD1 promoter is regulated by CodY (24). A clustalW analysis of the upstream region of oppD1, oppD2, and oppD of L. lactis IL1403 showed marked differences (data not shown).…”

Overall control of nitrogen metabolism in Lactococcus lactis by CodY, and possible models for CodY regulation in Firmicutes

“…In vitro experiments showed that isoleucine is the most important effector among BCAA for CodY repression (this work; den Hengst et al, 2005;Shivers & Sonenshein, 2004). Since the CodY signal in L. lactis is essentially mediated by isoleucine, excess of this amino acid in the environment leads to growth inhibition by blocking inappropriately CodY-dependent biosynthesis pathways.…”

“…Moreover, the CodY-box is found in the CodY-protected region from the oppD promoter region of L. lactis (two CodY-boxes) and from the ilvB, citB, dpp, fla-che, comK and sfrA promoter regions of B. subtilis (Bergara et al, 2003;den Hengst et al, 2005;Kim et al, 2003;Serror & Sonenshein, 1996a, b;Shivers & Sonenshein, 2004). Moreover, mutations and deletions carried out on the promoter regions of dppA, hut and ureA of B. subtilis and oppD of L. lactis and that affect CodY regulation are within or immediately adjacent to the CodY-box (Eda et al, 2000;Serror & Sonenshein, 1996a;Wray et al, 1997;Wray & Fisher, 1994).…”

“…For this purpose, we used radiolabelled fragments of 250 bp, corresponding to several L. lactis promoter regions. Since it was shown that isoleucine enhanced CodY affinity for its targets in L. lactis and in B. subtilis (den Hengst et al, 2005;Shivers & Sonenshein, 2004), we tested the effect of each single BCAA on the formation of the CodY-ilvD promoter region complex (Fig. 2).…”

“…In B. subtilis, CodY has been shown to directly interact with, in addition to GTP, BCAA, in particular isoleucine (Shivers & Sonenshein, 2004). Interestingly, studies in Lactococcus lactis have shown that in this bacterium, CodY responds to BCAA, but not GTP, suggesting that the activity of CodY is modulated differently between these two bacteria (den Hengst et al, 2005;Guedon et al, 2001b;Petranovic et al, 2004). In L. lactis, CodY has been found to repress genes involved in the utilization of proteins in the medium, including the cell-wall proteinase (prtP), peptide transporters (opp, opt), peptidases (pepC, pepN) and aminopeptidases (araT, bcaT) (Chambellon & Yvon, 2003;Guedon et al, 2001a, b;Sanz et al, 2001).…”