1 Regulación de metabolismo de carbono en bacterias Gram negativas.Caso: Escherichia coli.

2 Los microorganismos Versatilidad metabólicaUtilizan muchas fuertes diferentes de carbono Se adaptan continuamente a los cambios del medio Compiten con otros por nutrientes limitantes

3 Bacteria Tiene sensores que monitorean su alrededorPueden encender y apagar la utilización de un gran número de fuentes de carbono. Sienten gradientes de concentración de nutrientes. Se adaptan a cambos de fuerza osmótica, estrés, nutrientes, oxígeno (o falta de) y limitación de nutrientes.

4 Sistema PEP-PTS Sistema involucrado en:el transporte y la fosforilación de un gran número de carbohidratos, movimiento hacia estas fuentes de carbono (quimiotáxis) y regulación de muchas rutas metabólicas

5 Componentes del sistemaFosfoenolpiruvato (in) + Carbohidrato (out) Piruvato (in) + Carbohidrato-P (in)

6 Sistema fosfotransferasa (PTS).Energía libre de hidrólis (DG°’) del gupo fosfato del PEP es kcal/mol (-61.5 kJ/mol) La de un grupo fosfato de un carbohidrato tipico es de -3 kcal/mol (-12.5 kJ/mol). Desperdicio?????

7 Funciones del PTS Translocación y fosforilación de sustratos

8 Energética de la PTS PEP es equivalente al ATP como moneda energética en la célula Una molécula de ATP se forma a partir de una de PEP durante la glucólisis por la piruvato cinasa. Por carbohidratos que se acumulan por un sistema no-PTS se gastan más de un equivalente de ATP por unidad de monosacárido. Uno para el transporte y otro para la fosforilación por ATP

9 Reacciones del sistema PTS

10 Proteínas del sistema PTSLa enzima I (EI) y HPr son proteínas solubles y citoplásmicas Participan en la fosforilación de los carbohidratos transportados por PTS (proteínas generales del PTS). Las enzimas II (Ells) son específicas para cada carbohidrato y pueden ser: Una proteína sencilla unida a la membrana que tiene 3 dominios (A, B, y C), como para el manitol. Dos o más proteínas en la que al menos una es membranal (e.g., B y C) y una es soluble (IIA o enzima III [EIII]) como la IICBGlc_IIAGlc para glucosa en E. coli

11

12 Proteína Hpr Hpr por Histidine protein (proteína de histidina)Proteína pequeña monomérica de 9,000 a 10,000 Da excepto cuando hace un dominio con la fosofotransferasa específica del carbohidrato como FPr de S. typhimurium. Se fosforila en una histidina conservada por la P-EI (residuo de histidina 15 en E. coli).

13 La fosforila P-EI en la posición N-1 de un residuo de histidina (His-15 en E. coli HPr),Todas las HPrs secuenciadas o de aquellas que se ha deducido la secuencia tienen este residuo y su entorno altamente conservado.

14

15 <

16 <

17 FIG. 1. Carbohydrate transport and phosphorylation by the PTS and their coupling to glycolysis. Carbohydrates are transported and concomitantly phosphorylated by the PTS. The phosphorylated carbohydrate feeds into glycolysis, normally at the glucose-6-P or fructose-6-P level. Two phosphoenolpyruvate molecules are usually formed in glycolysis, one of which is used to drive the transport and initial phosphorylation of the carbohydrate. As a result, the phosphorylation state of the PTS proteins depends on both the concentration of extracellular carbohydrates and the ratio of internal phosphoenolpyruvate and pyruvate. Abbreviations for enzymes (in boldface type) are as follows: Pgi, phosphoglucose isomerase; Pfk, phosphofructokinase; Fba, fructose-1,6-bisphosphate aldolase; Tpi, triose-phosphate isomerase; Gap, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Pgm, phosphoglycerate mutase; Eno, enolase; Pyk, pyruvate kinase. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2006, p. 939–1031 Vol. 70, No. 4

18 FIG. 2. Mechanisms underlying CCR and inducer exclusion in enteric bacteria. The import of glucose and other PTS carbohydrates leads to net dephosphorylation of the PTS proteins (including EIIAGlc and the B domain of EIIBCGlc) and thereby to inducer exclusion and recruitment of the transcription regulator Mlc to the membrane. The upper left part of the figure shows that unphosphorylated EIIAGlc blocks the import of lactose, maltose, and melibiose and the phosphorylation of glycerol by binding to the respective transporter or kinase. The upper right part of the figure shows recruitment of Mlc by unphosphorylated EIIBCGlc, which prevents the regulator from binding to its target sites on the DNA. In the absence of glucose and in the presence of phosphoenolpyruvate, the PTS proteins are found mainly in the phosphorylated state. The central part of the figure shows that phosphorylated EIIAGlc activates adenylate cyclase (AC) but probably only in the presence of an unknown adenylate cyclase activation factor (ACAF). Adenylate cyclase binds phosphorylated as well as unphosphorylated EIIAGlc (see reference 632). The bottom part of the figure shows the effect of the activated transcription factors (free Mlc and Crp:cAMP) on the transcription of the genes encoding Crp, adenylate cyclase, Mlc, and EIIBCGlc, respectively. The inset shows the Crp:cAMP concentration dependence of crp transcription (deduced from data reported in references 310 and 311). The arrow indicates the physiological concentration of activated Crp in exponentially growing cells in the absence of PTS carbohydrates.

19 Mecanismo de quimiotáxisi en EMecanismo de quimiotáxisi en E. coli y el papel del sistema PTS en la quimiotáxis de carbohidratos. En ausencia de una molécula activa en la quimiotáxis (arriba izquierda), CheA se autofosforila en un residuo de histidina. Esta reacción es estimulada por CheW, que recluta a CheA en la proteína receptor MCP. La proteína P-CheA transfiere su grupo fosforilo a CheY y P-CheY se une al motor del flagelo FliM, haciendo que la rotación del flagelo evoque la rotación de las manecillas del reloj (CW), lo que hace que la bacteria de tumbos. En presencia de la moléculas activa quimiotáctiamente (arriba a la derecha), la actividad autofosforilante de CheA se inhibe, y como resultado de esto, la concentración de P-CheY disminuye. Las moléculas de FliM ya no pueden acomplejarse con CheY, lo que favorece la rotación del flagelo en contra de las manecillas del reloj. Esto resulta en un nado terso hacia donde se incrementa la concentración de la sustancia activa quimiotáctiamente. La presencia de un carbohidrato metabolizable eficientemente por el sistema PTS (o la ausencia de PEP), inducen respuestas similares ya que, en estas condiciones, EI está presente, principalmente en forma no fosforilada. De hecho, EI defosforilado inhibe la autofosforilación de CheA (centro) y, por lo tanto, también favorece el nado liso. La sensibilidad del sistema a las señales está regulada por la metilación y desmetilación de los MCPs (abajo), la que es catalizada por CheR y CheB, respectivamente. La metilación de MCP estimula la autofosoforilación de CheA y la desmetilación la inhibe. FIG. 3. Mechanism of chemotaxis in E. coli and role of the PTS in carbohydrate chemotaxis. In the absence of chemotactically active molecules (top left), CheA autophosphorylates at a histidyl residue. This reaction is stimulated by CheW, which recruits CheA to the MCP receptor protein. P CheA transfers its phosphoryl group to CheY, and P CheY binds the flagellar motor FliM, thereby evoking clockwise (CW) rotation of the flagella, which leads to tumbling of the bacterium. In the presence of chemotactically active molecules (top right), the autophosphorylation activity of CheA is inhibited, and as a result, the concentration of P CheY drops. FliM molecules will no longer be complexed with CheY, which favors counterclockwise (CCW) rotation of the flagella. This results in smooth swimming towards increasing concentrations of the chemotactically active substance. The presence of an efficiently metabolizable PTS carbohydrate (or the absence of PEP) induces a similar response, as under these conditions, EI is present mainly in an unphosphorylated form. In fact, dephospho-EI inhibits the autophosphorylation of CheA (center) and therefore also favors smooth swimming. The sensitivity of the system towards the signal is regulated through methylation and demethylation of the MCPs (bottom), which are catalyzed by CheR and CheB, respectively. MCP methylation stimulates the autophosphorylation of CheA, and demethylation inhibits it.

20 Fig. 1. Models of carbon catabolite repressionFig. 1. Models of carbon catabolite repression. The Åëgure 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 Åíow, while dashed lines show information pathways. A: CCR in enteric bacteria. Incoming carbon sources generate speciÅëc signals by which the activity of speciÅëc regulators is modulated. Concomitantly, metabolism of the internalized carbon sources determines the ratio of phosphoenolpyruvate to pyruvate, which inÅíuences, 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-speciÅëc 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.

21

22 Organización del operón PTSCRP : factor positivo de transcripción

23

24 œ

25 Fuente de carbono represoraEl modelo muestra a una célula bajo dos condiciones: 1, en presencia de una fuente de carbón represiva (fondo blanco), y 2, en ausencia de la fuente represiva de carbón (fondo gris). Lactosa maltosa Fuente de carbono represora Exclusión del inductor ATP CCW GK IIAGlc P + IIAGlc Cya GP FM cAMP + CheY/A P + HPr CAP BglG BglG, proteína antitermidadora del operón -glucósido CAP, proteína activadora por catabolito CCW, rotar en contra de las manecillas del reloj CheY/A, proteínas de quimiotaxis Cya, adenilato ciclasa EI, enzima fosfotransferasa I del sistema PTS FM, motor del flagelo GK, glicerol cinasa GP, glicógeno fosforilasa Hpr, fosfotransferasa del PTS con histidina + EI La regulación por activación (+) y represión (--|) se indica, en dónde los eventos de fosforilación se señalan por flechas sólidas y otras interacciones por flechas punteadas. El estado de fosforilación de IIAGlc se resalta. En condiciones de represión IIAGlc se encuentra desfosforilada y media la exclusión del inductor. En condicones no-represivas, IIAGlc estimula la síntesis de cAMP y dispara, indirectamente, la activación de los gnes reprimidos por catabolito por medio del complejo cAMP-CAP (CCR global). Figure 1. Carbon catabolite regulation in enteric Gram-negative bacteria. The model shows a bacterial cell under two conditions: (i) in the presence of a repressing carbon source (white background), and (ii) in the absence of a repressing carbon source (grey background). Regulation by activation (+) and repression (T) is indicated, whereby phosphorylation events are depicted by solid and other interactions by dashed arrows. The phosphorylation state of IIAGlc is highlighted. Under repressing conditions, IIAGlc is mainly dephosphorylated and mediates inducer exclusion. Under non-repressing conditions IIAGlc-P stimulates cAMP synthesis and triggers, indirectly, activation of catabolite-repressed genes by the cAMP-CAP complex (global CCR). Abbreviations are as follows: BglG, antiterminator protein of the β-glucoside operon; CAP, catabolite activator protein; CCW, counter clockwise; CheY/A, chemotaxis proteins; Cya, adenylate cyclase; EI, PTS phosphotransferase enzyme I; FM, flagellar motor; GK, glycerol kinase; GP, glycogen phosphorylase; HPr, histidine-containing PTS phosphotransferase. For further explanations see text. Antonie van Leeuwenhoek 82: 59–71, 2002. P PEP CCR global Piruvato Regulación del carbono por catabolito en bacterias entéricas Gram negativas.

26 galactosa maltosa SacT + Hpr CW GK Hpr HPr FM HPrK/P CheY/A CcpA -/+El esquema muestra una bacteria bajo dos condiciones: 1, en presencia de una fuente de carbono represora (fondo blanco), y 2, en ausencia de la misma fuente (fondo gris). Fosforilación flechas sólidas Fuente de carbono represora galactosa maltosa SacT Exclusión del inductor + P-his- Hpr CW (vuelta) GK FBP Hpr HPr -ser-P FM HPrK/P CheY/A CcpA ATP -/+ ADP La regulación por activación (+) y represión (--|), se indican en dónde los eventos de fosoforilación se muestran con flechas sólidas y otras interacciones con flechas punteadas. El estado de fosforilación de la HPr se relsalta. En condiciones represoras, Hpr está principalmente fosforilado en una serina (HPr-ser-P), mediando la exclusión del inductor y la represión/activación por catabolito carbonado dependiente de CcpA (CCR/CCA global). En condiciones no represivas, predomina HPr-his-P y activa el transporte dependiente de PTS, glicerol cinasa (GK) y reguladores sustrato específicos como sacT. Figure 2. Carbon catabolite regulation in low-GC Gram-positive bacteria. The model shows a bacterial cell under two conditions: (i) in the presence of a repressing carbon source (white background), and (ii) in the absence of a repressing carbon source (grey background). Regulation by activation (+) and repression (T) is indicated, whereby phosphorylation events are depicted by solid arrows and other interactions by dashed arrows. The phosphorylation state of HPr is highlighted. Under repressing conditions, HPr is mainly serine-phosphorylated (HPr-ser-P), mediating inducer exclusion and CcpA-dependent carbon catabolite repression/activation (global CCR/CCA). Under non-repressing conditions HPr-his-P predominates and activates PTS-dependent transport, glycerol kinase (GK), and substrate-specific regulators like SacT. Abbreviations are as follows: CcpA, catabolite control protein; CheY/A, chemotaxis proteins; cre, catabolite responsive element; CW, clockwise; EI, PTS phosphotransferase enzyme I; FM, flagellar motor; FBP, fructose-1,6-bisphosphate; GK, glycerol kinase; HPr, histidine-containing PTS phosphotransferase; HPrK/P, HPr kinase/phosphatase; SacT, antiterminator protein of the sucrose operon. For further explanations see text. P EI CcpA, proteína de control por catabolito CheY/A, proteína de quimiotéxis cre, elemento que responde al catabolito CW, a favor de las manecillas del reloj EI, enzima I del sistema PTS FM, motor del flajelo FBP, fructose-1,6-bifosfato GK, glicerol cinasa HPr, fosfotransferasa del sistema PTS que contiene histidina HPrK/P, HPr cinasa/fosfatasa SacT, proteína antiterminadora del operon de sacarosa cre PEP CCR/CCA global Piruvato Regulación por catabolito en bacterias Gram positivas con bajo contenido de GC

27 FIG. 4. Alignment of the first 55 amino acids of HPr proteins from firmicutes (B.s., B. subtilis; E.f., E. faecalis; L.c., L. casei; S.s., S. salivarius; S.c., S. carnosus), from a spirochete (T.p., T. pallidum), from proteobacteria with a Ser-46 region strongly resembling the corresponding sequence in HPr of firmicutes (X.f., Xylella fastidiosa; N.m., N. meningitidis), and from other gram-negative bacteria (E.c., E. coli; H.i., H. influenzae; V.c., V. cholerae) (V. cholerae possesses a second HPr in which the region around Ser-46 more strongly resembles the corresponding region in the HPr of firmicutes). The arrows indicate the amino acids His-15 and Ser-46. In HPrs from organisms, in which these residues are phosphorylated, they are shown in boldface type. Conserved regions around the phosphorylation sites are boxed. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2006, p. 939–1031 Vol. 70, No. 4

28 FIG. 5. The gene context of hprK in bacteria of the phylum FirmicutesFIG. 5. The gene context of hprK in bacteria of the phylum Firmicutes. The hprK gene is followed by lgt in all sequenced genomes of the firmicutes except in L. mesenteroides, Oenococcus oeni, and some clostridiae. In addition, there are other genes associated with hprK that appear to be conserved. They include two genes without a known function (in L. lactis, E. faecalis, and several streptococci [S. agalactiae, S. mutans, S. pneumoniae, S. pyogenes, S. suis, S. thermophilus, and S. uberis]) and a glycerol-3-phosphate dehydrogenase gene and a UTP-glucose-1-phosphate uridylyltransferase gene (in E. faecalis) followed by a thioredoxin reductase gene (in L. acidophilus, L. johnsonii, L. gasseri [these species lack the uridylyltransferase], L. brevis, L. plantarum, P. pentosaceus, L. mesenteroides, and O. oeni [the latter two species lack the lgt gene]). Staphylococci contain YvoF, a protein with a hexapeptide transferase motif (S. aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus). YvoF, together with YvoE (a pyrophosphatase), is also present in B. anthracis, B. cereus, B. thuringiensis, Exiguobacterium species, L. innocua, and L. monocytogenes, while YvoD, YvoE, and YvoF are found in the bacilli B. clausii, B. halodurans, B. licheniformis, B. stearothermophilus, B. subtilis, G. kaustophilus, and O. iheyensis.

29 FIG. 6. Mechanisms underlying CCR in firmicutesFIG. 6. Mechanisms underlying CCR in firmicutes. The uptake of glucose and other rapidly metabolizable PTS sugars (top left) leads to a net dephosphorylation of the PTS proteins. The center and bottom of the figure show that the high concentration of FBP present in cells growing on a rapidly metabolizable carbohydrate stimulates the HPr kinase activity of the bifunctional HprK/P and the formation of P-Ser-HPr. P-Ser-HPr interacts with CcpA, and the protein complex binds to the cre operator sites on the DNA. The promoter regions are indicated as 10 and 35. CCA occurs when the cre is located upstream from the promoter, while CCR requires a cre located within or downstream from the promoter. High concentrations of Pi present in resting cells favor the pyrophosphate-producing dephosphorylation of P-Ser-HPr by HprK/P. The top right part of the figure shows that P-Ser-HPr probably interacts with certain non-PTS carbohydrate transport systems, such as the maltose transporter from L. casei, and thereby inhibits their transport activity. Phosphorylation of LacS by P His-HPr stimulates the lactose/galactose exchange reaction (in S. thermophilus). In the presence of rapidly metabolizable PTS substrates, the low level of P His-HPr does not allow sufficient phosphorylation of GlpK, which leads to the inactivation of GlpK and to inducer exclusion. Pyr., pyruvate.

30 Domain structure of the transcription antiterminator LicT and the transcription activators LicR and LevR of B. Subtilis. FIG. 7. Domain structure of the transcription antiterminator LicT and the transcription activators LicR and LevR of B. subtilis. The N-terminal RNA or DNA binding domain and the two regulatory domains PRD1 and PRD2 together with their conserved histidyl residues are indicated. Histidyl residues, which have been shown to become phosphorylated, are in boldface type. When the phosphorylation exerts a positive effect on the activity of the regulator, they are shown in red, whereas blue indicates the sites of negative regulation. For LicT, its four conserved histidyl residues become phosphorylated. Phosphorylation in PRD1 inhibits, while phosphorylation in PRD2 stimulates, LicT activity. Most other antiterminators of the BglG/SacY family also contain four conserved histidines, but sometimes, not all of them can be phosphorylated. LevR contains an NtrC-like central domain and EIIAMan- and EIIBGat-like domains inserted between the complete PRD1 and a truncated PRD2. The positive site of phosphorylation is His-585, and the negative site is His-869. The conserved histidyl residues His-506 and His-567 in PRD1 and the EIIBGat phosphorylation site (Cys-718) do not seem to become phosphorylated. An identical organization can be observed in most other NifA/NtrC-type PRD-containing transcription activators. However, a few LevR-like regulators contain a complete PRD2 with an additional potentially phosphorylatable histidyl residue. The DNA binding domain of LicR resembles that of DeoR, and PRD1 and PRD2 are followed by an EIIBGat-like and EIIAMtl-like domain. An identical domain organization can be observed in other DeoR-type PRD-containing transcription activators. All four conserved histidines in PRD1 and PRD2 are sites of positive regulation, while LicR activity is inhibited by phosphorylation at His-559 in the EIIAMtl-like domain.

31 FIG. 8. Transcription regulation by the PTS via PRD-containing transcription antiterminators. (A) In the absence of the corresponding inducer, full-length transcription of several PTS-encoding genes/operons is inhibited owing to the formation of a terminator structure (t, yellow) on the nascent mRNA upstream from the start codon. Under these conditions, the corresponding antiterminator cannot bind to its RNA target, RAT (blue), because the EIIB is mainly phosphorylated and transfers its phosphoryl group to PRD1 of the antiterminator. The absence of a repressing sugar is expected to also allow phosphorylation at the activating domain (PRD2) by P His-HPr. However, the negative effect of phosphorylation at PRD1 is dominant. The RAT sequence can also form a stem-loop, which, however, was calculated to free less energy than the terminator t. Interestingly, in most antiterminator-controlled PTS operons, the two sites RAT and t overlap (green), and the formation of the terminator therefore prevents the formation of the RAT stem-loop and vice versa. (B) If an inducer is present, the EIIB as well as PRD1 of the corresponding antiterminator will be present mainly in an unphosphorylated form. Because antiterminator-controlled PTSs are usually low-capacity sugar transporters, there will be sufficient P His-HPr to guarantee activating phosphorylation in PRD2. The activated antiterminator binds to its RAT and thus favors the formation of the RAT stem-loop, thereby preventing the formation of the terminator stem-loop, as part of it (in green) is already used for the RAT stem-loop. (C) If, in addition to the inducing sugar, a repressing carbohydrate is present, the amount of P His-HPr will be low in the cells. In firmicutes, P-Ser-HPr will also be formed, which further lowers the amount of P His-HPr. These conditions prevent activating phosphorylation at PRD2, and most antiterminators are therefore inactive, although the presence of the inducer probably prevents the phosphorylation in PRD1. Dephosphorylation of PRD2 in the presence of a rapidly metabolizable PTS sugar therefore represents a CcpAindependent, P-Ser-HPr-dependent CCR mechanism. ptsH1 as well as licT(Pia) but not ccpA mutants are relieved from this type of CCR.

32 FIG. 9. The Dha PTS of E. coli. Phosphotransfer from PEP to Dha is mediated by five distinct proteins (EI, HPr, DhaM, DhaL, and DhaK). DhaM itself is composed of three PTS domains: a truncated EI, HPr, and an EIIA of the mannose class PTS. From the P EIIA domain of DhaM, the phosphoryl group is transferred to an ADP molecule tightly bound to DhaL and from there is transferred to a Dha molecule bound to DhaK. Expression of the E. coli dha operon is regulated by the transcription activator DhaR. In addition, the gene encoding the activator DhaR is subject to negative autoregulation. When Dha is present in the cell, it is rapidly phosphorylated, and the ultimate phosphoryl donor, DhaL, therefore carries predominantly ADP. The DhaL:ADP complex binds to DhaR and stimulates its regulator functions. DhaK without Dha interacts with DhaR and down-regulates its activity, while the complex with its substrate, which is formed when Dha is present in the cell, does not interact with DhaR (37). It should be noted that many other organisms do not contain the three-domain DhaM but possess only EIIADha instead. In these bacteria, HPr probably transfers its phosphoryl group directly to EIIADha.

33 FIG. 10. Gene organization downstream from rpoN in several bacteriaFIG. 10. Gene organization downstream from rpoN in several bacteria. The genes encode the sigma factor 54 (rpoN), the ribosomeassociated protein Y (yhbH, in yellow), EIIANtr (ptsN), an ATP-binding protein (yhbJ, in red), and the HPr paralog NPr (npr), respectively.

34 JOURNAL OF BACTERIOLOGY, June 1996, p. 3411–3417 Vol. 178, No. 12Cra y reg carbono JOURNAL OF BACTERIOLOGY, June 1996, p. 3411–3417 Vol. 178, No. 12

35 cAMP-independent mechanisms of catabolite repression were operative in E. coli (7, 15).the mechanisms of catabolite repression in evolutionarily divergent bacteria are not the same (17, 37, 38).

36 The catabolite repressor/activator (Cra) protein of enteric bacteria was initially characterized as the fructose repressor, FruR. Mutants defective in the cra gene (previously designated fruR) exhibited a pleiotropic phenotype, being unable to grow with gluconeogenic substrates as the sole carbon source (5, 13).

37 the cra gene controlled the transcriptional expression of numerous genes concerned with carbon and energy metabolism (4, 12, 14, 39).

38 Cra protein, a member of the LacI-GalR family, recognizes an imperfect palindromic DNA sequence to which it binds asymmetrically.

39 2 modos de acción de Cra If this Cra operator precedes the RNA polymerase binding site, it activates transcription of the downstream operon, but if it overlaps or follows the RNA polymerase binding site, it represses transcription.

40 The effects of Cra on transcription are counteracted by micromolar concentrations of fructose-1-phosphate and millimolar concentrations of fructose-1,6-bisphosphate, which promote catabolite repression of Cra-activated operons and catabolite activation of Cra-repressed operons.

41 Cra apparently controls the direction of carbon flow in ECra apparently controls the direction of carbon flow in E. coli and consequently influences the rates of utilization of dozens of exogenous carbon sources.

42 CENTRAL IMPORTANCE OF THE FRUCTOSE-SPECIFIC PHOSPHOTRANSFERASE SYSTEM (PTS)Fructose has important in the early evolution of carbohydrate metabolic pathways in bacteria. Fructose is the only sugar that feeds directly into the centrally important Embden- Meyerhof glycolytic pathway without isomerization or epimerization. The metabolism of fructose can be initiated by two distinct routes, and in E. coli both routes are mediated by the phosphotransferase system (PTS) (Fig. 1).

43 The sequence of phosphoryl transfer events in the PTS-catalyzed phosphorylation of fructose in E. coli. PEP, FIG. 1. The sequence of phosphoryl transfer events in the PTS-catalyzed phosphorylation of fructose in E. coli. PEP, the end product of glycolysis, is the immediate phosphoryl donor for the PTS phosphotransfer chain. This chain is initiated by autophosphorylation of Enzyme I (EI), which then transfers phosphate (P) either to the small, heat-stable phosphocarrier protein of the PTS, HPr (bottom scheme), or to its fructose-inducible paralog, FPr, a domain within the diphosphoryl transfer protein (DTP) which also contains the IIAFru domain (top scheme). HPr;P transfers the phosphoryl moiety to the IIAMan constituent of the mannose Enzyme II complex and then to the IIBMan protein in preparation for fructose phosphorylation on the 6-hydroxyl, catalyzed by the IICDMan permease complex (bottom scheme). FPr;P in DTP first transfers its phosphoryl moiety to the IIAFru moiety of DTP in preparation for IIB9BC phosphorylation and subsequent transport and phosphorylation of fructose on the 1-hydroxyl (top scheme). While IIFru phosphorylates fructose with high affinity and specificity, IIMan phosphorylates fructose with low affinity and specificity. Both fructose phosphates are converted to the common intermediate of glycolysis, fructose 1,6-bisphosphate (FBP), which can be further metabolized to yield PEP.

44 CENTRAL IMPORTANCE OF THE FRUCTOSE-SPECIFIC PHOSPHOTRANSFERASE SYSTEM (PTS)The fructose PTS of E. coli is unique in possessing its own HPr-like protein domain, FPr, encoded within the fructose (fru) catabolic operon. Bacteria in many evolutionarily divergent genera (e.g., Azospirillum, Fusobacterium, Lactobacillus, Listeria, Pseudomonas, Rhodobacter, Streptomyces, etc.) possess the high-affinity fructose-specific PTS, but they apparently cannot phosphorylate other sugars via the PTS (16, 22, 23, 32, 33, 42, 44).

45 in Haemophilus influenzae, the first bacterium for which a completely sequenced genome became available (10), genes only for a fructose-specific PTS permease are found. In E. coli three silent gene clusters (designated frv, frw, and frx) uniquely encode silent “backup” fructose PTSs of unknown physiological function (31).

46 Finally, the fru operon of enteric bacteria is regulated at the transcriptional level primarily by Cra, although the cAMP-CRP complex plays a secondary role (9).

47 ISOLATION AND PROPERTIES OF cra MUTANTSFirst isolated by selecting for strains that synthesized the protein products of the fructose (fru) catabolic operon at high constitutive levels. ptsH mutants of E. coli and Salmonella typhimurium lack the small phosphocarrier protein of the PTS, HPr, and consequently cannot utilize most PTS sugars (glucose, mannitol, N-acetyl glucosamine, etc.).

48 Función de Csr (carbon storage regulator)Esto se lleva al cabo usando una proteína pequeña que se une a RNA, CsrA, que reconoce y se une específicamente a mRNAs, en vez de a secuencias específicas de DNA. Fig. 1. Outline of central carbon metabolism in E. coli, its regulation by CsrA and relationship to the aromatic (shikimate) pathway. Negative, positive or no regulation by CsrA of indicated pathways or reactions is depicted as encircled 2, 1, or 3, respectively. The common aromatic precursors, PEP and E4P, are shown in bold boxes. Some compounds derived from the shikimate pathway of E. coli (native aromatics), recombinant strains of E. coli, or from semi-synthetic approaches (non-native aromatics and derivatives) are also shown.