Génétique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas cedex, France
Correspondence
Eric Guédon
eric.guedon{at}jouy.inra.fr
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ABSTRACT |
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Microarray datasets and supplementary tables are available with the online version of this paper.
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INTRODUCTION |
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CodY is a highly conserved repressor protein present in a variety of low-G+C species of Gram-positive bacteria (Ratnayake-Lecamwasam et al., 2001). In B. subtilis, CodY has been shown to bind GTP, which enhances its regulatory activity. It follows that in cases of nutrient limitation leading to a drop in GTP in the cell, CodY repression is alleviated, allowing the expression of many genes involved in cell adaptation to this poorer environmental condition. Many CodY-dependent genes are also regulated by additional mechanisms, allowing their control in response to other environmental factors (Brandenburg et al., 2002
; Debarbouille et al., 1999
; Ferson et al., 1996
; Kim et al., 2003
; Mader et al., 2004
; Mirel et al., 2000
; Nakano et al., 1991
; Oda et al., 2000
; Ogura et al., 2001
; Wray et al., 1997
; Yoshida et al., 2003
). 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
). CodY thus appears to be involved in external nitrogen supply, and its activity may be modulated by an intracellular pool of BCAA that would constitute an indicator of the nitrogen status of the cell (Guedon et al., 2001b
; Petranovic et al., 2004
). Compared to B. subtilis where the targets of CodY have been extensively studied, the global role of CodY appears to be limited in L. lactis.
In this study, more than 100 potential CodY-regulated genes have been characterized in L. lactis. Interestingly, most of them encode proteins involved in amino acid biosynthesis and in functions related to nitrogen supply, showing that L. lactis CodY globally controls nitrogen metabolism. To the best of our knowledge, L. lactis CodY is the first regulator shown to control nitrogen metabolism genes so extensively in a bacterium. A conserved 15 nt palindromic sequence located in the vicinity of the 35 box of CodY-regulated genes was characterized.
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METHODS |
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DNA manipulation procedures and strain construction.
Plasmids and total DNA were prepared as described previously (Sperandio et al., 2005). All enzymes for DNA technology were used according to the manufacturers' specifications. Electrotransformation of E. coli and L. lactis was performed as described by Maniatis et al. (1982)
and Holo & Nes (1989)
, respectively. The oligonucleotides used in this work were synthesized by Sigma Genosys (Table 1
). DNA sequencing was performed on both strands using a fluorescent sequencing procedure (Perkin-Elmer Biosystems).
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RNA isolation.
Total RNA was isolated from IL1403 wild-type and JIM8558 codY mutant strains cultivated in CDM+Cas. When the OD600 reached 0·4, cells of 25 ml portions of the cultures were pelleted and rapidly frozen in nitrogen and stored at 80 °C. Total RNA was extracted from cell pellets as described by Sperandio et al. (2005) by using the High Pure RNA Isolation Kit (Roche). RNA concentration was determined by absorbance at 260 nm, and quality of RNA preparations was checked (i) by determining the ratio A260/A280, (ii) by visualizing the integrity of the 23S and 16S rRNA bands on an agarose gel and (iii) by verifying the absence of DNA contamination by PCR.
DNA-microarray analysis and validation of data.
Genome-wide expression profiles were established by using commercial DNA microarray (Eurogentec, HO50C) containing most of the L. lactis IL1403 genes (2072 ORFs spotted in duplicate). Synthesis of CyDye fluorescently labelled cDNAs, DNA microarray hybridizations and washings were performed as previously described (Sperandio et al., 2005). DNA microarrays were scanned through two channels for the respective fluorescent dyes using a confocal laser scanner (Virtek) to monitor the fluorescence intensities. For each spot, signal and local background intensities were determined with Imagene Software (version 5.1; BioDiscovery Inc.). A local background-subtracted signal value was calculated for each spot. Expression profiles were performed with RNA extracted from two independent cultures of strains IL1403 and JIM8558 to reduce culture-dependent or growth artefacts. Each RNA was labelled twice in order to perform a Dye-Swap. Raw data from four slides were normalized with the preP software (Garcia de la Nava et al., 2003
) by using a local normalization (LOWESS method) to reduce fluorescence bias introduced by the labelling reactions as well as by the differences in fluorescence intensity between the two dyes. A ratio corresponding to the mean of normalized signal and a z-test were calculated to compare expression profiles of strain IL1403 and its derivative codY mutant as described by Garcia de la Nava et al. (2003)
. Logarithmic representations of the data are presented in Fig. 1
, allowing rapid visual inspection of data quality and reproducibility (Fig. 1a, b
) and evaluation of overall modifications of pattern expression induced by codY mutation (Fig. 1c
). Genes considered as significantly differentially expressed correspond to those for which (i) expression is, at least in one channel, twofold above the background, (ii) the P-value is lower than 0·35 and (iii) the differential expression level is higher than 2 (induced gene) or lower than 0·5 (repressed gene). Raw datasets are available as supplementary material with the online version of this paper. Additional analyses were performed to confirm the significance of the DNA-microarray data including (i) consistency with previous knowledge of the CodY regulated genes, (ii) consistency with the expression levels between genes belonging to the same clusters and (iii) additional measures by real-time quantitative PCR (RT-QPCR).
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Crude extract preparation and gel retardation assay.
The protein extracts used for the biochemical assays were prepared from B. subtilis codY carrying pHT315-Pxyl (JIM8168) or pHT315-Pxyl-codYlactis plasmid (JIM8169) (Petranovic et al., 2004
). In strain JIM8169, the L. lactis CodY regulator is overproduced from the inducible xylA promoter while the control strain JIM8168 does not contain the codY gene. Cells were grown in 250 ml LB medium in the presence of 20 mM xylose. At culture saturation, cells were harvested and washed once with TN buffer and then resuspended in 1·5 ml lysis buffer (Yoshida et al., 1999
). The cells were disrupted by sonication (30 min) with a Vibra Cell Disrupter (Bioblock Scientific). After centrifugation, 40 % glycerol was added to the supernatant and the preparation was used as the CodY-enriched (JIM8169) or control (JIM8168) extract.
DNA probes of about 250 bp corresponding to the promoter regions of cysD, gltA, ilvD, serC, thrA, pepN and asnB were amplified by PCR using specific primers (Table 1) and labelled at the 5' end with [
-32P]ATP by the T4 polynucleotide kinase (NEB). Fragments carrying ilvD, cysD and gltA promoter regions with specific substitutions were obtained by PCR using mutated primers (Table 1
). Unincorporated nucleotides were removed with the NucleoSpin PCR purification kit (Macherey Nagel). CodYDNA complexes were formed in 10 µl by incubating the 32P-labelled probes (10 fmol DNA) with different amounts of B. subtilis crude extract (2·520 µg) in binding buffer [20 mM Tris (pH 7·8), 100 mM KCl, 0·5 mM EDTA, 1 mM DTT, 10 % glycerol] in the presence of 0·1 g poly(dI-dC) l1 and 10 mM isoleucine (pH 7·8). Reaction mixtures were incubated at 25 °C for 10 min and electrophoresis was carried out at 4 °C in TAM buffer as previously described (Sperandio et al., 2005
).
Bioinformatic procedures.
iMoMi is a multigenomic relational database around which are implemented different utilities to facilitate the search of operator sequences (N. Pons and others, unpublished). A first utility allows automatic extraction of orthologous gene promoter regions from a set of genomes. In this work we used a set of 14 genomes from low-G+C % Gram-positive bacteria including L. lactis IL1403 (accession no. AE005176), Streptococcus agalactiae NEM316 (accession no. AL732656), Streptococcus pneumoniae R6 (AE007317), Streptococcus mutans UA159 (AE014133), Streptococcus pyogenes MGAS315 (AE014074), Streptococcus thermophilus CNRZ 1066 (CP000024), Staphylococcus aureus Mu50 (BA000017), Staphylococcus epidermidis ATCC 12228 (AE015929), Bacillus cereus ATCC 14579 (AE016877), B. subtilis 168 (AL009126), Listeria monocytogenes EGD-e (NC_003210), Enterococcus faecalis VE583 (AE016830), Lactobacillus plantarum WCFS1 (AL935263), Lactobacillus johnsonii NCC 533 (AE017198) and Clostridium acetobutylicum ATCC 824 (AE001437). A second utility allows the extraction of a promoter set from microarray data. It is then possible to achieve automatically a search of motifs by combining the two utilities, by first selecting a set of promoters from the microarray data (here from IL1403 versus its codY derivative) and then their orthologous genes in the selected set of genomes. The last step is to search motifs by an appropriate procedure; the MEME algorithm (Bailey & Elkan, 1994) was used in the present work. Finally, an extensive search for a motif in all promoter regions of the selected genomes (Dsouza et al., 1997
) was done.
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RESULTS |
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The reliability of the DNA-microarray data was tested by (i) corroborating the data with previous knowledge on CodY-regulated genes, (ii) checking the consistency of expression levels within each cluster and (iii) performing additional measures by RT-QPCR. First, except for araT, pepX and dtpT, all genes already known to be regulated by CodY, such as pepC, pepN, bcaT and the genes of the opp and opt operon, were retrieved by our DNA-microarray analysis (Chambellon & Yvon, 2003; Guedon et al., 2001a
, b
; Sanz et al., 2001
). Second, the change in the expression level of 75 clustered genes was consistent with their genetic organization. Only four genes out of these 23 clusters (argE, oppB, oppC and malQ) were rejected by our criteria, but manual inspection revealed that their scores were just below the threshold used. Last, the relative expression level of 17 genes was measured by RT-QPCR from new cultures in CDM+Cas of the wild-type and the codY mutant strains (Fig. 1d
). The ratio values obtained in these experiments were similar to that obtained previously by DNA microarray, with a Pearson correlation coefficient of 0·91.
Of the 110 genes, 21 were not tested by the above criteria. Among these, 13 had a highly significant differential expression (P-value <0·05), suggesting that they are also likely to be regulated by CodY. We decided to include them in further analysis. From the clustering of the 110 genes, we deduced that CodY regulates 61 transcriptional units either directly or indirectly. As expected for a repressor, most of these genes (104, 95 %) are negatively controlled. Among these, 56 are downregulated more than fourfold, indicating their tight control by CodY.
Analysis of the L. lactis CodY regulon
Analysis of the potential functions of the 110 genes reveals that 18 putative transcription units, which contain a total of 50 genes, encode proteins involved in amino acid biosynthesis pathways or are clustered with this class of genes. This set of repressed genes includes the pathway for biosynthesis of the sulfur amino acids, histidine, lysine, threonine, asparagine, aromatic amino acids, serine, glycine, glutamate and BCAA (Table 2). It also includes four genes organized in two clusters linked to the TCA cycle (pycA, gltA, citB, icd) that have, as a major role in L. lactis, glutamate and aspartate synthesis from central metabolism. Moreover, 19 genes encode proteins involved in peptide and amino acid transport (ctrA, gltS, oppDFCBA, optSABCDF, ydgCB, yibG, yshA) and peptide degradation (pepC, pepN). All these genes are repressed by CodY except two, gltS and yshA, encoding amino acid transporters. In addition to the above genes, involved in nitrogen supply, CodY also represses 17 genes from central and intermediary metabolism such as the glycerol assimilation pathway (glgKDF2, ypjH), organic acid fermentation (mleSP, lctO, aldB) and polysaccharide metabolism (amyL, ygjD-malQ-glgCDAP-apu, xylH). Last, the transcript level of six genes involved in regulatory processes is also modified by codY inactivation. Three are related to nitrogen metabolism (gadR, aldR, glnB), one encodes HslA, a histone-like DNA-binding protein, and two are still uncharacterized (yohC and ymhA).
Interestingly, CodY represses the expression of six genes (yfgC, yaiEF, yxdFG and yaiH) which might be involved in cell communication or in bacteriocin production. YfgC shares 31 % identity with the LacA lacticin RM, a lactococcal bacteriocin (Yarmus et al., 2000). YaiF, YxdF and YaiH have all the features of the CAAX amino-terminal protease family proteins, while YaiE and YxdG are transport and binding proteins. The CAAX proteases are membrane-bound metalloproteases involved in protein and/or peptide modification (Pei & Grishin, 2001
). In Lactobacillus plantarum, one such protein is associated with a cluster involved in bacteriocin production (Pei & Grishin, 2001
).
Specific CodY binding to the promoter region of regulated genes
In order to show that CodY directly regulates transcription of the newly identified CodY-dependent genes, we carried out gel retardation experiments with a codY B. subtilis crude protein extract from the JIM8169 cells producing high amounts of L. lactis CodY (Petranovic et al., 2004
). 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
). Whereas the migration of the ilvD probe was not modified in the presence of 10 µg CodY-containing extract, addition of isoleucine induced the formation of the CodY-ilvD promoter fragment complex, as observed by the mobility shift of the probe. The effect of leucine or valine was lower or insignificant, respectively. These results confirmed that isoleucine is the main signal for CodY regulation and that crude cell extracts can be used to test CodY targets in L. lactis. Further binding experiments were carried out in the presence of 10 mM isoleucine.
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First, the omission of asparagine from CDM increased the lag phase of L. lactis NCDO 2118 from 3 to 15 h. This lag was reduced to 9 h if isoleucine was removed as well, showing that isoleucine interfered negatively with asparagine biosynthesis. Second, L. lactis NCDO 2118 was not able to grow in CDM containing isoleucine, leucine, valine, glutamate, serine and methionine as nitrogen source (CDM6). Surprisingly, its growth could be restored by the addition of asparagine, histidine or lysine plus threonine, which are not required in CDM. Interestingly, if isoleucine was omitted from CDM6, the additional amino acids were no longer required, suggesting an isoleucine CodY-mediated growth repression.
Identification of a CodY regulatory box
We have shown that CodY regulator directly controls several promoter regions by band-shift experiments. Since most regulators recognize a motif to promote their binding to specific sites, we searched for sequence patterns conserved among CodY-regulated genes in order to detect a CodY regulatory box (CodY-box). For this purpose, the ExtractMotif program using the MEME algorithm (Bailey & Elkan, 1994; N. Pons and others, unpublished) was used to find a conserved motif in the region upstream of the start codon of the optS, ilvD, gltA, asnB, ctrA, gltB, yaiH and amtB genes. A conserved 15 nt sequence with AATTKTCAGAMWAWW as degenerate consensus was identified in the promoter region of these genes (Fig. 4
). This motif is found twice in the gltA and optS promoter regions. Interestingly, the motif is located in the vicinity of the potential promoter sequences and often overlaps them (Fig. 4
).
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To test the relevance of the CodY-box on CodY DNA binding, we used a gel retardation assay (Fig. 5). Replacement of the leading AA of the consensus and the A just preceding the consensus with CGG completely abolished the CodY binding to the gltA promoter, as did the replacement of the GAA with a TGG, while TC
GT and AT
GC substitutions modified significantly the migration of the complex. Moreover, while the addition of an excess of gltA unlabelled probe dramatically decreased CodY binding to the same labelled DNA fragment, no modification of the CodY binding was observed in the presence of an excess of gltA unlabelled probe carrying substitutions in the CodY-box (Fig. 5c
). Finally, single substitutions in the cysD and ilvD CodY-boxes also affected the ability of CodY to form a complex with its targets (data not shown). These experiments indicate that the motif identified by the bioinformatic approach is a CodY DNA-binding site.
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A second search was carried out to find sets of orthologous genes that contain the CodY motif in the genomes of L. lactis, Str. agalactiae, Str. pneumoniae, Str. mutans, Str. pyogenes, Staphylococcus aureus, Staph. epidermidis, B. cereus, B. subtilis, Listeria monocytogenes, E. faecalis and C. acetobutylicum (see Supplementary Table 2, available with the online version of this paper). Strikingly, CodY motifs are present upstream of almost all BCAA biosynthesis genes, specific BCAA transporters and oligopeptide transporters, which are known CodY targets in L. lactis and B. subtilis. Furthermore, several other amino acid biosynthesis genes, previously shown to be CodY-dependent in L. lactis, are also preceded by a CodY-box in most genomes (aspartokinase, aspartate
-semialdehyde dehydrogenase, homoserine dehydrogenase, phosphoserine aminotransferase, threonine synthase and O-acetylhomoserine thiolase). Among transporter genes preceded by the CodY motif are amtB, encoding ammonium transporter, and several amino acid transporters of unknown specificity. Last, a CodY motif binding site is present in the close vicinity of the 35 box of the promoters controlling the transcription of CodY in the bacteria of the genera Lactococcus, Streptococcus, Enterococcus, Staphylococcus and Clostridium.
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DISCUSSION |
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Overall control of nitrogen metabolism by CodY
As already described, CodY controls the major genes involved in peptide assimilation, including their transport and their further degradation by peptidases (Guedon et al., 2001b). Furthermore, CodY also controls the expression of five genes encoding transporters involved in the uptake of free amino acids. CodY thus exerts a control on most genes involved in external amino acid supply. Moreover, CodY also represses the expression of de novo amino acid biosynthesis genes. This control covers full sets of genes encoding amino acid biosynthesis pathways such as BCAA, glutamate-glutamine, histidine, serine, threonine, lysine and asparagine. It also applies to single steps, often located at the entry of the pathways, such as aroH, the first step of the common branch leading to aromatic amino acid biosynthesis, and hom and cysD, involved in methionine synthesis. Finally, the CodY control is indirect on cysteine and glycine biosynthesis pathways, which use serine as precursor, and on proline, arginine and alanine, which require glutamate for their biosynthesis. Among these genes, we have shown that CodY binds to the promoter regions of ilvD, gltA, thrA, pepN, ctrA, asnB and cysD, confirming the direct control of CodY on their transcription. Finally, CodY also represses glycogen metabolism and glycerol assimilation pathways that may fuel the cell with a carbon skeleton for amino acid biosynthesis upon sugar starvation. These results indicate that CodY controls all pathways for nitrogen supply in L. lactis by modulating de novo amino acid biosynthesis and the import of secondary nutritional sources.
Strikingly, BCAA biosynthesis genes are among the most tightly regulated by CodY in L. lactis and are also controlled by CodY in B. subtilis (Molle et al., 2003). BCAA are the intracellular effectors activating CodY repression in L. lactis (Guedon et al., 2001b
; Petranovic et al., 2004
) and in B. subtilis (Shivers & Sonenshein, 2004
). Thus, both in L. lactis and in B. subtilis, CodY exerts a feedback control on the pathway involved in the biosynthesis of its direct effectors. This control is direct since CodY binds to the promoter region of ilvB, ilvD and ybgE in B. subtilis (Shivers & Sonenshein, 2004
) and ilvD in L. lactis (this work). Furthermore, isoleucine strongly increases the affinity of CodY for these promoters, confirming that BCAA exerts a similar effect on CodY in L. lactis and B. subtilis (Shivers & Sonenshein, 2004
; this work).
The role of CodY in nitrogen supply is significantly extended in L. lactis compared to B. subtilis since in the latter, CodY controls the biosynthesis of BCAA, asparagine and glutamate precursors and amino acid and peptide transporters (Molle et al., 2003). Previously, the role of CodY from B. subtilis has been compared to that of Lrp from E. coli, the leucine-responsive regulatory protein (Molle et al., 2003
). In this bacterium, Lrp alters the expression of about one-tenth of the genes, including several genes involved in the synthesis of amino acids and their transport and the opp operon (Hung et al., 2002
; Tani et al., 2002
). However, this control is less strong, it might be positive (e.g. for leuD, serA and proB) and does not encompass all pathways. To our knowledge, L. lactis CodY represents the first example of such a global and tight control on nitrogen metabolism by a single transcriptional regulator in bacteria.
Isoleucine signal and nitrogen metabolism
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. Several studies in chemically defined media have reported a CodY-dependent isoleucine growth inihibition in L. lactis linked to repression of the biosynthesis of valine and leucine (Goupil-Feuillerat et al., 1997
), asparagine, histidine, lysine and threonine (this work) and glutamate (Lapujade et al., 1998
). In the latter case, the growth defect was shown to be the result of a reduced synthesis of 2-oxoglutarate associated with a low level of the glutamate synthase (Lapujade et al., 1998
). These results can now be explained by the CodY-mediated tight control of the gltA-citB-icd and gltBD operons in the presence of isoleucine. Last, CodY-dependent isoleucine inhibition has also been observed in complex media such as milk (Chambellon & Yvon, 2003
), showing that a strong imbalance of isoleucine leads to growth inhibition.
CodY targets in Gram-positive bacteria
Although extensively studied in B. subtilis, no consensus sequence has yet been found for the CodY-binding site and it has been suggested that CodY might not recognize a primary sequence but tertiary DNA structures (Serror & Sonenshein, 1996a). In this work, we have characterized a 15 nt palindromic motif with AATTTTCNGAAAATT as consensus sequence. This motif is present in the promoter region of 63 % of the CodY-dependent transcriptional units identified by transcriptome analysis in L. lactis and of 40 % of the known CodY B. subtilis targets. Furthermore, a CodY-box is also located in its own promoter region and in those of BCAA biosynthesis, amino acid and oligopeptide transporter genes of Gram-positive bacteria of the genera Staphylococcus, Bacillus, Listeria and Streptococcus, suggesting the general significance of this motif in bacteria containing the CodY regulator.
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
). To improve the relevance of our in silico analysis, biochemical experiments coupled with mutation analysis have been carried out on several CodY-dependent gene promoters of L. lactis. In each case, mutations affecting the characterized motif decreased or impaired the ability of CodY to form a complex with the promoter regions of its target genes, demonstrating the CodY operator role of the CodY-box.
Although many CodY-regulated gene promoters contain the CodY-box, 60 % and 30 % of the CodY targets from B. subtilis and L. lactis, respectively, have no CodY motif with less than three mismatches. This suggests that (i) additional degeneracy rules exist to define this motif, (ii) CodY may also recognize DNA structures as suggested by Serror & Sonenshein (1996a), or (iii) CodY indirectly controls their expression level. Notably, we have shown in this paper that the pepN promoter was a direct target of CodY, although it does not display the presently defined CodY-box, suggesting that additional work is required to fully understand the interaction of CodY with its targets.
Conclusion
We have shown that CodY controls amino acid supply in L. lactis, including protein degradation pathways, uptake of peptides and amino acids and de novo biosynthesis, providing here the first example of an overall control of nitrogen metabolism by a single regulator. Characterization of a CodY-box in L. lactis and its distribution in other Gram-positive bacteria suggest that CodY is generally involved in nitrogen control in Firmicutes. As CodY effector, isoleucine would have a predominant role in sensing nitrogen availability in these micro-organisms.
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ACKNOWLEDGEMENTS |
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Received 10 May 2005;
revised 8 September 2005;
accepted 9 September 2005.
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