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REVIEWS Control of key metabolic intersections in Bacillus subtilis Abraham L. Sonenshein Abstract | The remarkable ability of bacteria to adapt efficiently to a wide range of nutritional environments reflects their use of overlapping regulatory systems that link gene expression to intracellular pools of a small number of key metabolites. By integrating the activities of global regulators, such as CcpA, CodY and TnrA, Bacillus subtilis manages traffic through two metabolic intersections that determine the flow of carbon and nitrogen to and from crucial metabolites, such as pyruvate, 2‑oxoglutarate and glutamate. Here, the latest knowledge on the control of these key intersections in B. subtilis is reviewed. Glycolysis The metabolic pathway that converts glucose into pyruvate, with the concomitant production of ATP and NADH. Pentose-phosphate pathway The metabolic pathway by which glucose-6-phosphate is oxidized to ribose-5-phosphate, with the concomitant production of NADPH. Citric acid cycle The metabolic pathway that oxidizes acetyl CoA to carbon dioxide. 2-oxoglutarate–glutamate– glutamine cycle The metabolic pathway that connects carbon and nitrogen metabolism and permits ammonium ions to be incorporated into organic molecules. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA. e-mail: [email protected] doi:10.1038/nrmicro1772 Published online 5 November 2007 Most culturable bacteria thrive in the rich media that are used to study their behaviour in the laboratory. In the natural environment, however, nutrients are often present in extremely dilute concentrations (for example, in the sea), inaccessible because of the paucity of water (for example, in the desert) or only transiently available (for example, in soil or the mammalian oral cavity and gastrointestinal tract). Moreover, the quality of the available nutrients can be highly variable. As a result, bacteria have evolved remarkably sophisticated adaptation systems that allow them to take advantage of the wide range of sources of the essential elements carbon, nitrogen, phosphorus and sulphur. This enables them to feed the central metabolic pathways — glycolysis, the pentose-phosphate pathway, the citric acid cycle and the 2‑oxoglutarate–glutamate–glutamine cycle — from which all of the precursors that are required for the synthesis of the cell’s macromolecules (DNA, RNA, proteins, peptidoglycan and lipid bilayers) are derived. With the exception of peptidoglycan, these precursors and polymers are universal in the biological world and most of the biochemical pathways that are involved in their synthesis are highly conserved. The interactions between bacteria and eukaryotic host organisms are often commensal or symbiotic, but are sometimes parasitic, which causes serious harm to the host. However, not all pathogenic bacteria are constitutively virulent; many reserve the traits that we perceive as virulent for conditions of stress, particularly nutritional stress1. It is possible that these bacteria synthesize proteases, lipases and other virulence factors that damage eukaryotic cells because they lack nutrients or, in the case of intestinal pathogens, because they need to be excreted into the environment in order to colonize new hosts or niches. nature reviews | microbiology Rigorous biochemical, genetic and molecular analyses carried out over the past 50 years have given us a detailed view of the intricate reactions that generate the central metabolites in bacterial cells, the genes that encode the enzymes that carry out these reactions and many of the mechanisms that regulate the expression of these genes2. The genes that are required for the utilization of nutritional sources are typically regulated by the availability of the substrate, just as the genes that are required for the biosynthesis of a particular cellular constituent are typically regulated by the accumulation of the end-product. As far as we know, virtually all bacteria have additional layers of control that coordinate the use of nutrients of the same type (for example, carbon or nitrogen) using global regulators. Recent work has established that some of these global regulators are also integrated into a larger regulatory scheme by which the cell (Bacillus subtilis in this Review) coordinates the flow through key metabolic intersections in response to a small number of specific signalling metabolites (TABLE 1). Here, the goal is to summarize our current knowledge about the regulation of the genes that encode the enzymes that mediate the flow of carbon from pyruvate to the citric acid cycle and carbonoverflow pathways, as well as the enzymes that interconvert 2‑oxoglutarate, glutamate, glutamine and certain other amino acids in B. subtilis. It is beyond the scope of this Review to consider the regulation of sugar transport, glycolysis, gluconeogenesis and the pentose-phosphate pathway. For detailed information about these pathways, the reader is referred to excellent reviews by Deutscher and collegues3,4 and Aymerich and colleagues5. Carbon overflow in B. subtilis B. subtilis is a Gram-positive, spore-forming bacterium that has been the subject of intense investigation since volume 5 | december 2007 | 917 © 2007 Nature Publishing Group REVIEWS Table 1 | Bacillus subtilis regulators of central metabolism genes Regulatory Target genes or proteins Metabolites protein sensed Direct or indirect sensing Mediator Effect of metabolite Citrate Direct None Inactivation Specific regulators CcpC citZCH, citB and ccpC RocR rocABC, rocDEF and rocG Ornithine Direct None Activation GltC gltAB and gltC 2-oxoglutarate and glutamate Direct None Activation by 2oxoglutarate and inactivation by glutamate RocG GltC None Direct None Inactivation GlnR glnRA, ureABC and tnrA Glutamine Indirect Glutamine synthetase by an unknown mechanism Activation Global regulators CcpA Many carbon-metabolism genes FBP and glucose-6phosphate Direct and indirect FBP stimulates HPr kinase to Activation phosphorylate Hpr and Crh, which interact with CcpA; the interaction of FBP and glucose-6-phosphate with the P-HPr–CcpA complex CodY Many carbon- and nitrogen-metabolism genes, transport genes, sporulation genes and competence genes. GTP and BCAAs Direct None Activation TnrA Many nitrogenmetabolism genes Glutamine Indirect Glutamine synthetase–glutamine complex interacts with TnrA Inactivation A summary of the regulatory proteins that are implicated in the control of some central metabolism genes. See main text for details and references. BCAA, branched-chain amino acid; FBP, fructose-1,6-bisphosphate. Global regulator A protein that controls many genes and operons in response to a specific signal. Sporulation A developmental programme in some microorganisms in response to unfavourable environmental conditions that results in spores that are highly resistant to environmental stresses. LacI protein family The proteins that are related in sequence and function to the classical repressor of the E. coli lac operon. Phosphoenolpyruvatedependent phosphotransferase transport system A multi-protein phosphorelay system that couples the phosphorylation of sugars to their transport across the cytoplasmic membrane. the early 1950s. The attraction of sporulation as a prototypical system of cellular differentiation and the ease of genetic manipulation made B. subtilis an early choice for detailed investigation. At present, B. subtilis is the second most intensively studied bacterium, after Escherichia coli, and is a useful paradigm for most of the Gram-positive bacterial world6. If growing in a medium that contains an excess of glucose (the preferred carbon source for many bacteria), B. subtilis metabolizes a large proportion of the glucose only as far as pyruvate and acetyl CoA, and subsequently converts these compounds to by-products of metabolism (also known as fermentation products), including lactate, acetate and acetoin, which are excreted into the extracellular environment (FIG. 1). The enzymes of glycolysis depend on the cofactor NAD+ to take up electrons and hydrogen atoms that are released by substrate oxidation; the conversion of pyruvate to lactate has the advantage of regenerating NAD+ from its reduced form, NADH, which is a step that is essential for continued glycolysis. The conversion of acetyl CoA to acetate is coupled to the synthesis of ATP by the activities of the enzymes phosphotransacetylase and acetate kinase. Thus, these overflow pathways enable the cell to maintain redox balance and generate ATP without using the cytochrome system. When the glucose has been fully consumed, the cells reintroduce the by-products into central metabolism (using lactate dehydrogenase, acetoin dehydrogenase and acetyl CoA synthetase) and metabolize them further through the citric acid 918 | december 2007 | volume 5 cycle, so generating additional ATP and reducing power (FIG. 1). Unsurprisingly, the genes that encode the enzymes that are involved in overflow metabolism are regulated by proteins that sense the nutritional status of the cell. In B. subtilis, CcpA activates the expression of the genes that are required for the synthesis of acetate, lactate and acetoin7–12 (FIG. 1). CodY also contributes to the activation of the acetate- and lactate-synthesis pathways, as discussed below (FIG. 1). Moreover, the re-utilization enzymes for acetate and acetoin are repressed by CcpA and CodY11–16 (FIG. 1). As described in more detail below, these two regulators function together to integrate the expression of carbon-overflow genes with overall B. subtilis metabolism. The global regulators CcpA and CodY CcpA is a member of the LacI protein family and a global regulator of carbon-metabolism pathways in many Gram-positive bacteria 17. In B. subtilis, CcpA contributes to the regulation of more than 100 genes, the products of which, in most cases, are involved in carbon acquisition or metabolism10–12,18. CcpA can be either a positive or negative regulator of transcription, and its activity is determined by a complex interaction with two other proteins and at least one signalling metabolite4. HPr, a component of the phosphoenolpyruvate-dependent phosphotransferasetransport system, and the closely related protein Crh, are phosphorylated on a specific serine residue by HPr kinase if cells are grown in media that contain glucose www.nature.com/reviews/micro © 2007 Nature Publishing Group REVIEWS as a DNA-binding protein is stimulated by interaction with either of two ligands, GTP or a BCAA (branchedchain amino acid; particularly isoleucine or valine)40,41. These two ligands have additive effects on CodY activity 41. Consensus sequences for CodY-binding sites (the ‘CodY box’) have been proposed, but adherence to this consensus is variable42,43. Taking these properties of CcpA and CodY into account, we can see that the decision to convert pyruvate to excretable overflow products is made by two regulatory proteins (CcpA and CodY) that respond to three metabolites (FBP, GTP and BCAAs). The same regulatory proteins and metabolites control the re-use of the overflow products if the supply of glucose is exhausted. Glucose GTP BCAAs CodY CcpA FBP CcpA Lactate Fatty acids Pyruvate Acetolactate Acetoin Acetyl CoA Acetyl~ P Acetate GTP CcpA CodY BCAAs Citric acid cycle Figure 1 | Interactions of the global regulators CcpA and CodY with carbonoverflow metabolism in Bacillus subtilis. In the presenceNature of excess glucose, B. subtilis Reviews | Microbiology metabolizes a large proportion of the glucose only as far as pyruvate and acetyl CoA. These molecules are then converted to by-products of metabolism, such as lactate, acetate and acetoin, using carbon-overflow pathways. CcpA activates the expression of the genes that are required for the synthesis of these by-products, and CodY contributes to the activation of the acetate- and lactate-synthesis pathways. Additionally, the re-utilization enzymes for acetate and acetoin are repressed by both CcpA and CodY (indicated by blunt arrows). In turn, CcpA is regulated by fructose-1,6-bisphosphate (FBP). CodY is subject to regulation by GTP and branched-chain amino acids (BCAAs). Competence The ability of bacteria to take up extracellular DNA. or other rapidly metabolized carbon sources19–21. The phosphorylated proteins can independently bind to CcpA 22 and increase its affinity for cre sites — the 12-base pair sequences that are the preferred binding site for CcpA23–25. The activity of HPr kinase is stimulated by ATP and fructose‑1,6-bisphosphate (FBP)26,27, which links the activity of CcpA to the availability of a glycolytic intermediate4. FBP and glucose‑6-phosphate can also bind directly to CcpA in complex with phosphorylated HPr 24,28. Both mechanisms appear to contribute to CcpA activity. B. subtilis CodY, and its homologues in other Grampositive bacteria, defines a unique family of regulatory proteins29. CodY was first discovered as a repressor of the genes which, through their products, help cells to adapt to poor nutritional availability30–36. That is, many genes that are repressed by CodY encode proteins that allow cells to move to new, potentially nutritionally richer locations, to degrade extracellular macromolecules and transport the breakdown products into the cell for degradation. CodY also represses the development of genetic competence and sporulation as adaptations to nutrient limitation37–40. The activity of CodY nature reviews | microbiology Global regulators and the citric acid cycle The first three enzymes of the citric acid cycle — citrate synthase, aconitase and isocitrate dehydrogenase — form the tricarboxylic acid (TCA) branch (FIG. 2). Together with pyruvate dehydrogenase, they drive the conversion of pyruvate to 2‑oxoglutarate; although citrate synthase is generally regarded as the ratelimiting enzyme of the pathway, the irreversibility of isocitrate dehydrogenase forces the pathway towards 2‑oxoglutarate. The regulation of the TCA-branch enzymes is intricate and they respond to multiple metabolic signals at the levels of gene expression and enzyme activity. The genes that encode citrate synthase (citZ) and isocitrate dehydrogenase (citC) are joined in an operon by citH (also called mdh), which encodes malate dehydrogenase44,45. The promoter of the citZ gene drives the expression of all three genes, but, as citC and citH also have gene-specific promoters, the control of citC and citH by repression at the citZ promoter is incomplete 46. The aconitase gene (citB) is encoded elsewhere on the chromosome in a monocistronic transcription unit47. Transcription from the citZ and citB promoters is repressed by CcpC, a member of the LysR family of regulatory proteins48,49 (FIG. 2). CcpC has only one other known target in the B. subtilis chromosome: its own promoter50. In Listeria monocytogenes, CcpC has been implicated in the regulation of a glutamine-transport gene as well as citB51. CcpC is inactivated as a repressor of citZ and citB by interaction with citrate48,52. Although it is logical for the synthesis of aconitase to increase as its substrate, citrate, accumulates, it seems bizarre that citrate synthase would be induced by the accumulation of its end-product. It is worth noting, however, that such a phenomenon is as much the rule as the exception. The lac operon of E. coli is induced by allolactose, the synthesis of which depends on β‑galactosidase activity53, and the histidine-utilization (hut) operon of Klebsiella aerogenes is induced by urocanate, the product of the first enzyme of the pathway54. Although CcpC is a specific regulator of the TCAbranch genes, two other modes of regulation are layered on top of this primary regulatory mechanism. First, the citZCH operon is repressed directly by CcpA12,49 and, second, CodY is also a repressor of the citB gene55. volume 5 | december 2007 | 919 © 2007 Nature Publishing Group REVIEWS Glucose FBP Valine CcpA Pyruvate Acetyl CoA Aspartate Oxaloacetate Citrate synthase citZ Citrate CcpC Aconitase citB Isocitrate Isoleucine GTP CodY 2-oxoglutarate Nature Figure 2 | Interactions of global regulators with the citric acidReviews cycle in| Microbiology Bacillus subtilis. In B. subtilis, three regulatory proteins (CcpC, CcpA and CodY) and four metabolites (citrate, fructose-1,6-bisphosphate (FBP), GTP and isoleucine or valine) function together to determine the extent to which pyruvate and acetyl CoA enter the tricarboxylic acid branch of the citric acid cycle. CcpA is an indirect repressor of citB by its control of citrate synthesis49,52. Therefore, the full expression of citZ, citB and citC requires the antagonism of at least three repressor proteins (FIG. 2). This result is achieved for CcpC by the accumulation of citrate; for CodY, the intracellular concentrations of GTP, isoleucine or both must drop below a critical level; and for CcpA, the FBP pool must decrease. These compounds do in fact change in concentration if cells experience general nutrient limitation56. Thus, in B. subtilis, three regulatory proteins (CcpC, CcpA and CodY) and four metabolites (citrate, FBP, GTP and isoleucine or valine) function together to determine the extent to which pyruvate and acetyl CoA enter the TCA branch of the citric acid cycle (FIG. 2). A fifth metabolite also plays a part posttranslationally. Citrate synthase is subject to feedback inhibition by 2‑oxoglutarate, which acts competitively with respect to the substrate oxaloacetate (H.J. Kim and A.L.S., unpublished observations). This observation helps to explain the inverse correlation between aconitase synthesis and the 2‑oxoglutarate pool57 and the discovery that the repression of citB expression by arginine depends on the conversion of arginine to 2‑oxoglutarate52. Based on these facts, it is now possible to understand why the TCA branch is strongly repressed during the growth of cells in a rich medium that contains glucose and a mixture of amino acids. Glucose generates FBP, which activates CcpA, and therefore contributes to repression of the 920 | december 2007 | volume 5 citZCH operon. Any synthesis of citrate synthase will be negated by the presence of a pool of 2‑oxoglutarate, which is produced by the catabolism of amino acids. As a result, the pool of citrate will be low, CcpC will be highly active as a repressor and citZ transcription will be severely limited. Moreover, citB will be repressed strongly by the combined activities of CcpC and CodY; in this case the richness of the medium will allow efficient synthesis of GTP, and the presence of isoleucine and valine in the medium will help to keep CodY highly active. The complexity of this system of regulation can be exacerbated by the repression of the ccpC gene by both CcpA and CcpC 50,52, but the regulation of CcpC synthesis by CcpA does not seem to be a major contributor to the control of the gene expression of TCA-branch enzymes52. In addition, two other global regulators have been implicated in the regulation of citB transcription. AbrB, a protein that is best known as a repressor of genes that are expressed early in stationary phase, is a direct positive regulator of citB55 and TnrA — the global nitrogen metabolism regulatory protein (discussed below) — is also a positive regulator that, perhaps, acts indirectly52. The link between carbon and nitrogen metabolism The metabolite 2‑oxoglutarate stands at the crossroads between carbon metabolism and nitrogen metabolism (FIG. 3). As one of the substrates of glutamate synthase (GOGAT), 2‑oxoglutarate provides the de novo carbon skeleton for the two most important nitrogen‑containing compounds in the cell, glutamate and glutamine. Moreover, as the product of glutamate metabolism by glutamate dehydrogenase, 2‑oxoglutarate can be the entry point into central metabolism for the carbon skeletons of several amino acids (including arginine, ornithine, proline and histidine), which are catabolized to glutamate. In addition, 2‑oxoglutarate is a substrate (or product) of most of the amino-transferases that interconvert amino acids and their keto acids. Each of the pathways to and from 2‑oxoglutarate is subject to operon-specific and global regulation (FIG. 3). In B. subtilis, the de novo synthesis of glutamate from 2‑oxoglutarate is catalysed uniquely by GOGAT, the heterodimeric product of the gltAB operon (FIG. 3). The operon-specific regulator of gltAB is GltC58,59 — a LysR family member, the in vitro activity of which is determined by its interaction with two different ligands; 2‑oxoglutarate activates GltC, whereas glutamate acts as a competitive inhibitor with 2‑oxoglutarate60. The two ligands are thought to cause GltC to adopt different conformations that lead to binding to the gltAB promoter at different positions, so that transcription is either activated or repressed60. Recent work has shown that GltC activity is also regulated by direct interaction with glutamate dehydrogenase (discussed below)61. The synthesis of GOGAT and glutamine synthetase is repressed by TnrA, the principal global regulator of nitrogen-metabolism genes in B. subtilis62 (FIG. 3). TnrA is only active if cells are trying to grow with slowly metabolizable nitrogen sources. Under such conditions, TnrA www.nature.com/reviews/micro © 2007 Nature Publishing Group REVIEWS Glucose Arginine uptake Intracellular arginine FBP RocR Ornithine CcpA GTP Other amino acids Glutamate dehydrogenase CodY CcpA rocG GltC 2-oxoglutarate Glutamate gltAB CodY Glutamate synthase TnrA GTP BCAAs NH4+ Histidine Glutamine synthetase Glutamine BCAAs Figure 3 | Bacillus subtilis global regulators at the intersection between carbon and nitrogen metabolism. The intersection between carbon and nitrogen metabolism is regulated by at least six proteins (GltC, TnrA, RocG,| Microbiology RocR, CcpA Nature Reviews and CodY) that respond to 2‑oxoglutarate, glutamine, ornithine, fructose-1,6-bisphosphate (FBP), GTP and branchedchain amino acids (BCAAs). represses some genes, but also activates operons such as nrgAB, nasBC and nasDEF, the products of which mediate the uptake of ammonium ion and the use of secondary nitrogen sources such as nitrate and nitrite62‑64. If the favoured nitrogen source, glutamine, is available, TnrA is inactivated, as it is pulled into a complex with glutamine synthetase and glutamine65. The GOGAT genes, gltAB, are also repressed by CodY36, probably by indirect mechanisms. The synthesis of 2‑oxoglutarate from glutamate by glutamate dehydrogenase depends on the operonspecific activator RocR66. Other amino acids that are converted to glutamate, such as arginine, proline and histidine, can also be sources of 2‑oxoglutarate. The glutamate dehydrogenase (rocG) gene lies upstream of an operon (rocABC) that is involved in arginine– ornithine–proline metabolism67, but is co-regulated with rocABC through binding of RocR to an enhancer element. This element lies upstream of rocA but downstream of rocG68,69. Ornithine is the co-activator; without ornithine, the affinity of RocR for its binding sites is greatly reduced67. A second, weaker promoter for rocG provides a low level of rocG expression in the absence of ornithine70. Commichau and colleagues61 have recently shown that RocG has a second activity as a regulatory protein — it directly contacts and inhibits GltC. This discovery sheds light on two previously mysterious physiological observations. The transcription of gltAB is stimulated by glucose and requires CcpA; in other words, a ccpA mutant is a glutamate auxotroph70–72 and is repressed if cells are grown in a medium that contains arginine or ornithine73. Consequently, the induction of the rocG gene by ornithine (owing to its effect on RocR) leads to inhibition of gltAB transcription by RocG; the repression of rocG by CcpA, nature reviews | microbiology if glucose is present, can explain the dependence of gltAB transcription on CcpA and glucose. The CcpA-dependent repression of rocG when cells are exposed to glucose70 (FIG. 3) makes sense because glutamate, and all other amino acids that can be converted to glutamate, are alternative carbon sources. CcpA also represses the hut operon directly74. CodY collaborates with CcpA to repress the hut operon33 and also represses the uptake of arginine (B. Belitsky, personal communication), γ‑aminobutyrate32 and other amino acids and peptides36. Glutamine is essential for cell growth, is the preferred nitrogen source of B. subtilis and is the second substrate of GOGAT (FIG. 3). In addition, in B. subtilis, glutamine synthetase is virtually the only enzyme that assimilates ammonium ions into organic compounds. The glutamine synthetase gene (glnA) is the second gene of the glnRA operon75. GlnR is both an autorepressor76,77 and a negative regulator of the urease31,78 and tnrA79 genes. The activity of GlnR is stimulated by glutamine synthetase and glutamine80–82. In Streptococcus pneumoniae, which lacks TnrA, GlnR represses the synthesis of glutamine and glutamate (glutamate is synthesized by anabolic glutamate dehydrogenase), the enzyme glucose‑6-phosphate dehydrogenase and the uptake of glutamine 83 . Glutamine synthetase is necessary for the repressing activity of S. pneumoniae GlnR83. The glutamate dehydrogenase gene is also repressed by CodY in this bacterium83. Gram-positive bacteria that lack TnrA might generally use GlnR to carry out some of the global roles that are assumed by TnrA in B. subtilis. In summary, the intersection between carbon and nitrogen metabolism is regulated by at least six proteins — GltC, TnrA, RocG, volume 5 | december 2007 | 921 © 2007 Nature Publishing Group REVIEWS Aspartate Glucose Threonine α-Ketobutyrate Pyruvate TnrA CcpA CodY α-Ketoisocaproate α-Ketoisovalerate Leucine Valine α-Keto-β-methylvalerate Isoleucine Figure 4 | The metabolic context of the Bacillus subtilis branched-chain amino | Microbiology acid biosynthetic pathway. CcpA is a positive regulator ofNature the ilvReviews genes; TnrA and CodY are negative regulators. RocR, CcpA and CodY — that respond to 2‑oxoglutarate, glutamine, ornithine, FBP, GTP and BCAAs. A regulatory network for metabolic intersections As discussed, CcpA, TnrA and CodY are global regulators that sense diverse intracellular metabolites (FBP, glutamine, GTP and BCAAs) and provide a top layer of general nutritional regulation that determines the rate of expression of the central metabolic genes, the products of which determine the flux through two crucial metabolic intersections. This integrates these pathways with each other and with the general state of the cell. Thus, this information provides at least a rudimentary idea of how B. subtilis has evolved to regulate crucial decision points in central metabolism by taking into account several different metabolic signals. One interpretation of this is that the compounds that are particularly important to the cell include pyruvate and 2‑oxoglutarate. Each molecule of pyruvate that the cell generates has several potential fates: it can be used directly for the synthesis of alanine, valine and leucine; it can be converted to oxaloacetate to initiate the pathway for the synthesis of aspartate, asparagine, methionine, diaminopimelate, lysine, threonine and isoleucine; it can be reduced to lactate; or it can be converted to acetyl CoA for metabolism through the citric acid cycle, biosynthesis of fatty acids or by catabolism to acetate. It is clearly in the interest of the cell that pyruvate is distributed to as many potential fates as are appropriate to meet the cell’s specific and overall metabolic needs84. Similarly, the distribution of 2‑oxoglutarate to several potential pathways must be balanced. On the one hand, if the cell is in carbon excess and needs to generate glutamate, glutamine and other amino acids, molecules of 2‑oxoglutarate, which are produced by the citric acid cycle, must be siphoned off by GOGAT, whereas catabolism of the existing amino-acid pools must be avoided. On the other hand, if the cell finds itself in nitrogen excess and, therefore, needs to send carbon skeletons to the citric acid cycle for the biosynthesis of other compounds, or for gluconeogenesis, the activities of GOGAT and glutamine synthetase must be reduced. 922 | december 2007 | volume 5 The unexpected role of BCAA biosynthesis A key element that balances the distribution of pyruvate and 2‑oxoglutarate is the pathway for the biosynthesis of BCAAs. B. subtilis has four transcription units that are devoted to BCAA biosynthesis85. The ilvA, ilvD and ybgE genes are present in monocistronic transcription units. The other seven genes (ilvBHI and leuABCD) are found in one large operon. All of these transcription units are induced by the presence of glucose in a CcpAdependent manner11,86–88; all but ilvA are repressed by CodY36,41. As a result, even though CcpA and CodY cooperate to repress or activate genes of the carbonoverflow, amino-acid-degradation and citric acid cycle pathways, they regulate BCAA biosynthesis antagonistically (FIG. 4). Moreover, TnrA is also involved in BCAA regulation, serving as a repressor during conditions of nitrogen limitation89 (FIG. 4). The mechanism by which CcpA and CodY have opposing effects on ilv gene expression has been explored in detail. The two proteins bind to partially overlapping sites just upstream of the –35 region of the ilvB promoter. CcpA acts partly as an indirect positive regulator by competing with CodY for binding and partly as a direct positive regulator of the ilvB operon87,88. Both mechanisms are consistent with the finding that a ccpA mutant is a partial BCAA auxotroph86, in which auxotrophy is suppressed by a codY mutation87. The net result of these antagonistic effects is that when cells are shifted to a glucose-containing medium, CcpA stimulates the production of BCAAs, making CodY a more avid DNA-binding protein. Therefore, under such conditions, CodY is a stronger repressor of the genes that it represses (such as the aconitase and acetyl CoA synthetase genes) and a stronger activator of the acetate kinase and lactate dehydrogenase genes (FIG. 5). Of course, the greater production of BCAAs in a glucose medium also means that CodY will be a more effective competitor with CcpA for binding to the ilvB promoter; presumably the two proteins quickly create a steady-state condition, in which the rate of ilvB-operon expression reflects this competition. By mediating the creation of different steady-state levels of BCAA biosynthesis, under different conditions of nutrient availability, CcpA and CodY determine the extent to which CodY is active, thereby determining the impact of CodY on the many genes that it controls. In other words, if cells are exposed to glucose, CcpA regulates not only its own target genes but also those genes that are targets of CodY. The impact of TnrA on ilv gene regulation has not been studied in as much detail. Nonetheless, it can be concluded that when nitrogen is limiting TnrA is active, binds far upstream of the ilvB operon promoter and represses transcription by twofold to threefold through an unknown mechanism89. Thus, if carbon is in excess and nitrogen is limiting, TnrA partially counterbalances the effect of CcpA on BCAA biosynthesis, making CodY less active as a repressor of amino-acid utilization pathways, while itself turning on the same, and other, utilization pathways for alternative nitrogen sources (FIGS 3,4). www.nature.com/reviews/micro © 2007 Nature Publishing Group REVIEWS these factors explains why the BCAAs seem to be widely used by bacteria to monitor their physiological state. aa Glucose Uncharged tRNA CcpA aa CodY ilvB BCAAs GTP + ATP aa RelA pppGpp ppGpp tRNA and rRNA synthesis Nature Reviews | Microbiology Figure 5 | Intersection of RelA, CcpA and CodY in the regulation of the Bacillus subtilis ilvB operon. If bacteria are limited for amino acids, the stringent response is induced. Uncharged tRNAs bind to the A site of the ribosome and activate the ribosome-associated enzyme RelA, which catalyses the formation of pppGpp from GTP and ATP. The breakdown product of pppGpp, ppGpp, reduces the rate of transcription of ribosomal RNA (rRNA) and tRNA genes and also affects the transcription of many other genes. In a relA mutant strain, GTP accumulates to a higher-than-normal level, so increasing the activity of CodY. If cells are growing in a glucose-containing medium, CcpA stimulates the expression of ilvB, which leads to an increase in the branched-chain amino acid (BCAA) pool and greater activation of CodY (shown in the shaded area). Homolactic fermentation The production of lactic acid (lactate) as the sole pyruvatederived product during growth on sugars, such as glucose. Why are BCAAs useful intracellular signals? BCAAs seem to have a key regulatory role, by different mechanisms, in both Gram-positive and Gramnegative bacteria. Even though CodY, in Gram-positive bacteria, and the leucine-responsive protein (Lrp) in Gram-negative bacteria, are dissimilar in sequence and structure, they have many of the same functional roles. Lrp regulates a large number of functionally diverse operons, responds to the presence of a BCAA (mainly leucine, but also responds to serine and alanine), controls both stationary-phase genes and virulence and interacts with other regulatory proteins90–92. Does it make sense for the cell to monitor intracellular BCAA pools as opposed to pools of other amino acids? Isoleucine, leucine and valine are among the most abundant amino acids in proteins; maintaining their pools is essential for high-level protein synthesis. Moreover, cells should be able to monitor their ability to synthesize BCAAs because one of the immediate precursors of the BCAAs, α‑ketoisovaleric acid, is also a precursor of pantothenic acid and coenzyme A (FIG. 4). In addition, the branched chain α‑keto acids are precursors of the branched-chain fatty acids, which in B. subtilis constitute ~90% of membrane fatty acids during growth at 37°C93. Perhaps, some combination of nature reviews | microbiology How does the stringency factor RelA fit in? If bacteria are limited for amino acids, uncharged tRNAs bind to the A site of the ribosome and activate the ribosome-associated enzyme RelA, which catalyses the formation of pppGpp from GTP and ATP94. The breakdown product of pppGpp, ppGpp, is a global signalling compound that reduces the rate of transcription of ribosomal RNA and tRNA genes and also activates or inhibits the transcription of many other genes94. In E. coli, two proteins, RelA and SpoT, contribute to pppGpp synthesis; SpoT also has pppGpp hydrolase activity94. However, in B. subtilis, and some other Gram-positive bacteria, a single protein, RelA, has both RelA and SpoT activities95. Induction of the stringent response in B. subtilis leads to a dramatic decrease in the expression of genes that are involved in RNA and protein synthesis, and an increase in the expression of the ilvB operon, gabP, ureABC, appDAB, several rap-phr operons and spo0A96, all of which are members of the CodY regulon31,36. Interestingly, relA mutants of B. subtilis are also defective in sporulation97 and the development of genetic competence38. Part of the explanation for this phenotype could be the hyperaccumulation of GTP. Three decades ago, Freese and co-workers98 showed that for B. subtilis sporulation to be initiated the intracellular pool of GTP must decrease by a factor of 3–5. More recently, Ochi and colleagues38 showed that a B. subtilis relA mutant is incapable of mediating the decrease in the intracellular GTP pool that normally occurs at the transition from exponential growth to stationary phase; an artificial reduction in the GTP pool or the introduction of a codY mutation into the relA mutant strain restores sporulation and competence. As a crucial factor in sporulation and at least two key competence genes are repressed by CodY37,40, the simplest explanation for these results is that, in a relA mutant, the GTP pool remains high, even under the conditions of amino-acid limitation that would normally lead to competence and sporulation, so keeping CodY in its active state and competence and sporulation repressed. The effects of ppGpp synthesis on the GTP pool might also explain the role of ppGpp in B. subtilis ribosomal-RNA synthesis99. In addition, relA mutants are partial BCAA auxotrophs95. The hyperactivity of CodY in a relA mutant might explain this auxotrophy by causing the CodY-dependent repression of BCAA biosynthesis to be unusually strong (FIG. 5). CcpA and CodY in other Gram-positive bacteria CcpA. CcpA is found in virtually all low-G+C Grampositive bacteria and has a similar role to B. subtilis CcpA in controlling metabolism in many of these species. Typically, a ccpA mutant grows slowly in glucose medium, overexpresses utilization genes for alternative carbon sources and is defective in the expression of lactate dehydrogenase and other carbon-overflow pathways100–106. The non-pathogenic bacterium Lactococcus lactis normally carries out homolactic fermentation; a volume 5 | december 2007 | 923 © 2007 Nature Publishing Group REVIEWS Two-component regulatory system A system that responds to an environmental stimulus and regulates gene expression accordingly. Composed of a histidine-kinase sensor, usually situated in the outer membrane, that phosphorylates a response regulator in the cytoplasm, which, in turn, activates transcription from selected promoters. ccpA mutant underexpresses the lactate dehydrogenase gene and produces a mixed acid fermentation pattern instead103. Purified Streptococcus bovis CcpA binds in vitro to a cre site, but only in the presence of the serine-phosphorylated form of HPr102. The control of CcpA activity by serine-phosphorylated HPr is also highly conserved in Gram-positive bacteria. In at least two strains of S. pneumoniae, a ccpA (regM) mutant is attenuated for virulence in three models of infection — bacteraemia100, nasopharyngeal carriage101 and pneumonia101. In the D39 strain, disruption of ccpA results in a reduction in the synthesis of capsular polysaccharide100. However, a direct mechanistic connection between CcpA activity and virulence has not yet been discovered. The CcpA protein of L. monocytogenes controls carbon use, but is not the mediator of the carbon catabolite repression of most virulence-gene expression104,105. One exception might be the internalin genes (inlA and inlB), which are transcribed from several promoters, one of which appears to be under the control of CcpA105. The major regulator of L. monocytogenes virulence is PrfA, a positive regulator of virtually all virulence genes107. Although CcpA is not a factor in the regulation of PrfA synthesis or activity104, the common components of the phosphotransferase system — HPr and enzyme I — do seem to have an important role in modulating PrfA activity108, perhaps owing to their ability to determine the phosphorylation state of phosphotransferasesystem permeases and other transporters 105,108. All PrfA-dependent genes are overexpressed in a mutant that is defective in HPr kinase105. In Staphylococcus aureus, the role of CcpA in metabolism and virulence-gene expression is only beginning to be explored. However, considerable work in the related bacterium Staphylococcus xylosus has established that CcpA is the primary regulator of sugar use and is activated by the serine-phosphorylated form of HPr109. In an S. aureus ccpA mutant, growth in glucose medium is slightly impaired and lactate excretion is delayed106. The mutant expresses lower levels of the virulenceregulating RNA molecule RNAIII, but higher levels of the virulence factors α‑toxin, protein A and serotype-5 capsule106. Canonical cre sites can be discerned upstream of the α‑toxin and protein-A coding regions, but not near the agr (RNAIII-encoding) or capsule genes106. Given the complex, overlapping regulatory systems that control S. aureus virulence, it is not surprising that some of these effects are small; it might prove difficult to determine the contribution of CcpA to the regulation of many of the virulence genes in this species. CcpA has a complex role in regulating the synthesis of Clostridium perfringens enterotoxin. On the one hand, a ccpA mutant is derepressed for enterotoxin gene expression during exponential growth phase; on the other hand, the mutant is highly defective in sporulation, which is normally required for the induction of enterotoxin expression110. In addition, CcpA is needed for the glucose-dependent repression of capsule synthesis and for the high-level expression of the collagenase gene110. 924 | december 2007 | volume 5 CodY. CodY is also a highly conserved protein in lowG+C Gram-positive bacteria. Global analysis — predominantly using transcription microarrays — has revealed that CodY regulons in a wide range of Gram-positive bacteria have much in common. Typically, CodY represses the synthesis of the BCAAs, histidine and arginine and transporters for amino acids, peptides and sugars, and activates the transcription of carbon-overflow pathways and guanine nucleotide synthesis36,43,111,112. In L. lactis, a staple of the dairy industry, CodY represses the synthesis of amino acids, peptide transporters, peptidases and amino-acid catabolism enzymes42,43,113–115 in response to BCAAs, but not to GTP116,117. This selective response to BCAAs seems to be unique to the CodY proteins from the Streptococcus–Lactococcus–Enterococcus family of bacteria. In the past few years, a number of studies have sought to link the activity of CodY to the control of virulence in pathogenic species. In the first such study, Malke, Ferretti and colleagues112 showed that a large number of Streptococcus pyogenes genes are regulated, directly or indirectly, by CodY. Some of these genes, such as fasX and mga, encode positive regulators of virulence and are themselves positively regulated by CodY112,118. The genes that depend on FasX and Mga for their expression are therefore part of the CodY regulon. Others, such as the CovRS two-component regulatory system, are negative regulators of virulence genes and are repressed by CodY112,118. It is unclear which of the virulence-associated genes are direct targets of CodY, although it is intriguing that the gene for a magnesium transporter is preceded by a CodY box, as CovS, the sensor protein of the CovRS two-component system, is activated by magnesium118. Clostridium difficile, the primary causative agent of antibiotic-associated diarrhoea, secretes two toxins that are the primary virulence factors. Expression of the toxin genes depends on an alternative RNA-polymerase sigma factor, TcdR119, the synthesis of which, as well as the synthesis of the toxin proteins, is restricted to stationary phase if cells are grown in rich medium120–122. The tcdR gene is strongly repressed by CodY, which links virulence-factor synthesis to conditions of nutrient limitation123. C. difficile CodY binds tightly to the tcdR promoter region in the presence of GTP and BCAAs. In two clinical isolates of S. aureus, a large number of virulence genes, including regulatory genes and genes that encode toxins, proteases, capsule biosynthesis and biofilm matrix polysaccharide (polysaccharide intercellular antigen), have proved to be derepressed in a codY mutant (C. Majerczyk et al., unpublished observations). Surprisingly, in a third isolate, the absence of CodY led to the failure to produce a biofilm124. More complex roles for CodY have been suggested by recent studies that used L. monocytogenes and Bacillus anthracis. In a mouse model, the growth of L. monocytogenes biofilms and its virulence are both dependent on the RelA protein125. Adherence induces ppGpp accumulation; in the absence of RelA, the bacteria can adhere but cannot multiply125. The severe virulence defect seems to be attributable, at least in part, to the higher GTP pool in relA mutants, as the www.nature.com/reviews/micro © 2007 Nature Publishing Group REVIEWS introduction of a codY mutation partially suppresses the virulence defect of the relA single mutant111. However, CodY might also be a positive regulator of virulence, as a L. monocytogenes codY mutant is not as virulent as its wild-type parent111 and a codY mutation augments the virulence defect that is caused by a ccpC mutation (M. Mittal et al., unpublished observations). Perhaps some aspects of pathogenesis (such as growth in a host) are activated by CodY and others (such as toxin synthesis) are repressed. In B. anthracis, CodY is essential for the synthesis of the three major toxin proteins and for virulence in a mouse model of anthrax (W. van Schaik et al., unpublished observations). It remains to be seen whether this effect is direct or indirect. Conclusions Why would a pathogenic bacterium evolve to have some or all of its virulence genes under the control of CcpA and CodY? The answer to this question depends on our 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Hammer, B. K. & Swanson, M. S. Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp. Mol. Microbiol. 33, 721–731 (1999). Neidhardt, F. C. et al. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (ASM, Washington DC, 1996). 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DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene citB | citC | citZ | mdh Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=genomeprj Bacillus anthracis | Bacillus subtilis | Clostridium difficile | Clostridium perfringens | Escherichia coli | Lactococcus lactis | Listeria monocytogenes | Staphylococcus aureus | Staphylococcus xylosus | Streptococcus pneumoniae | Streptococcus pyogenes Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=protein CcpA | CcpC | CodY | Crh | GltC | HPr | pppGpp | RocG | RocR | TnrA FURTHER INFORMATION Abraham L. Sonenshein’s homepage: http://www.tufts.edu/ sackler/microbiology/faculty/sonenshein/index.html All links are active in the online pdf volume 5 | december 2007 | 927 © 2007 Nature Publishing Group