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

Eric Guédon, Brice Sperandio, Nicolas Pons, Stanislav Dusko Ehrlich and Pierre Renault

Génétique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas cedex, France

Correspondence
Eric Guédon
eric.guedon{at}jouy.inra.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CodY, a pleiotropic transcriptional regulator conserved in low G+C species of Gram-positive bacteria, was previously described to be the central regulator of proteolysis in Lactococcus lactis. In this study, over 100 potential CodY targets were identified by DNA-microarray analysis. Complementary transcriptional analysis experiments were carried out to validate the newly defined CodY regulon. Moreover, the direct role of CodY in the regulation of several target genes was demonstrated by gel retardation experiments. Interestingly, 45 % of CodY-dependent genes encode enzymes involved in amino acid biosynthesis pathways, while most of the other genes are involved in functions related to nitrogen supply. CodY of L. lactis represents the first example of a regulator in Gram-positive bacteria that globally controls amino acid biosynthesis. This global control leads to growth inhibition in several amino-acid-limited media containing an excess of isoleucine. A conserved 15 nt palindromic sequence (AATTTTCNGAAAATT), the so-called CodY-box, located in the vicinity of the –35 box of target promoter regions was identified. Relevance of the CodY-box as an operator for CodY was demonstrated by base substitutions in gel retardation experiments. This motif is also frequently found in the promoter region of genes potentially regulated by CodY in other Gram-positive bacteria.


Abbreviations: BCAA, branched-chain amino acid(s); RT-QPCR, real-time quantitative PCR

Microarray datasets and supplementary tables are available with the online version of this paper.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In prokaryotes, the metabolism of amino acids is regulated by many specific mechanisms, including biochemical control of enzymes and of their synthesis in response to the availability of particular metabolites. In addition to these targeted regulatory mechanisms, more broad-range regulatory systems have been described, allowing a coordination of the whole cell machinery in response to the environment. This higher hierarchical mechanism might be carried out at the transcriptional level by global regulators, also called pleiotropic regulators. Examples of such regulators are now available for different bacteria, the paradigm being Escherichia coli, where seven regulatory proteins (CRP, FNR, IHF, FIS, ArcA, NarL and Lrp) directly modulate the expression of 51 % of the genes (Arfin et al., 2000; Hung et al., 2002; Kang et al., 2005; Martinez-Antonio & Collado-Vides, 2003; Zheng et al., 2004). Broad-range regulators have also been described in Gram-positive bacteria, examples being Bacillus subtilis CcpA and CodY (Blencke et al., 2003; Molle et al., 2003; Moreno et al., 2001; Yoshida et al., 2001). CcpA is involved in catabolic repression and controls the expression of about 10 % of B. subtilis genes (Blencke et al., 2003; Moreno et al., 2001; Yoshida et al., 2001). The role exerted by this global regulator can be compared to that of CRP in E. coli (Moreno et al., 2001). CodY represses the expression of a wide variety of genes involved in macromolecular degradation, nutrient transport, amino acid catabolism, branched-chain amino acid (BCAA) biosynthesis, genetic competence, antibiotic synthesis, motility and chemotaxis (Bergara et al., 2003; Debarbouille et al., 1999; Ferson et al., 1996; Fisher et al., 1996; Inaoka & Ochi, 2002; Inaoka et al., 2003; Kim et al., 2003; Lazazzera et al., 1999; Mirel et al., 2000; Molle et al., 2003; Serror & Sonenshein, 1996a, b; Shivers & Sonenshein, 2004; Slack et al., 1995; Wray et al., 1997). In controlling expression of these late-exponential-phase and early-stationary-phase genes, the regulation by CodY allows cells to adapt to general nutrient limitation (Molle et al., 2003). The role of CodY has been compared with that of Lrp from E. coli, although these regulators do not share any structural similarities (Molle et al., 2003; Shivers & Sonenshein, 2004).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth conditions, bacterial strains and media.
Escherichia coli TG1 was used for plasmid propagation (Gilson, 1984). E. coli and B. subtilis strains were grown in Luria–Bertani (LB) medium at 37 °C under vigorous aeration (Maniatis et al., 1982). L. lactis strains were grown at 30 °C without aeration in M17 or in a chemically defined medium (CDM) containing 0·5 g trehalose l–1 as carbon source and all amino acids except aspartic acid and glutamic acid as nitrogen source (Sissler et al., 1999; Terzaghi & Sandine, 1975). When required, 1·8 % Casitone (Sigma) was added (CDM+Cas) (Guedon et al., 2001a). CDM6 that derived from CDM contains only glutamate, isoleucine, leucine, methionine, serine and valine as source of amino acids (Lapujade et al., 1998). To test the growth, precultures of L. lactis in CDM were washed twice in Ringer's solution and inoculated at 1 % in fresh CDM or its derivatives. Growth was measured kinetically with a Microbiology Reader Bioscreen C (Labsystems). When required, X-Gal (20 µg ml–1), erythromycin (5 µg ml–1 for L. lactis, 100 µg ml–1 for E. coli, 1 µg ml–1 for B. subtilis), ampicillin (100 µg ml–1 for E. coli), spectinomycin (200 µg ml–1 for B. subtilis) and neomycin (5 µg ml–1 for B. subtilis) were added.

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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Table 1. Oligonucleotide primers used for PCR amplification

The BamHI restriction sites designed for codY gene deletion in L. lactis strain IL1403 are in bold. The substitution bases designed into the CodY-box of the gltA, cysD and ilvD genes are underlined.

 
The chromosomal codY gene deletion of L. lactis IL1403 was constructed as follows. DNA fragments of 611 bp and 662 bp carrying, respectively, the upstream and the downstream region of the codY gene were generated by PCR. These fragments contain a BamHI restriction site at their 3' and 5' ends, respectively, designed in codY up and codY down primers (Table 1). The PCR-amplified products were digested with BamHI, ligated and cloned into the pGEM-T easy cloning vector (Promega). This plasmid was fused to the lactococcal thermosensitive vector pGhost 9 at the SpeI site to yield pJIM1080 codY deletion vector. In L. lactis IL1403, the codY gene was replaced by the inactive copy carried by pJIM1080 by double crossing over as described by Biswas et al. (1993), to give strain JIM8558. PCR amplification and Southern blotting were performed to verify the effective codY gene deletion in strain JIM8558.

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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Fig. 1. (a, b, c) Logarithmic-scale plots of spot intensities determined from microarray hybridizations. Comparison of logarithmic plot of spot mean normalized intensities of array hybridizations with two independent samples (a) from the wild-type strain (WT1 versus WT2) and (b) from the codY mutant strain (codY1 versus codY2). Each spot intensity corresponds to the mean of four different spot normalized intensity values. (c) Logarithmic plot of spot mean normalized intensities of array hybridizations from the wild-type and the codY mutant strains (WT versus codY). Each spot intensity corresponds to the mean of eight different normalized intensity values. (d) Scatter plot comparative analysis of microarray and RT-PCR experiments. Each data point represents the logarithmic ratio of the signal intensity determined for 17 CodY-dependent genes (ahpF, asnB, ctrA, cysD, glgD, glpF2, gltA, gltS, ilvD, lctO, optS, serC, yaiF, yncA, yohC, yshA, yxdG) for the wild-type and the codY mutant strains, as determined by both RT-QPCR and microarray analyses. R2, Pearson correlation coefficient.

 
Twenty genes (pi127, pi241, pi319, pi326, fhuD, mesJ, purC, purD, purE, purF, purH, purK, purL, purM, purN, ptsH, rpmGA, ypgD, yphC and ypdC) were excluded as probable artefacts (they belong to large clusters not differentially expressed, with expression levels just above the threshold; independent additional measures did not confirm DNA-microarray data). These 20 genes are thus not presented in Table 2.


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Table 2. CodY-regulated genes identified by DNA-microarray experiments

 
RT-PCR amplification.
RT-QPCR was performed using first-strand DNA as template. cDNAs were synthesized from 20 µg RNA samples as described above but without using aminoallyl-dUTP. Specific primers for each studied gene (Table 1) were designed using eprimers software (http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html). PCR was performed using the ABsolute QPCR SYBR Green MIX (ABgene) and was run on an ABI 7700 instrument (Applied Biosystems) as described previously (Sperandio et al., 2005). For each strain, cDNA was synthesized from RNA extracted from three independent cultures and performed in triplicate for each gene. Internal control and normalization of the results were done with the tuf gene encoding the elongation factor TU.

Crude extract preparation and gel retardation assay.
The protein extracts used for the biochemical assays were prepared from B. subtilis {Delta}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 [{gamma}-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). CodY–DNA complexes were formed in 10 µl by incubating the 32P-labelled probes (10 fmol DNA) with different amounts of B. subtilis crude extract (2·5–20 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) l–1 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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Determination of CodY-regulated genes
In order to identify genes regulated by CodY, the expression of 2072 genes was quantified by the use of commercial L. lactis DNA microarrays. The experiment was carried out by comparing the expression profiles from L. lactis IL1403 and its derivative deleted for the codY gene grown in repressive conditions for CodY. For this purpose, total RNA was isolated from the two strains grown in CDM+Cas and harvested in the mid-exponential growth phase. Fig. 1 presents an overall view of the modifications of the expression pattern consequent to codY inactivation. Table 2 presents 110 out of the 130 genes that displayed (i) a significant signal, (ii) a differential trancript level higher than twofold and (iii) a P-value lower than 0·35. Twenty genes were rejected on the basis of experiments and analysis performed to validate the DNA-microarray data (see Methods).

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 {Delta}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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Fig. 2. Effect of BCAA on CodY DNA binding to ilvD promoter region. A labelled DNA fragment containing the ilvD promoter region (10 fmol) was incubated with or without 10 µg CodY-enriched crude extract from B. subtilis cells in the presence or not of 10 mM of one of the three BCAA (I, isoleucine; L, leucine;V, valine) and analysed on non-denaturing polyacrylamide gel electrophoresis.

 
Probes corresponding to ilvD, ctrA, gltA, asnB, thrA, pepN and cysD (Fig. 3a, b, c, d, e, f, respectively, and not shown for cysD) were retarded in the presence of 2·5 µg CodY-containing extract and the shift reached a maximum with 20 µg total proteins (lanes 2–5). As control, no band shift was detected with 20 µg crude extract of the {Delta}codY B. subtilis control strain JIM8168 (lane 6). Finally, no band shift could be detected if the CodY-independent metA promoter was used as probe in the presence of CodY-containing extract (data not shown). These data demonstrate the formation of a specific CodY complex with seven promoter regions and suggest the direct control of these regions by CodY.



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Fig. 3. Gel mobility shift assay for CodY binding to different regulated gene promoter regions. The promoter regions of CodY-dependent genes were PCR amplified and labelled. Radiolabelled DNA probes (10 fmol) [ilvD (a), ctrA (b), gltA (c), asnB (d), thrA (e), pepN (f)] were incubated with B. subtilis crude extracts at various concentrations, and analysed by non-denaturing PAGE (see Methods for details). Lane 1 contains no protein.Lanes 2–5 contain 2·5, 5, 10 and 20 µg CodY-enriched crude extract, respectively. Lane 6 contains 20 µg control crude extract.

 
Effect of isoleucine-dependent CodY repression on amino acid metabolism
The repression of CodY on such a large panel of genes involved in nitrogen supply as a function of the concentration of isoleucine, as single signal, is surprising. Indeed, in the presence of an excess of isoleucine, it might be expected that CodY would interfere with the biosynthesis of many other amino acids, and therefore have a negative effect on growth. Growth inhibition phenomena due to isoleucine (hereafter designated isoleucine inhibition) were therefore sought.

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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Fig. 4. Sequence alignment of promoter regions of CodY-dependent genes. The ATG start codon of each gene is indicated in italics. The potential –10 and –35 boxes for each promoter are underlined. The identified CodY-boxes are shaded. The degenerate and the strict palindromic consensus are deduced from the alignment of the CodY-boxes. K (T or G), M (A or C), W (A or T), N (A, T, G or C).

 
We next searched for the strict palindromic AATTTTCNGAAAATT sequence (three mismatches accepted) in all potential promoter regions of the L. lactis IL1403 genome (Bolotin et al., 2001). Interestingly, 40 out of the 64 promoter regions of CodY-regulated genes contain at least one CodY-box (Table 2). Moreover, these newly found motifs are located in most cases less than 20 nt from the predicted –35 or –10 boxes of the promoters, a location well in agreement with their potential regulatory role and therefore strengthening their significance.

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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Fig. 5. In vitro analysis of the role of the CodY-box in the DNA-binding ability of CodY. DNA probes (10x10–15 mol) were incubated without (–) or with (+) CodY-enriched crude extract (b, 20 µg; c, 5 µg) in the presence of 10 mM isoleucine and analysed by non-denaturing PAGE. (a) Sequence of the wild-type (gltAwt) and mutated (gltAM1, M2, M3, M4) gltA CodY DNA-binding box. (b) Gel mobility shift assay for CodY binding to gltAwt, gltAM1, gltAM2, gltAM3 and gltAM4 promoter regions. (c)Gel mobility shift assay for CodY binding to gltAWT promoter region in the presence of an excess (10x10–13 mol) of unlabelled gltAwt or gltAM3 probes. ~, Contains, as negative control, 10 µg control crude extract (without CodYlactis).

 
CodY-box in B. subtilis and other Gram-positive bacteria
To check for the existence of the CodY-box in bacteria other than L. lactis, we carried out a search for the occurrence of the AATTTTCNGAAAATT sequence (three mismatches accepted) in the intergenic regions of the B. subtilis genome. This search provided 248 hits in front of 228 genes (20 genes have two potential CodY-boxes). Importantly, among the 67 promoter regions determined as CodY targets by Chip-to-Chip experiments (Molle et al., 2003), 25 displayed at least one CodY-box (see Supplementary Table 1, available with the online version of this paper).

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 {beta}-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.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CodY is a transcriptional regulator found in many low G+C species of Gram-positive bacteria. Most studies of this regulator have been carried out in B. subtilis and L. lactis. In the former, CodY was shown to repress expression of a wide variety of genes involved in macromolecular degradation, nutrient transport, amino acid catabolism, BCAA biosynthesis, genetic competence, antibiotic synthesis and motility and chemotaxis (Molle et al., 2003). In the latter, CodY was found to be the central regulator for proteolysis and peptide assimilation pathways (Chambellon & Yvon, 2003; Guedon et al., 2001a, b; Sanz et al., 2001). In this work, over 100 potential CodY targets were found by DNA microarrays, and a consensus for the CodY DNA-binding site was characterized.

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.


   ACKNOWLEDGEMENTS
 
B. Sperandio and N. Pons have a grant from the Ministère de la Recherche et de l'Education Nationale. We thank J. P. Furet for his assistance and helpful advice on RT-QPCR experiments. Commercial DNA microarrays were used in the frame of a project coordinated by Jamila Anba and Pierre Renault, ‘Impact des OGM’, granted by the French Ministry for Research and Education.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Arfin, S. M., Long, A. D., Ito, E. T., Tolleri, L., Riehle, M. M., Paegle, E. S. & Hatfield, G. W. (2000). Global gene expression profiling in Escherichia coli K12. The effects of integration host factor. J Biol Chem 275, 29672–29684.[Abstract/Free Full Text]

Bailey, T. L. & Elkan, C. (1994). Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2, 28–36.[Medline]

Bergara, F., Ibarra, C., Iwamasa, J., Patarroyo, J. C., Aguilera, R. & Marquez-Magana, L. M. (2003). CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis. J Bacteriol 185, 3118–3126.[Abstract/Free Full Text]

Biswas, I., Gruss, A., Ehrlich, S. D. & Maguin, E. (1993). High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175, 3628–3635.[Abstract]

Blencke, H. M., Homuth, G., Ludwig, H., Mader, U., Hecker, M. & Stulke, J. (2003). Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis: regulation of the central metabolic pathways. Metab Eng 5, 133–149.[CrossRef][Medline]

Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, S. D. & Sorokin, A. (2001). The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res 11, 731–753.

Brandenburg, J. L., Wray, L. V., Jr, Beier, L., Jarmer, H., Saxild, H. H. & Fisher, S. H. (2002). Roles of PucR, GlnR, and TnrA in regulating expression of the Bacillus subtilis ure P3 promoter. J Bacteriol 184, 6060–6064.[Abstract/Free Full Text]

Chambellon, E. & Yvon, M. (2003). CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids. Appl Environ Microbiol 69, 3061–3068.[Abstract/Free Full Text]

Debarbouille, M., Gardan, R., Arnaud, M. & Rapoport, G. (1999). Role of bkdR, a transcriptional activator of the sigL-dependent isoleucine and valine degradation pathway in Bacillus subtilis. J Bacteriol 181, 2059–2066.[Abstract/Free Full Text]

den Hengst, C. D., Curley, P., Larsen, R., Buist, G., Nauta, A., van Sinderen, D., Kuipers, O. P. & Kok, J. (2005). Probing direct interactions between CodY and the oppD promoter of Lactococcus lactis. J Bacteriol 187, 512–521.[Abstract/Free Full Text]

Dsouza, M., Larsen, N. & Overbeek, R. (1997). Searching for patterns in genomic data. Trends Genet 13, 497–498.[CrossRef][Medline]

Eda, S., Hoshino, T. & Oda, M. (2000). Role of the DNA sequence downstream of the Bacillus subtilis hut promoter in regulation of the hut operon. Biosci Biotechnol Biochem 64, 484–491.[CrossRef][Medline]

Ferson, A. E., Wray, L. V., Jr & Fisher, S. H. (1996). Expression of the Bacillus subtilis gabP gene is regulated independently in response to nitrogen and amino acid availability. Mol Microbiol 22, 693–701.[CrossRef][Medline]

Fisher, S. H., Rohrer, K. & Ferson, A. E. (1996). Role of CodY in regulation of the Bacillus subtilis hut operon. J Bacteriol 178, 3779–3784.[Abstract/Free Full Text]

Garcia de la Nava, J., van Hijum, S. & Trelles, O. (2003). PreP: gene expression data pre-processing. Bioinformatics 19, 2328–2329.[Abstract/Free Full Text]

Gilson, T. J. (1984). Studies on the Epstein-Barr virus genome. PhD thesis, University of Cambridge.

Goupil-Feuillerat, N., Cocaign-Bousquet, M., Godon, J. J., Ehrlich, S. D. & Renault, P. (1997). Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis. J Bacteriol 179, 6285–6293.[Abstract/Free Full Text]

Guedon, E., Renault, P., Ehrlich, S. D. & Delorme, C. (2001a). Transcriptional pattern of genes coding for the proteolytic system of Lactococcus lactis and evidence for coordinated regulation of key enzymes by peptide supply. J Bacteriol 183, 3614–3622.[Abstract/Free Full Text]

Guedon, E., Serror, P., Ehrlich, S. D., Renault, P. & Delorme, C. (2001b). Pleiotropic transcriptional repressor CodY senses the intracellular pool of branched-chain amino acids in Lactococcus lactis. Mol Microbiol 40, 1227–1239.[CrossRef][Medline]

Holo, H. & Nes, I. F. (1989). High-frequency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55, 3119–3123.

Hung, S. P., Baldi, P. & Hatfield, G. W. (2002). Global gene expression profiling in Escherichia coli K12. The effects of leucine-responsive regulatory protein. J Biol Chem 277, 40309–40323.[Abstract/Free Full Text]

Inaoka, T. & Ochi, K. (2002). RelA protein is involved in induction of genetic competence in certain Bacillus subtilis strains by moderating the level of intracellular GTP. J Bacteriol 184, 3923–3930.[Abstract/Free Full Text]

Inaoka, T., Takahashi, K., Ohnishi-Kameyama, M., Yoshida, M. & Ochi, K. (2003). Guanine nucleotides guanosine 5'-diphosphate 3'-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis. J Biol Chem 278, 2169–2176.[Abstract/Free Full Text]

Kang, Y., Weber, K. D., Qiu, Y., Kiley, P. J. & Blattner, F. R. (2005). Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function. J Bacteriol 187, 1135–1160.[Abstract/Free Full Text]

Kim, H. J., Kim, S. I., Ratnayake-Lecamwasam, M., Tachikawa, K., Sonenshein, A. L. & Strauch, M. (2003). Complex regulation of the Bacillus subtilis aconitase gene. J Bacteriol 185, 1672–1680.[Abstract/Free Full Text]

Lapujade, P., Cocaign-Bousquet, M. & Loubiere, P. (1998). Glutamate biosynthesis in Lactococcus lactis subsp. lactis NCDO 2118. Appl Environ Microbiol 64, 2485–2489.[Abstract/Free Full Text]

Lazazzera, B. A., Kurtser, I. G., McQuade, R. S. & Grossman, A. D. (1999). An autoregulatory circuit affecting peptide signaling in Bacillus subtilis. J Bacteriol 181, 5193–5200.[Abstract/Free Full Text]

Mader, U., Hennig, S., Hecker, M. & Homuth, G. (2004). Transcriptional organization and posttranscriptional regulation of the Bacillus subtilis branched-chain amino acid biosynthesis genes. J Bacteriol 186, 2240–2252.[Abstract/Free Full Text]

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Martinez-Antonio, A. & Collado-Vides, J. (2003). Identifying global regulators in transcriptional regulatory networks in bacteria. Curr Opin Microbiol 6, 482–489.[CrossRef][Medline]

Mirel, D. B., Estacio, W. F., Mathieu, M., Olmsted, E., Ramirez, J. & Marquez-Magana, L. M. (2000). Environmental regulation of Bacillus subtilis {sigma}D-dependent gene expression. J Bacteriol 182, 3055–3062.[Abstract/Free Full Text]

Molle, V., Nakaura, Y., Shivers, R. P., Yamaguchi, H., Losick, R., Fujita, Y. & Sonenshein, A. L. (2003). Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis. J Bacteriol 185, 1911–1922.[Abstract/Free Full Text]

Moreno, M. S., Schneider, B. L., Maile, R. R., Weyler, W. & Saier, M. H., Jr (2001). Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Mol Microbiol 39, 1366–1381.[CrossRef][Medline]

Nakano, M. M., Xia, L. A. & Zuber, P. (1991). Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis. J Bacteriol 173, 5487–5493.[Medline]

Oda, M., Kobayashi, N., Ito, A., Kurusu, Y. & Taira, K. (2000). cis-acting regulatory sequences for antitermination in the transcript of the Bacillus subtilis hut operon and histidine-dependent binding of HutP to the transcript containing the regulatory sequences. Mol Microbiol 35, 1244–1254.[CrossRef][Medline]

Ogura, M., Yamaguchi, H., Yoshida, K., Fujita, Y. & Tanaka, T. (2001). DNA microarray analysis of Bacillus subtilis DegU, ComA and PhoP regulons: an approach to comprehensive analysis of B. subtilis two-component regulatory systems. Nucleic Acids Res 29, 3804–3813.[Abstract/Free Full Text]

Pei, J. & Grishin, N. V. (2001). Type II CAAX prenyl endopeptidases belong to a novel superfamily of putative membrane-bound metalloproteases. Trends Biochem Sci 26, 275–277.[CrossRef][Medline]

Petranovic, D., Guedon, E., Sperandio, B., Delorme, C., Ehrlich, D. & Renault, P. (2004). Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator. Mol Microbiol 53, 613–621.[CrossRef][Medline]

Ratnayake-Lecamwasam, M., Serror, P., Wong, K. W. & Sonenshein, A. L. (2001). Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev 15, 1093–1103.[Abstract/Free Full Text]

Sanz, Y., Lanfermeijer, F. C., Renault, P., Bolotin, A., Konings, W. N. & Poolman, B. (2001). Genetic and functional characterization of dpp genes encoding a dipeptide transport system in Lactococcus lactis. Arch Microbiol 175, 334–343.[CrossRef][Medline]

Serror, P. & Sonenshein, A. L. (1996a). Interaction of CodY, a novel Bacillus subtilis DNA-binding protein, with the dpp promoter region. Mol Microbiol 20, 843–852.[Medline]

Serror, P. & Sonenshein, A. L. (1996b). CodY is required for nutritional repression of Bacillus subtilis genetic competence. J Bacteriol 178, 5910–5915.[Abstract/Free Full Text]

Shivers, R. P. & Sonenshein, A. L. (2004). Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids. Mol Microbiol 53, 599–611.[CrossRef][Medline]

Sissler, M., Delorme, C., Bond, J., Ehrlich, S. D., Renault, P. & Francklyn, C. (1999). An aminoacyl-tRNA synthetase paralog with a catalytic role in histidine biosynthesis. Proc Natl Acad Sci U S A 96, 8985–8990.[Abstract/Free Full Text]

Slack, F. J., Serror, P., Joyce, E. & Sonenshein, A. L. (1995). A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon. Mol Microbiol 15, 689–702.[Medline]

Sperandio, B., Polard, P., Ehrlich, S. D., Renault, P. & Guédon, E. (2005). Sulfur amino acids metabolism and its control in Lactococcus lactis IL1403. J Bacteriol 187, 3762–3778.[Abstract/Free Full Text]

Tani, T. H., Khodursky, A., Blumenthal, R. M., Brown, P. O. & Matthews, R. G. (2002). Adaptation to famine: a family of stationary-phase genes revealed by microarray analysis. Proc Natl Acad Sci U S A 99, 13471–13476.[Abstract/Free Full Text]

Terzaghi, B. & Sandine, W. E. (1975). Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29, 807–813.

Wray, L. V., Jr & Fisher, S. H. (1994). Analysis of Bacillus subtilis hut operon expression indicates that histidine-dependent induction is mediated primarily by transcriptional antitermination and that amino acid repression is mediated by two mechanisms: regulation of transcription initiation and inhibition of histidine transport. J Bacteriol 176, 5466–5473.[Abstract]

Wray, L. V., Jr, Ferson, A. E. & Fisher, S. H. (1997). Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H. J Bacteriol 179, 5494–5501.[Abstract/Free Full Text]

Yarmus, M., Mett, A. & Shapira, R. (2000). Cloning and expression of the genes involved in the production of and immunity against the bacteriocin lacticin RM. Biochim Biophys Acta 1490, 279–290.[Medline]

Yoshida, K. I., Shibayama, T., Aoyama, D. & Fujita, Y. (1999). Interaction of a repressor and its binding sites for regulation of the Bacillus subtilis iol divergon. J Mol Biol 285, 917–929.[CrossRef][Medline]

Yoshida, K., Kobayashi, K., Miwa, Y. & 9 other authors (2001). Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis. Nucleic Acids Res 29, 683–692.[Abstract/Free Full Text]

Yoshida, K., Yamaguchi, H., Kinehara, M., Ohki, Y. H., Nakaura, Y. & Fujita, Y. (2003). Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box. Mol Microbiol 49, 157–165.[CrossRef][Medline]

Zheng, D., Constantinidou, C., Hobman, J. L. & Minchin, S. D. (2004). Identification of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucleic Acids Res 32, 5874–5893.[Abstract/Free Full Text]

Received 10 May 2005; revised 8 September 2005; accepted 9 September 2005.



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