Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
List of types of proteins wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Protein moonlighting wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
Bacterial microcompartment wikipedia , lookup
Signal transduction wikipedia , lookup
Magnesium transporter wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Protein phosphorylation wikipedia , lookup
FEMS Microbiology Letters 209 (2002) 141^148 www.fems-microbiology.org MiniReview Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization Reinhold Bru«ckner b a; , Fritz Titgemeyer b a Mikrobiologie, Universita«t Kaiserslautern, Paul-Ehrlich-Strasse 23, D-67663 Kaiserslautern, Germany Lehrstuhl fu«r Mikrobiologie, Friedrich-Alexander-Universita«t Erlangen-Nu«rnberg, Staudtstrasse 5, D-91058 Erlangen, Germany Received 4 October 2001 ; received in revised form 12 February 2002; accepted 12 February 2002 First published online 12 March 2002 Abstract Carbon catabolite repression (CCR) in bacteria is generally regarded as a regulatory mechanism to ensure sequential utilization of carbohydrates. Selection of the carbon sources is mainly made at the level of carbohydrate-specific induction. Since virtually all carbohydrate catabolic genes or operons are regulated by specific control proteins and require inducers for high level expression, direct control of the activity of regulators or control of inducer formation is an efficient measure to keep them silent. By these mechanisms, bacteria are able to establish a hierarchy of sugar utilization. In addition to the control of induction processes by CCR, bacteria have developed global transcriptional regulation circuits, in which pleiotropic regulators are activated. These global control proteins, the catabolite gene activator protein (CAP), also known as cAMP receptor protein, in Escherichia coli or the catabolite control protein (CcpA) in Gram-positive bacteria with low GC content, act upon a large number of catabolic genes/operons. Since practically any carbon source is able to trigger global transcriptional control, expression of sugar utilization genes is restricted even in the sole presence of their cognate substrates. Consequently, CAP- or CcpA-dependent catabolite repression serves as an autoregulatory device to keep sugar utilization at a certain level rather than to establish preferential utilization of certain carbon sources. Together with other autoregulatory mechanisms that are not acting at the gene expression level, CCR helps bacteria to adjust sugar utilization to their metabolic capacities. Therefore, catabolic/metabolic balance would perhaps better describe the physiological role of this regulatory network than the term catabolite repression. 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Global transcriptional control ; Signal transduction; Metabolic balance ; Autoregulation ; Inducer exclusion 1. Introduction The term carbon catabolite repression (CCR) is currently in use to describe the general phenomenon in microorganisms whereby the presence of a carbon source in the medium can repress expression of certain genes and operons, whose gene products are often concerned with the utilization of alternative carbon sources [1]. In the vast majority of documented cases, the preferred carbon source is glucose with the famous Escherichia coli glucose^lactose diauxie as the classical example [2]. On the contrary, the lactic acid bacterium Streptococcus thermophilus prefers lactose over glucose [3], indicating that adaptation to special ecological niches may result in the choice of practi- * Corresponding author. Tel. : +49 (631) 205 2199 ; Fax : +49 (631) 205 3799. E-mail address : [email protected] (R. Bru«ckner). cally any carbohydrate as favored substrate. While the ¢nal outcome of CCR is uniform, reduced expression of certain genes and operons, the mechanisms leading to repression may be quite diverse. The presence of a repressing carbon source can result in lower concentrations of inducers speci¢c for alternate routes of catabolism, in altered activities of speci¢c regulators, or in the activation of global control proteins, such as the catabolite gene activator protein (CAP) in enteric bacteria or the catabolite control protein (CcpA) in low-GC Gram-positive bacteria. Of these mechanisms, global regulatory circuits mediated by CAP or CcpA were in the focus of interest, implicating their outstanding importance. In a relatively recent controversy over the contribution of di¡erent CCR mechanisms to E. coli glucose^lactose diauxie, it has been demonstrated that inhibition of lactose permease and therefore prevention of induction is the cause for lac operon repression in the presence of both sugars rather than CAP-mediated control [2]. This example from the best studied bac- 0378-1097 / 02 / $22.00 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 5 9 - 1 FEMSLE 10407 3-5-02 142 R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148 terial species E. coli demonstrates that many aspects of CCR remain to be uncovered, especially in less analyzed bacteria. In this review, we will discuss major mechanisms of CCR and emphasize the importance of autoregulatory processes in CCR to balance carbohydrate uptake and metabolic capacities. Since a wealth of information is available from enteric bacteria, especially E. coli, and Gram-positive bacteria with low GC content, most of the data will be from these groups of organisms. 2. Mechanisms preventing carbohydrate-speci¢c induction The mode of carbohydrate uptake in bacteria may be quite diverse depending on the proteins involved in transport. The most striking di¡erence in transport processes is the entry of carbohydrates either in phosphorylated or non-phosphorylated form, a distinction that is especially important for CCR. Carbohydrate transport and concomitant phosphorylation is achieved by components of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS) [6]. The system consists of sugar-speci¢c PTS permeases, also referred to as enzymes II (EII), and two general PTS proteins, enzyme I (EI) and histidine-containing protein (HPr), that participate in the phosphorylation of all PTS-transported carbohydrates. The speci¢c permeases are composed of up to four protein domains (EIIA, B, C, D), at least one of which is membrane-bound. These protein domains may be detached or fused as a single polypeptide chain. E. coli glucose permease consisting of the membrane protein EIICBglc and the cytoplasmic EIIAglc is perhaps the best known example for a composite PTS permease. The PTS phosphoryl transfer chain starts with EI and PEP, proceeds via HPr, EIIA, and EIIB to the sugar, which is transported by EIIC, and results in sugar phosphates that may be metabolized immediately. On the contrary, carbohydrates that are internalized in non-phosphorylated form independently from the PTS need phosphorylation by kinases prior to metabolism. We will in the following refer to the proteins responsible for these transport processes as non-PTS permeases. If more than one carbohydrate is present in the growth medium, bacteria normally take up and utilize only one carbon source at the same time and leave the other substrate in the medium for later use. Prevention of the substrate-speci¢c induction of catabolic genes has been identi¢ed as the major cause of preferential sugar utilization. By processes like inducer exclusion, inducer expulsion [4], or the control of the activity of regulators by phosphorylation [5], the presence of preferred substrates results in the lack of expression of alternative pathways. Inducer exclusion is a regulatory phenomenon whereby a carbohydrate inhibits uptake of another carbon source. In E. coli, inducer exclusion is mediated by the glucose- speci¢c enzyme IIA (EIIAglc ) of the PTS (Table 1; Fig. 1A) [6]. When a PTS substrate, for example glucose, is present, the phosphate group of PTS proteins is drained to the incoming sugar. Consequently, EIIAglc exists predominantly in its unphosphorylated form. This form of EIIAglc binds to non-PTS sugar permeases, that are speci¢c for lactose, maltose, melibiose, and ra⁄nose. As a result, transport of these sugars is inhibited and formation of inducers, e.g., allolactose for lac induction, is prevented. Due to the lack of speci¢c intracellular inducers, the respective operons remain poorly expressed. Interestingly, EIIAglc ^permease interactions are enhanced in the presence of the cognate substrates, ensuring economical use of the regulatory protein [6]. In addition, EIIAglc is able to bind to glycerol kinase preventing the production of glycerol 3-phosphate, the inducer of the glycerol catabolic genes. Since glycerol enters E. coli by facilitated di¡usion and glycerol 3-phosphate is not a substrate for the glycerol facilitator, phosphorylation is required for entrapping glycerol inside the cell. Consequently, repression of glycerol kinase activity by EIIAglc also reduces glycerol uptake. A recent genome-wide survey of E. coli proteins to detect a consensus sequence for EIIAglc binding revealed several novel candidates [7]. Therefore, EIIAglc may carry out more regulatory interactions than recognized so far. In low-GC Gram-positive bacteria, another PTS component, the phosphocarrier protein HPr, is involved in inducer exclusion (Table 1; Fig. 1B). A form of HPr (PSer-HPr), phosphorylated at serine at position 46 by HPr kinase [8,9], was shown in vivo to be essential for repression of non-PTS sugar transport in Lactobacillus casei [10] and Lactococcus lactis [11]. Although these data clearly implicate P-Ser-HPr in inducer exclusion, direct biochemical evidence for the proposed inhibition of permeases is still missing. Another form of inducer control involving glycerol kinase has recently been uncovered in Bacillus subtilis [12]. Glycerol kinase activity is stimulated upon PEP-dependent phosphorylation by phospho-histidine HPr (P-His-HPr) and lack of this phosphorylation, for instance in the presence of PTS substrates, results in prevention of glycerol uptake and repression of the glycerol utilization operon. Besides inducer exclusion, inducer expulsion is believed to contribute to CCR in some Gram-positive bacteria [4]. The phenomenon was detected during sugar uptake studies using non-metabolizable carbohydrates. Addition of rapidly metabolizable carbon sources led to a fast expulsion of the accumulated sugars. Genetic evidence in L. lactis and in L. casei showed that P-Ser-HPr does not mediate inducer expulsion in these organisms [10,11]. Therefore, the mechanism of inducer expulsion remains to be elucidated and its physiological relevance for CCR remains elusive. Another mode to control carbohydrate-speci¢c induction, whose physiological signi¢cance is documented, depends on a specialized duplicated protein domain, the PTS FEMSLE 10407 3-5-02 R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148 143 Table 1 Proteins involved in carbon catabolite repression Proteina Function Target(s) Process Organismsb CAP CcpA Cya HPrK/P EIIAglc activation repression/activation production of cAMP phosphorylation of HPr at serine-46 allosteric inhibition global transcriptional control global transcriptional control global signal transduction global signal transduction inducer exclusion E lGþ E lGþ E P-EIIAglc P-Ser-HPrc stimulation transcriptional corepressor CAP sites CcpA sites (cre) ^ HPr sugar permeases, glycerol kinase Cya CcpA, sugar permeases E lGþ P-His-HPr control of protein activities global signal transduction global transcriptional control and inducer exclusion speci¢c transcriptional control E, lGþ GlkA glucose phosphorylation, interaction with regulator(s) ? global signal transduction hGþ PRD regulators, glycerol kinase unknown CAP, catabolite activator protein ; CcpA, carbon catabolite protein ; Cya, adenylate cyclase; HPrK/P, HPr kinase/phosphatase ; EIIAglc , glucose-speci¢c enzyme IIA; HPr, histidine-containing phosphocarrier protein ; GlkA, glucose kinase ; cre, catabolite responsive element; PRD, PTS regulation domain ; E, enteric bacteria ; lGþ and hGþ , Gram-positive bacteria with low and high GC content, respectively. a Only proteins that act globally and whose contribution to CCR has been de¢ned in vivo are included in the list. The phosphoryl group of EIIAglc and P-His-HPr originate from PEP via the PTS phosphotransfer chain EI-HPr-EIIA. In the presence of PTS sugars these proteins exist predominantly in non-phosphorylated form. In the absence of PTS sugar transport, they remain phosphorylated. On the contrary, P-Ser-HPr is produced at the expense of ATP by HPrK/P in the presence of glycolytic substrates, regardless whether these were transported by the PTS or by non-PTS permeases. b For clarity, individual species within the group of Gram-positive bacteria are not listed. Inducer control mechanisms may vary, but CcpA and HPrK/P appear to be uniformly present. c In B. subtilis and, most likely, in other bacilli, the HPr homologue Crh participates as P-Ser-Crh in CcpA corepression [48]. regulation domain (PRD), which is found in antiterminators and activators [5]. PRD proteins mediate sugar-speci¢c induction mostly of PTS carbohydrate catabolic operons. The RNA- or DNA-binding activity of their Nterminal e¡ector domains is controlled through two PRDs by multiple PTS-mediated phosphorylations in a complex manner. The cognate PTS permeases, acting negatively upon the regulators, are needed for sugar-speci¢c induction. Most but not all PRD-containing regulators require P-His-HPr-mediated phosphorylation for activity. This positive regulation provides the opportunity to control the induction process by CCR. If phosphorylation of PRD is prevented, for example by the transport of another PTS substrate, induction does not occur. Therefore, the ¢nal result of PRD-mediated regulation is the same as for inducer exclusion, prevention of sugar-speci¢c induction. In addition to the mechanisms of induction prevention presented above, competition among PTS permeases for phosphoryl transfer from P-His-HPr would be another way to govern hierarchical sugar utilization [6]. A specialized PTS-mediated regulation through a PTS EIIA-like protein domain that is fused to a non-PTS lactose transporter is found in S. thermophilus [13]. Phosphorylation of this PTS domain by P-His-HPr is not involved in establishing a hierarchy of sugar utilization, but it modulates lactose transport in an autoregulatory control circuit [13]. 3. Transcriptional control by global regulators One of the best studied consequences of the availability of carbohydrates is the activation of global transcriptional control systems, which are mechanistically di¡erent in enteric bacteria and low-GC Gram-positive bacteria. While in enteric bacteria an activation mechanism that uses the CAP protein is realized [14], and CCR is actually the result of diminished activation of promoters for catabolic genes or operons, a negative regulation mechanism by CcpA is operative in low-GC Gram-positive bacteria [15]. In E. coli, CAP activates transcription at more than 100 promoters and is in some cases also involved in repression. The activation process is well understood in structural and mechanistic detail, at least at simple CAP-dependent promoters [14]. CAP needs the allosteric e¡ector cAMP in order to bind e⁄ciently to DNA. Global regulation by CAP responds to the intracellular amount of cAMP (Fig. 1A) and by means of autoregulation to CAP levels [16]. The intracellular cAMP level, in turn, is adjusted by adenylate cyclase, whose activity depends on the phosphorylated form of EIIAglc (P-EIIAglc ) [6]. Besides controlling carbohydrate catabolic genes, CAP is directly involved in the modulation of a large number of other cellular processes. It exerts also indirect control by in£uencing expression of global regulators such as FIS [17], or by contributing to c factor selectivity [18]. Therefore, the role of CAP goes far beyond regulation of sugar utilization. On the other hand, sugar utilization is controlled by further pleiotropic regulators. Several glycolytic, gluconeogenic and glucose-related genes are controlled by Cra (formerly FruR) and Mlc [19,20]. Control of mRNA stability of some of these genes by CsrA adds another level of regulatory complexity [21]. A striking example of the multi-level regulation is the central ptsHIcrr operon, encoding FEMSLE 10407 3-5-02 144 R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148 HPr, EI, and EIIAglc , which is controlled by CAP, Cra, and Mlc. In low-GC Gram-positive bacteria, CcpA is of central importance for global transcription control in CCR (Table 1; Fig. 1B) [15]. Genome-wide analysis of CCR in B. subtilis estimated about 300 genes to be regulated by CcpA [22]. CcpA functions mainly as a repressor of transcription, but activation is also documented. The regulator requires the co-repressor P-Ser-HPr to bind e⁄ciently to its operator sequence cre (catabolite responsive element). Consequently, CcpA activity responds to the level of P-Ser-HPr. In some organisms, the ccpA gene is subject to autoregulation [23,24]. Compared to the profound knowledge on CAP activity, relatively few mechanistic details of CcpA action have been worked out. It appears that further work is needed to fully understand this important regulator. Two other proteins, ccpB and ccpC, participate in glucose repression in B. subtilis [25,26]. Understanding their contributions to global regulation and their interplay with CcpA will be required to get a comprehensive view on CCR in this organism. 4. Signal transduction leading to CCR Fig. 1. Models of carbon catabolite repression. The ¢gure shows regulatory circuits in enteric and low-GC Gram-positive bacteria. The schemes highlight the equivalent roles of two PTS proteins, EIIAglc in enteric bacteria and HPr in Gram-positive bacteria. Their state of phosphorylation and di¡erent phosphorylated forms trigger and coordinate the major responses of carbon regulation. Solid lines indicate catalytic interactions/activities and carbon £ow, while dashed lines show information pathways. A: CCR in enteric bacteria. Incoming carbon sources generate speci¢c signals by which the activity of speci¢c regulators is modulated. Concomitantly, metabolism of the internalized carbon sources determines the ratio of phosphoenolpyruvate to pyruvate, which in£uences, via EI and HPr, the phosphorylation state of the major signal distribution factor EIIAglc . Non-phosphorylated EIIAglc exerts inducer exclusion of non-PTS permeases by allosteric regulation (inhibition), while phosphorylated EIIAglc stimulates adenylate cyclase, thereby triggering global transcriptional control by CAP. B: CCR in low-GC Gram-positive bacteria. Besides carbohydrate-speci¢c induction processes, incoming carbon sources generate glycolytic intermediates that stimulate HPrK/P leading to the phosphorylation of HPr at serine-46. An elevated amount of P-Ser-HPr has three consequences: (i) global transcriptional control by CcpA, (ii) inducer exclusion of non-PTS permeases, and (iii) feedback inhibition of EI-dependent phosphorylation of HPr resulting in reduced PTS transport activity and diminished activity of PRD-containing activators. From the descriptions of major mechanisms of CCR it is apparent that the phosphorylation state of two PTS components, EIIAglc and HPr, determines regulatory responses in E. coli as well as in low-GC Gram-positive bacteria (Fig. 1). However, there is an important di¡erence concerning mutations in the general PTS for both types of regulations. Since EIIAglc /P-EIIAglc is part of the regular PEP-PTS phosphoryl chain, defects in either of the two general PTS components will abolish CCR in E. coli. On the other hand, the regulatory P-Ser-HPr is produced by HPr kinase in an ATP-dependent manner and PTS phosphoryl transfer is not required to trigger P-Ser-HPr-dependent CCR. P-His-HPr-dependent processes, however, require a functional PTS. Consequently, only mutations in ptsH, the gene encoding HPr, are able to block CCR completely in low-GC Gram-positive bacteria. EI de¢ciency leaves P-Ser-HPr-dependent regulation intact, provided a sugar is able to enter the cell by non-PTS transport [27]. How the ratio of P-EIIAglc to EIIAglc could be adjusted according to sugar availability is immediately apparent considering PTS-mediated glucose transport. If glucose is transported, the phosphoryl group of P-EIIAglc is delivered to incoming glucose via EIICBglc , the major PTS glucose transporter. Dephosphorylation of EIIAglc would be the consequence. Similarly, any PTS sugar could indirectly e¡ect the phosphorylation state of EIIAglc by draining phosphate groups to the incoming sugar. This scenario, however, provides no ready explanation for strong CCR by non-PTS carbohydrates such as glucose 6-phosphate. Surprisingly, glucose 6-phosphate and several other non-PTS substrates caused dephosphorylation of EIIAglc FEMSLE 10407 3-5-02 R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148 to various extents [28]. To explain these unexpected results, the authors proposed the PEP to pyruvate ratio to be involved in the control of EIIAglc phosphorylation state. The PEP/pyruvate ratio in turn is in£uenced by metabolism of carbohydrates. Another surprise came, when cAMP and P-EIIAglc levels were compared [28]. As P-EIIAglc is an e¡ector of adenylate cyclase, dephosphorylation of EIIAglc should result in low cAMP production. However, P-EIIAglc levels and the amount of cAMP did not correlate. These results are consistent with previous studies questioning this simpli¢ed regulatory model [29]. Despite long lasting e¡orts to clarify signal transduction leading to CAP-dependent CCR in E. coli, it appears that a major factor contributing to regulation of adenylate cyclase escaped detection. Recently, the glycolytic pathway and its intermediates were implicated in additional regulatory events [30,31]. The dominant mechanism of glycerol kinase repression was found to be the allosteric inhibition by fructose 1,6bisphosphate (FBP) rather than inhibition by EIIAglc . Blocking glycolysis resulted in a markedly reduced level of the main glucose transporter EIICBglc caused by rapid decay of the EIICBglc encoding ptsG mRNA. Hence, glycolysis and glycolytic intermediates could be more important for signal transduction in E. coli than anticipated. Glycolytic intermediates such as FBP are of central importance for signal transduction in low-GC Gram-positive bacteria [9]. A protein kinase, HPr kinase, is activated by elevated levels of FBP to carry out ATP-dependent phosphorylation of HPr at the serine at position 46. High concentrations of inorganic phosphate (Pi ) stimulate the reverse reaction, dephosphorylation of Ser-46, catalyzed by the same enzyme [32]. Therefore, the enzyme is actually bifunctional, an HPr kinase/phosphatase (HPrK/P). How kinase/phosphatase activities are balanced is not yet known. However, recent determination of the X-ray structure of an active fragment of L. casei HPrK/P suggested direct competition of Pi and ATP for binding to HPrK/P [33]. It has been shown in L. lactis that cells taking up rapid metabolizable sugars have a high level of FBP and a low amount of Pi [34]. This situation would therefore favor kinase activity. In addition ATP levels may be involved in regulation [32]. Due to the involvement of glycolysis in the stimulation of HPrK/P kinase activity, it is immediately apparent that PTS and non-PTS sugars are able to cause CCR in this group of organisms (Fig. 1B). Another pathway of signal transduction leading to CCR appears to be operative in Streptomyces coelicolor, a Gram-positive bacterium with high GC content, that lacks the HPrK/P-dependent regulatory system. A central function has been attributed to glucose kinase GlkA, that is also essential for glucose metabolism [35,36]. A global regulatory role for GlkA, distinct from its catalytic function, was proposed, since complementation of a glkA mutant with a heterologous glucose kinase or activation of a cryptic glucose kinase could restore glucose fermentation but 145 not CCR. Moreover, it was reported that CCR exerted by carbon sources that are not metabolized via GlkA was abolished in the glkA mutant. The ¢nding that GlkA belongs to the ROK protein family [37], which consists of transcriptional regulators, open reading frames of unknown function and sugar kinases, supported further the idea of a general regulatory role for GlkA. It had been suggested that kinases of this family would exert their regulatory function interacting with transcription factors. A search for GlkA-binding factors and attempts to demonstrate a regulatory modi¢cation of GlkA did not give conclusive results [38]. It should be noted that the PTS components HPr and an EIIAglc -like protein are present in S. coelicolor [39]. The EIIAglc -like protein could complement EIIAglc of E. coli in glucose uptake and inducer exclusion demonstrating its potential in CCR (Titgemeyer, unpublished). Mutation in ptsH, however, showed no effect on glucose repression [40]. Therefore, the true role of the PTS and GlkA and their interplay in CCR of streptomycetes as well as the nature of the central transcriptional regulator remain to be elucidated. A special regulatory role for a glucose kinase of the ROK protein family was also proposed in the low-GC Gram-positive bacteria Staphylococcus xylosus and Bacillus megaterium [41,42]. Further analysis of GlkA in S. xylosus showed that the enzyme contributes to CCR by its catalytic function as part of a non-PTS glucose utilization system [43,44]. 5. Autoregulatory aspects of CCR The concept of preferred and, therefore, repressing carbon sources seems to implicate that other carbohydrates, whose utilization genes are repressed in the presence of the favored substrate, would not be able to trigger CCR. However, any carbohydrate entering glycolysis in Grampositive bacteria will inevitably activate HPrK/P and turn on CcpA-dependent regulation and inducer exclusion. In E. coli, carbohydrates provoke an equivalent response by modulating cAMP and P-EIIAglc levels. Therefore, CCR results in autoregulatory restriction of sugar utilization, an aspect which has received relatively little attention. The series of autoregulatory events in CCR of E. coli is best illustrated discussing the re¢ned model of lac regulation. As already mentioned, repression of the lac operon in the presence of glucose is due to inducer exclusion and dependent on the lac repressor [2]. When E. coli cells grow on lactose, one could assume that lac operon expression would be at its maximum. Two autoregulatory circuits, however, restrict lac expression under these conditions. First, in contrast to a widespread belief, the level of cAMP in lactose-consuming E. coli remains even slightly lower than in glucose-grown cells, leading to an only moderate activation of the lac promoter by CAP [2]. Secondly, since about half of EIIAglc exists in non-phosphorylated FEMSLE 10407 3-5-02 146 R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148 form in lactose-consuming cells, inducer exclusion is operative and reduced lactose permease activity is the consequence [45]. Taken together, the apparent induction of the lac operon by lactose is the result of enhanced transcription due to the relief from repression, which is partially counteracted by moderate CAP activation and restricted inducer uptake. The ¢nal lac expression level is de¢ned by global autoregulatory CCR. In low-GC Gram-positive bacteria, no complete view on autoregulatory aspects of one operon is available, but some cases of autoregulatory CCR are documented [12,13]. Since CcpA-dependent regulation is triggered by glycolytic intermediates, all catabolic genes for glycolytic sugars harboring cre for CcpA regulation are subject to CcpA-mediated autoregulation. In addition, inducer control may be exerted, provided the cognate sugar permease is sensitive to inhibition by P-Ser-HPr. A third autoregulatory consequence of HPrK/P-mediated HPr phosphorylation has recently been documented [46]. When a mutant allele of the L. casei hprK/P gene encoding an HPrK/P enzyme with strongly reduced phosphatase was expressed in B. subtilis, growth on PTS sugars was abolished. The elevated level of P-Ser-HPr in the mutant strain apparently blocked PTS phosphate transfer. Since P-Ser-HPr was found to be a poor substrate for in vitro PEP-dependent phosphorylation by EI, this inhibition was anticipated, but in vivo evidence was lacking. These results obtained with mutant HPrK/P strongly suggest that phosphorylation of HPr by HPrK/P reduces PTS phosphotransfer also in the wild-type situation, thereby slowing down PTS-dependent sugar transport. In addition, regulatory phosphorylations by P-His-HPr enhancing the activity of glycerol kinase and PRD-containing regulators are reduced. 6. Importance of autoregulatory restriction of sugar utilization To assess the signi¢cance of autoregulation in CCR, it is again instructive to look at certain mutants. Inactivation of the lactose repressor abolishes speci¢c regulation as well as glucose^lactose diauxie, but the cells grow well in the presence of lactose [2]. In a mutant strain with a lactose permease that is insensitive to EIIAglc -mediated inhibition, lactose became inhibitory for growth, when lac gene expression was additionally stimulated by exogenous cAMP [45]. Thus, loss of speci¢c lac regulation is easily tolerated by E. coli, but the absence of CCR is detrimental. Likewise, growth of an HPrK/P-de¢cient strain of S. xylosus was inhibited by glucose in a concentration-dependent manner [47]. In addition, the mutant strain produced elevated levels of methylglyoxal, indicating unbalanced metabolism. The amount of methylglyoxal, however, was not high enough to explain the inhibiting e¡ect of glucose on growth [47]. Thus, the reason for the severe growth retardation of S. xylosus is not clear at the moment, but accu- mulation of toxic metabolites would be a conceivable consequence of unrestricted sugar uptake. Under the same conditions, growth of a CcpA-de¢cient S. xylosus mutant was only slightly impaired [23]. Therefore, loss of a regulatory mechanism other than CcpA control must be responsible for glucose sensitivity in the S. xylosus HPrK/P mutant strain. Further analysis of this phenomenon indicated that the HPrK/P-mediated regulatory events described above are not involved [27]. Interestingly, mycoplasmas, cell wall-less organisms related to low-GC Gram-positive bacteria, retained the gene encoding HPrK/P in their small genomes [9], but have no ccpA gene. This observation may suggest that a common HPrK/P-dependent regulation exists in low-GC Grampositive bacteria, whose nature has not yet been recognized. In conclusion, autoregulatory CCR seems to be important to protect cells from adverse e¡ects caused by the uptake of excess carbohydrate. It will be interesting to analyze further examples of this aspect of CCR. 7. Conclusions The complex regulatory network responsible for CCR serves bacteria to achieve two distinct goals: (i) to utilize one carbon source preferentially from a mixture in the growth medium, and (ii) to limit carbohydrate uptake and utilization according to the cell’s metabolic capacities. In both cases the same control mechanisms are operative, but the strength of the regulatory response di¡ers greatly. The choice of the carbohydrate is made at the level of sugar-speci¢c induction. Repression of alternate carbohydrate utilization genes ensures that only a minimal set of genes is expressed to obtain a certain amount of a carbon source. Thus, hierarchical sugar utilization is meant to use carbon and energy sources economically. If only one carbon source is available, CCR is not absent, but is the inevitable consequence of carbohydrate metabolism acting as a built-in autoregulatory device to limit carbohydrate consumption. It is apparent that this form of regulation should not result in complete repression, otherwise carbon supply would stop. Considering the growth defects of mutants altered in CCR, especially the glucose sensitivity of an S. xylosus HPrK/P-de¢cient strain, autoregulatory CCR appears to be devoted to balance carbohydrate uptake and catabolic/metabolic capacities of the cell. Therefore, autoregulatory limitation of sugar consumption by CCR is indispensable when carbon sources are plentiful and by far more important for bacteria than the ability to utilize certain substrates preferentially. Acknowledgements We would like to thank J. Deutscher for providing information prior to publication. Work in our laboratories FEMSLE 10407 3-5-02 R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148 was supported by the Deutsche Forschungsgemeinschaft (DFG) within the priority program, Molecular Analysis of Regulatory Networks in Bacteria (to R.B. ; BR947/41), by an individual grant (to R.B. ; BR947/3), and to F.T. by grant SFB473, Signal Mechanisms of C-Regulation. References [1] Stu«lke, J. and Hillen, W. (1999) Carbon catabolite repression in bacteria. Curr. Opin. Microbiol. 2, 195^201. [2] Inada, T., Kimata, K. and Aiba, H. (1996) Mechanism responsible for glucose-lactose diauxie in Escherichia coli : challenge to the cAMP model. Genes Cells 1, 293^301. [3] van Den Bogaard, P.T., Kleerebezem, M., Kuipers, O.P. and de Vos, W.M. (2000) Control of lactose transport, L-galactosidase activity, and glycolysis by CcpA in Streptococcus thermophilus: evidence for carbon catabolite repression by a non-phosphoenolpyruvate-dependent phosphotransferase system sugar. J. Bacteriol. 182, 5982^5989. [4] Saier Jr., M.H., Chauvaux, S., Cook, G.M., Deutscher, J., Paulsen, I.T., Reizer, J. and Ye, J.J. (1996) Catabolite repression and inducer control in Gram-positive bacteria. Microbiology 142, 217^230. [5] Stu«lke, J., Arnaud, M., Rapoport, G. and Martin-Verstrate, I. (1998) PRD ^ a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria. Mol. Microbiol. 28, 865^874. [6] Postma, P.W., Lengeler, J.W. and Jacobson, G.R. (1993) Phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev. 6, 543^594. [7] Sondej, M., Sun, J., Seok, Y.J., Kaback, H.R. and Peterkofsky, A. (1999) Deduction of consensus binding sequences on proteins that bind IIAGlc of the phosphoenolpyruvate :sugar phosphotransferase system by cysteine scanning mutagenesis of Escherichia coli lactose permease. Proc. Natl. Acad. Sci. USA 96, 3525^3530. [8] Galinier, A., Kravanja, M., Engelmann, R., Hengstenberg, W., Kilho¡er, M.C., Deutscher, J. and Haiech, J. (1998) New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression. Proc. Natl. Acad. Sci. USA 95, 1823^ 1828. [9] Reizer, J., Hoischen, C., Titgemeyer, F., Rivolta, C., Rabus, R., Stu«lke, J., Karamata, D., Saier, M.H.J. and Hillen, W. (1998) A novel protein kinase that controls carbon catabolite repression in bacteria. Mol. Microbiol. 27, 1157^1169. [10] Dossonnet, V., Monedero, V., Zagorec, M., Galinier, A., Pe¤rezMart|¤nez, G. and Deutscher, J. (2000) Phosphorylation of HPr by the bifunctional HPr Kinase/P-Ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion. J. Bacteriol. 182, 2582^2590. [11] Monedero, V., Kuipers, O.P., Jamet, E. and Deutscher, J. (2001) Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis. J. Bacteriol. 183, 3391^3398. [12] Darbon, E., Servant, S.P., Jamet, E. and Deutscher, J. (2002) Antitermination by GlpP, catabolite repression via CcpA, and inducer exclusion elicited by P-GlpK dephosphorylation control Bacillus subtilis glpFK expression.. Mol. Microbiol. 43, 1039^1052. [13] Gunnewijk, M.G., van den Bogaard, P.T., Veenho¡, L.M., Heuberger, E.H., de Vos, W.M., Kleerebezem, M., Kuipers, O.P. and Poolman, B. (2001) Hierarchical control versus autoregulation of carbohydrate utilization in bacteria. J. Mol. Microbiol. Biotechnol. 3, 401^ 413. [14] Busby, S. and Ebright, R.H. (1999) Transcription activation by catabolite activator protein (CAP). J. Mol. Biol. 293, 199^213. [15] Henkin, T.M. (1996) The role of CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis. FEMS Microbiol. Lett. 135, 9^15. 147 [16] Ishizuka, H., Hanamura, A., Inada, T. and Aiba, H. (1994) Mechanism of the down-regulation of cAMP receptor protein by glucose in Escherichia coli: role of autoregulation of the crp gene. EMBO J. 13, 3077^3082. [17] Nasser, W., Schneider, R., Travers, A. and Muskhelishvili, G. (2001) CRP modulates ¢s transcription by alternate formation of activating and repressing nucleoprotein complexes. J. Biol. Chem. 276, 17878^ 17886. [18] Colland, F., Barth, M., Hengge-Aronis, R. and Kolb, A. (2000) c factor selectivity of Escherichia coli RNA polymerase: role for CRP, IHF and Lrp transcription factors. EMBO J. 19, 3028^3037. [19] Saier Jr., M.H. and Ramseier, T.M. (1996) The catabolite repressor/ activator (Cra) protein of enteric bacteria. J. Bacteriol. 178, 3411^ 3417. [20] Plumbridge, J. (2001) Regulation of PTS gene expression by the homologous transcriptional regulators, Mlc and NagC, in Escherichia coli (or how two similar repressors can behave di¡erently). J. Mol. Microbiol. Biotechnol. 3, 371^380. [21] Romeo, T. (1998) Global regulation by the small RNA-binding protein CsrA and the non-coding mRNA molecule CsrB. Mol. Microbiol. 29, 1321^1330. [22] Moreno, M.S., Schneider, B.L., Maile, R.R., Weyler, W. and Saier, M.H. (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. [23] Egeter, O. and Bru«ckner, R. (1996) Catabolite repression mediated by the catabolite control protein CcpA in Staphylococcus xylosus. Mol. Microbiol. 21, 739^749. [24] Mahr, K., Hillen, W. and Titgemeyer, F. (2000) Carbon catabolite repression in Lactobacillus pentosus : analysis of the ccpA region. Appl. Environ. Microbiol. 66, 277^283. [25] Chauvaux, S., Paulsen, I.T. and Saier Jr., M.H. (1998) CcpB, a novel transcription factor implicated in catabolite repression in Bacillus subtilis. J. Bacteriol. 180, 491^497. [26] Jourlin-Castelli, C., Mani, N., Nakano, M.M. and Sonenshein, A.L. (2000) CcpC, a novel regulator of the LysR family required for glucose repression of the citB gene in Bacillus subtilis. J. Mol. Biol. 295, 865^878. [27] Jankovic, I. and Bru«ckner, R. (2002) Carbon catabolite repression by the catabolite control protein CcpA in Staphylococcus xylosus. J. Mol. Microbiol. Biotechnol. 4, 309^314. [28] Hogema, B.M., Arents, J.C., Bader, R., Eijkemans, K., Yoshida, H., Takahashi, H., Aiba, H. and Postma, P.W. (1998) Inducer exclusion in Escherichia coli by non-PTS substrates : the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc . Mol. Microbiol. 30, 487^498. [29] Notley-McRobb, L., Death, A. and Ferenci, T. (1997) The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase. Microbiology 143, 1909^1918. [30] Holtman, C.K., Pawlyk, A.C., meadow, N.D. and Pettigrew, D.W. (2001) Reverse genetics of Escherichia coli glycerol kinase allosteric regulation and glucose control of glycerol utilization in vivo. J. Bacteriol. 183, 3336^3344. [31] Kimata, K., Tanaka, Y., Inada, T. and Aiba, H. (2001) Expression of the glucose transporter gene, ptsG, is regulated at the mRNA degradation step in response to glycolytic £ux in Escherichia coli. EMBO J. 20, 3587^3595. [32] Kravanja, M., Engelmann, R., Dossonnet, V., Blu«ggel, M., Meyer, H.E., Frank, R., Galinier, A., Deitscher, J., Schnell, N. and Hengstenberg, W. (1999) The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase. Mol. Microbiol. 31, 59^66. [33] Fieulaine, S., Morera, S., Poncet, S., Monedero, V., GueguenChaignon, V., Galinier, A., Janin, J., Deutscher, J. and Nessler, S. (2001) X-Ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain. EMBO J. 20, 3917^3927. FEMSLE 10407 3-5-02 148 R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148 [34] Thompson, J. and Torchia, D.A. (1984) Use of 31 P nuclear magnetic resonance spectroscopy and 14 C £uorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis. J. Bacteriol. 158, 791^800. [35] Angell, S., Lewis, C.G., Buttner, M.J. and Bibb, M.J. (1994) Glucose repression in Streptomyces coelicolor A3(2): a likely regulatory role for glucose kinase. Mol. Gen. Genet. 244, 134^143. [36] Kwakman, J.H.J.M. and Postma, P.W. (1994) Glucose kinase has a regulatory role in carbon catabolite repression in Streptomyces coelicolor. J. Bacteriol. 176, 2694^2698. [37] Titgemeyer, F., Reizer, J., Reizer, A. and Saier Jr., M.H. (1994) Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria. Microbiology 140, 2349^2354. [38] Mahr, K., van Wezel, G.P., Svensson, C., Krengel, U., Bibb, M.J. and Titgemeyer, F. (2000) Glucose kinase of Streptomyces coelicolor A3(2): large-scale puri¢cation and biochemical analysis. Antonie Van Leeuwenhoek 78, 253^261. [39] Parche, S., Nothaft, H., Kamionka, A. and Titgemeyer, F. (2000) Sugar uptake and utilisation in Streptomyces coelicolor: a PTS view to the genome. Antonie Van Leeuwenhoek 78, 243^251. [40] Butler, M.J., Deutscher, J., Postma, P.W., Wilson, T.J., Galinier, A. and Bibb, M.J. (1999) Analysis of a ptsH homologue from Streptomyces coelicolor A3(2). FEMS Microbiol. Lett. 177, 279^288. [41] Wagner, E., Marcandier, S., Egeter, O., Deutscher, J., Go«tz, F. and Bru«ckner, R. (1995) Glucose kinase-dependent catabolite repression in Staphylococcus xylosus. J. Bacteriol. 177, 6144^6152. [42] Wagner, A., Ku«ster-Scho«ck, E. and Hillen, W. (2000) Sugar uptake and carbon catabolite repression in Bacillus megaterium strains with inactivated ptsHI. J. Mol. Microbiol. Biotechnol. 2, 587^592. [43] Fiegler, H., Bassias, J., Jankovic, I. and Bru«ckner, R. (1999) Identi¢cation of a gene in Staphylococcus xylosus encoding a novel glucose uptake protein. J. Bacteriol. 181, 4929^4936. [44] Jankovic, I., Egeter, O. and Bru«ckner, R. (2001) Analysis of catabolite control protein A-dependent repression in Staphylococcus xylosus by a genomic reporter gene system. J. Bacteriol. 183, 580^586. [45] Hogema, B.M., Arents, J.C., Bader, R. and Postma, P.W. (1999) Autoregulation of lactose uptake through the LacY permease by enzyme IIAGlc of the PTS in Escherichia coli K-12. Mol. Microbiol. 31, 1825^1833. [46] Monedero, V., Poncet, S., Mijakovic, I., Fieulaine, S., Dossonnet, V., Martin-Verstraete, I., Nessler, S. and Deutscher, J. (2001) Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch o¡ carbon metabolism. EMBO J. 20, 3928^3937. [47] Huynh, P.L., Jankovic, I., Schnell, N.F. and Bru«ckner, R. (2000) Characterization of an HPr kinase mutant of Staphylococcus xylosus. J. Bacteriol. 182, 1895^1902. [48] Galinier, A., Haiech, J., Kilho¡er, M.C., Jaquinod, M., Stu«lke, J., Deutscher, J. and Martin-Verstraete, I. (1997) The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression. Proc. Natl. Acad. Sci. USA 94, 8439^8444. FEMSLE 10407 3-5-02