codY Mutant Research Articles

Identification of CodY Targets in Bacillus anthracis by Genome-Wide In Vitro Binding Analysis

In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.

The Stringent Response of Staphylococcus aureus and Its Impact on Survival after Phagocytosis through the Induction of Intracellular PSMs Expression

The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rshSyn, codY and rshSyn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rshSyn and rshSyn, codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psmα1-4 and/or psmβ1,2 could complement the survival of the rshSyn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis.

CodY-mediated regulation of Streptococcus pyogenes exoproteins

BackgroundThe production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene.ResultsWe compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain.ConclusionsOverall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.

CodY orchestrates the expression of virulence determinants in emetic Bacillus cereus by impacting key regulatory circuits

Bacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcR-dependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.

The impact of CodY on virulence determinant production in community‐associated methicillin‐resistant Staphylococcus aureus

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection.

CodY regulation is required for full virulence and heme iron acquisition inBacillus anthracis

Capsule and toxin are the major virulence factors of Bacillus anthracis. The B. anthracis pleiotropic regulator CodY activates toxin gene expression by post-translationally regulating the accumulation of the global regulator AtxA. However, the role of CodY on B. anthracis capsulation and virulence of encapsulated strains has been unknown. The role of CodY in B. anthracis virulence was studied in mouse and guinea pig models. Spore outgrowth and dissemination of the vegetative cells was followed in mice by bioluminescent imaging. We also determined the state of capsulation and the iron requirement for growth of the codY mutant. In all models tested, the codY mutant strain was strongly attenuated compared to the wild-type strain and, in mice, also compared to the atxA strain. The disruption of codY did not affect either ex vivo or in vivo capsulation, whereas atxA deletion affected ex vivo capsulation only. The disruption of codY led to a delayed initiation of dissemination but similar kinetics of subsequent spread of the bacilli. The codY mutant cannot grow on heme iron as sole iron source, whereas the parental and complemented strains can. The lack of CodY-mediated transcription weakens virulence by controlling iron acquisition and synthesis of toxin, but without modifying capsulation.

Dissecting Complex Metabolic Integration Provides Direct Genetic Evidence for CodY Activation by Guanine Nucleotides

The global regulator CodY controls the expression of dozens of metabolic genes and genes mediating adaptation to nutrient availability in many low-G+C Gram-positive bacteria. Branched-chain amino acids L-isoleucine, L-leucine, and L-valine (ILV) activate CodY both in vivo and in vitro, and genes that direct their synthesis (ilv, ybgE, and ywaA) are highly repressed by CodY, creating a potential negative feedback loop. The nucleoside triphosphate GTP also activates CodY in vitro, but the evidence for activation by GTP in vivo is limited and indirect. We constructed a Bacillus subtilis strain (ybgE bcd ywaA) that is unable to convert branched-chain α-keto acids to ILV or to use ILV as a precursor for branched-chain fatty acid synthesis. Unexpectedly, the strain was not viable on rich medium. Supplementing rich medium with short, branched-chain fatty acids or derepressing expression of genes for de novo ILV synthesis bypassed the original lethality, restoring growth and showing that the lack of viability was due to insufficient intracellular production of the precursors of branched-chain fatty acids. Spontaneous extragenic suppressor mutants that arose in the triple mutant population proved to have additional mutations in guaA or guaB or codY. Expression of ILV biosynthetic genes in codY mutants was increased. The gua mutations caused guanine/guanosine auxotrophy and led to partial derepression of direct CodY-repressed targets, including ILV biosynthetic genes, under conditions similar to those that caused the original lethality. We conclude that a guanine derivative, most likely GTP, controls CodY activity in vivo.

The global nutritional regulator CodY is an essential protein in the human pathogen Streptococcus pneumoniae

CodY is a global regulator highly conserved in low-G+C Gram-positive bacteria. It plays a key role in the adaptation of Bacillus subtilis to nutritional limitation through repression of a large gene set during exponential growth and relief of repression upon starvation. In several pathogenic bacteria, CodY regulates major virulence genes. Our interest in Streptococcus pneumoniae CodY originates from our observations that the oligopeptide permease Ami was involved in repression of competence for genetic transformation. We hypothesized that peptide uptake through Ami feeds amino acid pools, which are sensed by CodY to repress competence. As our initial attempts at inactivating codY failed, we launched an in-depth analysis into the question of the essentiality of codY. We report that codY cannot be inactivated unless a complementing ectopic copy is present. We obtained genetic evidence that a recently published D39 codY knock-out contains additional mutations allowing survival of codY mutant cells. Whole genome sequencing revealed mutations in fatC, which encodes a ferric iron permease, and amiC. This combination of mutations was confirmed to allow tolerance of codY inactivation. The amiC mutation is in itself sufficient to account for the strong derepression of competence development observed in D39 codY cells.

Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

The Global Regulator CodY Regulates Toxin Gene Expression inBacillus anthracisand Is Required for Full Virulence

In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis, the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis. Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA, as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally.

Characterization of the codY gene and its influence on biofilm formation in Bacillus cereus

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease ( approximately 130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.

Positive regulation of Bacillus subtilis ackA by CodY and CcpA: establishing a potential hierarchy in carbon flow

Conversion of pyruvate to acetate via the phosphotransacetylase-acetate kinase pathway generates ATP and is a major overflow pathway under conditions of carbon and nitrogen excess. In Bacillus subtilis, this pathway is positively regulated by CcpA, a global regulator of carbon metabolism genes. Transcription of the acetate kinase gene (ackA) proved to be activated as well by a second global regulatory protein, CodY. Expression of an ackA-lacZ fusion was reduced in a codY mutant strain. CodY was found to bind in vitro to two sites in the ackA promoter region and to stimulate ackA transcription in a run-off transcription assay. This is the first known case of direct positive regulation by CodY. CodY and CcpA were found to bind to neighbouring sites and their effects were additive both in vivo and in vitro. Surprisingly, positive regulation by CodY, unlike repression, responded primarily to only one type of effector molecule. That is, branched-chain amino acids (BCAAs) served as more potent co-activators of CodY-dependent ackA transcription than did GTP. Given the roles of CcpA and CodY in regulating genes whose products determine the metabolic fate of pyruvate, these two proteins may act together to mediate a hierarchical conversion of pyruvate to its many potential products.

Linking the nutritional status of Streptococcus pyogenes to alteration of transcriptional gene expression: The action of CodY and RelA

In this investigation, we identify the CodY protein from Streptococcus pyogenes as a pleiotropic transcription regulator with global features. The notion that acquisition of nutrients by this polyauxotrophic organism is the primary event occurring during the establishment of infection and that virulence expression is a result of this quest, led us to study the action of codY and relA genes on transcriptional gene expression under different nutritional conditions using complex and chemically defined media. Real-time reverse transcription PCR was used to determine the extent to which inactivation of codY and relA affects the mRNA levels of selected transcription factors, virulence genes, transporters, and genes encoding metabolic enzymes. The results show that CodY and RelA did not affect the expression of each other but that both exhibited strong negative autoregulatory properties. Genes negatively controlled by the relA-directed stringent response to amino acid starvation included, besides relA itself, transporters, metabolic enzymes, and at least two virulence genes ( graB and speH). The expression of many genes of all four groups studied proved to be subject to direct or indirect control by CodY, often in a nutritional status-dependent fashion. One of the most important results implicates CodY in growth phase-dependent positive transcriptional regulation of pel/ sagA and mga, loci that themselves positively affect the expression of numerous virulence factors. Increasing the cellular activity of nicotinamidase in both a codY mutant and wild-type background induced extensive transcriptional reprogramming, altering, among others, the growth phase-dependent transcription pattern of the genes for cysteine protease ( speB) and several transporters. Inasmuch as CodY influenced the expression of other regulators ( pel/sagA, mga, covRS, ropB, pyrR), its action is amplified and expands the complex regulatory network that governs gene expression in S. pyogenes.

Proteome Analyses of Heme-Dependent Respiration inLactococcus lactis: Involvement of the Proteolytic System

Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria. We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available. To provide insights into this phenomenon, we compared the proteomic profiles of L. lactis under fermentative and respiratory growth conditions in rich medium. We identified 21 proteins whose levels differed significantly between these conditions. Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism. Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids. A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism. Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions. This induction was independent of CodY, the major regulator of proteolytic functions in L. lactis. We also observed that pepO1 induction is redox sensitive. In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions. These observations suggest that new regulators activate proteolysis in L. lactis, which help to maintain the energetic needs of L. lactis during respiration.

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.

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.

Réseau de régulation de la transcription des gènes du système protéolytique de lactococcus lactis

Network of regulation of gene transcription of the proteolytic system of Lactococcus lactis. The proteolytic system of lactococci that allows degradation of caseins and proteins of milk is complex. Milk proteins contain all amino acids necessary for growth of lactic acid bacteria. The proteolytic system consists of an extracellularly located proteinase, transport systems for di-tripep- tides and oligopeptides and a multitude of intracellular peptidases. Expression of 13 genes was fol- lowed by transcriptional fusions in presence of different peptide sources. Transcription of 6 genes is repressed in media containing peptides and that of 4 genes ( pepN, pepC, prtP and opp-pepO1 operon) by dipeptides containing one of the 3 branched amino acids (isoleucine, leucine and valine). Repres- sion of gene transcription required that regulatory peptides are translocated into the cell and degraded in amino acids. Cell factors involved in this regulation were identified in derepressed mutants obtained by random mutagenesis by transposition. DtpT, a di-tripeptides transporter and CodY, homologous of the Bacillus subtilis pleiotropic regulator of transcription were the most frequently inactivated proteins. pepC, pepN and opp-pepO1transcription is not repressed in codY and dtpT mutant. These genes of the proteolytic system belong to a same regulon since their expression is repressed by CodY regulator depending on intracellular concentration of branched amino acids or derivative products of them. Lactococcus lactis / proteolytic system / transcription / regulation

Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression.

The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. subtilis.

Role of the DNA sequence downstream of the Bacillus subtilis hut promoter in regulation of the hut operon.

To identify the role of the downstream region of a hut promoter in regulation of the Bacillus subtilis hut operon, three single-base substitutions (+9G-->A, +14C-->T, and +23T-->G) were introduced into the hut operon. Analysis of expression of the hut operon containing each of these three single-base substitutions and the hut-lacZ fusions with the single-base substitutions at position +14 showed that the position at +14 and probably the position at +23 were required for amino acid repression at the hut promoter, while the position at +14 was not required for catabolite repression at the hut promoter. The position at +9 was required for a histidine-dependent increase of activity of the hut promoter. Analysis of expression of the hut-lacZ fusions and the hut operon in the codY mutant indicated that the position at +14 and probably the position at +23 were involved in CodY-mediated amino acid repression at the hut promoter and that CodY was not required for catabolite repression at the hut promoter.

Role of CodY in regulation of the Bacillus subtilis hut operon

Bacillus subtilis mutants deficient in amino acid repression of the histidine utilization (hut) operon were isolated by transposon mutagenesis. Genetic characterization of these mutants indicated that they most likely contained transposon insertions within the codVWXY operon. The codY gene is required for nutritional regulation of the dipeptide permease (dpp) operon. An examination of hut expression in a delta codY mutant demonstrated that amino acid repression exerted at the hutOA operator, which lies immediately downstream of the hut promoter, was defective in a delta codY mutant. The codY gene product was not required for amino acid regulation of either hut induction or the expression of proline oxidase, the first enzyme in proline degradation. This indicates that more than one mechanism of amino acid repression is present in B. subtilis. An examination of dpp and hut expression in cells during exponential growth in various media revealed that the level of CodY-dependent regulation appeared to be related to the growth rate of the culture.