On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain.

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA(RED). We have applied ultrafast spectroscopy on the photoaccumulated AppA(RED) state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA(RED), the FAD singlet excited state FAD(RED)* decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH(*), which subsequently decays to the original AppA(RED) molecular ground state in 60 ps. Thus, FAD(RED)* is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA(RED) that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA(RED), the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA(RED) ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA(RED) is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.

Glucose-6-phosphate-dependent phosphoryl flow through the Uhp two-component regulatory system.

Expression of the UhpT sugar-phosphate transporter in Escherichia coli is regulated at the transcriptional level via the UhpABC signalling cascade. Sensing of extracellular glucose 6-phosphate (G6P), by membrane-bound UhpC, modulates a second membrane-bound protein, UhpB, resulting in autophosphorylation of a conserved histidine residue in the cytoplasmic (transmitter) domain of the latter. Subsequently, this phosphoryl group is transferred to a conserved aspartate residue in the response-regulator UhpA, which then initiates uhpT transcription, via binding to the uhpT promoter region. This study demonstrates the hypothesized transmembrane signal transfer in an ISO membrane set-up, i.e. in a suspension of UhpBC-enriched membrane vesicles, UhpB autophosphorylation is stimulated, in the presence of [gamma-(32)P]ATP, upon intra-vesicular sensing of G6P by UhpC. Subsequently, upon addition of UhpA, very rapid and transient UhpA phosphorylation takes place. When P approximately UhpA is added to G6P-induced UhpBC-enriched membrane vesicles, rapid UhpA dephosphorylation occurs. So, in the G6P-activated state, UhpB phosphatase activity dominates over kinase activity, even in the presence of saturating amounts of G6P. This may imply that maximal in vivo P approximately UhpA levels are low and/or that, to keep sufficient P approximately UhpA accumulated to induce uhpT transcription, the uhpT promoter DNA itself is involved in stabilization/sequestration of P approximately UhpA.

Autoregulation of lactose uptake through the LacY permease by enzyme IIAGlc of the PTS in Escherichia coli K-12.

Bacterial growth on one or more carbon sources requires careful control of the uptake and metabolism of these carbon sources. In Escherichia coli, the phosphorylation state of enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in this control in two ways. The unphosphorylated form of IIAGlc causes 'inducer exclusion', the inhibition of uptake of a number of non-PTS carbon sources, including lactose uptake by the lactose permease. The phosphorylated form of enzyme IIAGlc probably activates adenylate cyclase. In cells growing on lactose, enzyme IIAGlc was approximately 50% dephosphorylated, suggesting that lactose could inhibit its own uptake. This inhibition could be demonstrated by comparing lactose uptake rates in the wild-type strain and in a mutant in which the lactose carrier was insensitive to inducer exclusion. In this deregulated mutant strain, lactose was consumed much faster, and large amounts of glucose were excreted. It was shown that enzyme IIAGlc was dephosphorylated more strongly and that the cAMP level was lower in the mutant, most probably causing the observed decrease in lac expression level. When the lac expression level in the mutant strain was increased to that of the parent strain by adding exogenous cAMP, growth on lactose was slower, suggesting that enzyme IIAGlc-mediated inhibition of lactose uptake and downregulation of the lac expression level protected the cells against excessive lactose influx. An even stronger increase in the lac expression level in a mutant lacking enzyme IIAGlc caused complete growth arrest. We conclude that the autoregulatory mechanism that controls lactose uptake is an important mechanism for the cells in adjusting the uptake rate to their metabolic capacity.

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.

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]-[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.

Inducer exclusion by glucose 6-phosphate in Escherichia coli.

The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA(Glc) as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA(Glc) leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-beta-D-thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA(Glc) or were insensitive to inducer exclusion because enzyme IIA(Glc) could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA(Glc). In a mutant insensitive to enzyme IIA(Glc)-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA(Glc)-mediated catabolite repression which embodies repression by non-PTS carbon sources.

Escherichia coli is unable to produce pyrroloquinoline quinone (PQQ).

Many bacteria can synthesize the cofactor pyrroloquinoline quinone (PQQ), a cofactor of several dehydrogenases, including glucose dehydrogenase (GCD). Among the enteric bacteria, Klebsiella pneumoniae has been shown to contain the genes required for PQQ biosynthesis. Escherichia coli and Salmonella typhimurium were thought to be unable to synthesize PQQ but it has been reported that strain EF260, a derivative of E. coli FB8, can synthesize PQQ after mutation and can oxidize glucose to gluconate via the GCD/PQQ pathway (F. Biville, E. Turlin & F. Gasser, 1991, J Gen Microbiol 137, 1775-1782). We have re-investigated this claim and conclude that it is most likely erroneous. (i) Strain EF260, isolated originally by Biville and coworkers, was unable to synthesize a holo-enzyme GCD unless PQQ was supplied to the growth medium. No GCD activity could be detected in membrane fractions. (ii) The amount of PQQ detected in the growth medium of EF260 was very low and not very different from that found in a medium with its parent strain or in a medium containing no cells. (iii) EF260 cells were unable to produce gluconate from glucose via the PQQ/GCD pathway. (iv) Introduction of a gcd::Cm deletion in EF260, eliminating GCD, did not affect glucose metabolism. This suggested a pathway for glucose metabolism other than the PQQ/GCD pathway. (v) Glucose uptake and metabolism in EF260 involved a low-affinity transport system of unknown identity, followed most likely by phosphorylation via glucokinase. It is concluded that E. coli cannot synthesize PQQ and that it lacks genes required for PQQ biosynthesis.

BglF, the sensor of the E. coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein.

The Escherichia coli BglF protein is a sugar permease that is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). It catalyses transport and phosphorylation of beta-glucosides. In addition to its ability to phosphorylate its sugar substrate, BglF has the unusual ability to phosphorylate and dephosphorylate the transcriptional regulator BglG according to beta-glucoside availability. By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and thus its ability to bind RNA and antiterminate transcription of the bgl operon. BglF has two phosphorylation sites. The first site accepts a phosphoryl group from the PTS protein HPr; the phosphoryl group is then transferred to the second phosphorylation site, which can deliver it to the sugar. We provide both in vitro and in vivo evidence that the same phosphorylation site on BglF, the second one, is in charge not only of sugar phosphorylation but also of BglG phosphorylation. Possible mechanisms that ensure correct phosphoryl delivery to the right entity, sugar or protein, depending on environmental conditions, are discussed.

Catabolite repression by glucose 6-phosphate, gluconate and lactose in Escherichia coli.

While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIA(Glc) of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of beta-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP* protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these non-PTS carbon sources.

Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells.

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTP gamma S), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t1/2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca2+ ions inhibited the enzyme (half-maximal effect at 0.3 microM), whereas InsP3 had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells.

Isolation and characterization of a mutation that alters the substrate specificity of the Escherichia coli glucose permease.

We isolated 10 mannitol-positive mutants from a mannitol-negative Escherichia coli strain. These mutations mapped within ptsG, encoding the glucose permease (EIIGlc), and resulted in a G-320-to-V substitution that allows EIIGlc to transport mannitol. Gly-320 lies within a putative transmembrane helix of EIIGlc that may be involved in substrate recognition.

A soluble factor and GTP gamma S are required for Dictyostelium discoideum guanylate cyclase activity.

Amoeba of Dictyostelium discoideum show a rapid, transient cGMP synthesis in response to chemotactic stimulation. Using Mg(2+)-GTP as a substrate, guanylate cyclase (E.C. 4.6.1.2.) activity is found exclusively in the particulate fraction of Dictyostelium cells. Here we show that the activity is dependent on the presence of the non-hydrolysable GTP-analogue GTP gamma S, which itself is only a poor substrate for the enzyme under the prevailing conditions. Evidence is presented that a transient exposure of the enzyme to GTP gamma S is sufficient to constitutively activate the enzyme. GTP gamma S-dependent activity is found to require a factor that can be separated from the enzyme by washing the particulate fraction with low salt buffer. Addition of the soluble cell fraction to these washed membranes restores enzyme activity.

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells.

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C.D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51-62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2'-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTP gamma S, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.

Intracellular localization of secretable cAMP in relaying Dictyostelium discoideum cells.

Dictyostelium discoideum cells synthesize and secrete the chemoattractant cAMP within minutes after chemotactic stimulation. During development, this signal-relay process is instrumental in cell aggregation, pattern formation, and differentiation. Cyclic AMP is known to accumulate inside the cell before secretion. In this study we investigated the subcellular localization of the nascent cAMP. After chemotactic stimulation at 0 degrees C and subsequent accumulation of intracellular cAMP, the newly synthesized chemoattractant could be released by gently opening cells in two different ways. Both methods make the cytosolic compartment accessible, whereas intracellular compartments surrounded by a membrane remain largely intact. The first method involved rapid lysis by forced passage through a 5-micron pore-size Nuclepore filter. The second technique was electropermeabilization under carefully controlled conditions that ensured the formation of small, stable pores in the plasma membrane. These pores allowed the passage of small molecules, such as cAMP, but not of macromolecules. To confirm the selectivity for the plasma membrane of both methods, we showed that a typical vesicular cell compartment, the lysosome, remained intact. Both procedures immediately released all intracellularly accumulated cAMP. We interpret our results as strong evidence for accumulation of nascent cAMP in the cytosolic compartment rather than in a vesicular compartment before it is secreted. This implies that cAMP secretion takes place via a trans-membrane transport mechanism, rather than by exocytosis.

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates.

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.

Guanine nucleotides modulate the function of chemotactic cyclic AMP receptors in Dictyostelium discoideum.

Guanosine di- and triphosphates specifically decrease the affinity of chemotactic cAMP receptors in isolated Dictyostelium discoideum membranes. The K0.5 was increased from 50 nM to 150 nM. Receptors were shown to be heterogeneous in dissociation kinetics. In the absence of guanine nucleotides three dissociation processes could be resolved, having first order rate constants of 8.7 X 10(-4), 1.3 X 10(-2), and higher than 0.1 s-1. Guanine nucleotides decreased the affinity for cAMP by transforming the slowest dissociating receptor form (KD is 8 nM) to forms dissociating more rapidly. Our data indicate that a guanine nucleotide binding protein (G-protein) is involved in the transduction of the cAMP signal in D. discoideum.

Isolation and properties of cyclic AMP-dependent protein kinase from Dictyostelium discoideum.

Cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) in Dictyostelium discoideum was shown to be developmentally controlled. No activity was measured in vegetative cells, but activity increased rapidly during differentiation. A simple procedure for the isolation of the catalytic subunit of the kinase from aggregating cells is presented. The cyclic AMP-dependent holoenzyme could be reconstituted by adding purified D. discoideum cyclic AMP-binding protein. Molecular weight, kinetic parameters, pH dependence and affinity for cyclic AMP were determined for the enzyme. Most properties are similar to those of cyclic AMP-dependent kinase from mammalian cells.

Two (completely) rate-limiting steps in one metabolic pathway? The resolution of a paradox using bacteriorhodopsin liposomes and the control theory.

Proton pumping by bacteriorhodopsin and charge-compensating ion movement can both and simultaneously behave as the rate-limiting step in light-driven proton uptake into bacteriorhodopsin liposomes. This apparently excessive control exerted on the net proton influx is possible because of the negative (-1) 'control coefficient' of the net proton influx with respect to the proton leaks. Furthermore, the property of bacteriorhodopsin that it is inhibited by the membrane potential is responsible for the transfer of part of the control on the net proton influx from the first, irreversible, step in the pathway (i.e. bacteriorhodopsin) to the second, reversible, step (i.e., charge-compensating ion movement).

Linear relations between proton current and pH gradient in bacteriorhodopsin liposomes.

The dependence of proton movement across the membrane of bacteriorhodopsin liposomes on the pH gradient was investigated. Under the appropriate experimental conditions, endogenous proton (or hydroxyl) leakage, proton movement catalyzed by protonophore or nigericin, and light-driven proton translocation depend linearly on the pH gradient. This justifies the use of linear proton flux vs. protonmotive force relations in a recent mosaic thermodynamic description of ion translocation in bacteriorhodopsin liposomes [Westerhoff, H. V., Scholte, B. J., & Hellingwerf, K. J. (1979) Biochim. Biophys. Acta 547, 544-560]. Since bacteriorhodopsin liposomes are a model system for all biological energy transducing systems in which proton pumps are involved, these findings also explain linear relations between proton flux and protonmotive force observed in and postulated for those systems. In cases where the membrane potential is not clamped at a low value, an initial phase of rapid proton movement occurs, followed by a phase of slower proton movement. The rate of proton movement during the slow phase is again linear with the pH gradient. Such a linear relation is not observed for the fast phase. Since the rapid proton movement phase is also observed in liposomes without bacteriorhodopsin, it is not due (only) to dissociation of scalar protons from bacteriorhodopsin. We suggest that during the initial phase of proton movement, the proton flux is not yet electrically compensated by the fluxes of other ions.

Mosaic nonequilibrium thermodynamics describes biological energy transduction.

A procedure, called "mosaic nonequilibrium thermodynamics," for describing ion movement and energy transduction in biological membranes is tested in a model system: bacteriorhodopsin liposomes. The important steps in the theoretical derivations are summarized; one of the experimental tests of the postulated fundamental flow-force relationships is shown. Furthermore, how the quantitative method, even if used only qualitatively, facilitates analysis and understanding of experimental results (in this case, the effect of medium composition on the development of pH gradient and membrane potential in the bacteriorhodopsin liposomes) is shown. The main advantage of this method lies in its quantitative description of the effect of variation of system parameters on the performance of, in this case, the reconstituted proton pump bacteriorhodopsin. As an example, the method is shown to explain quantitatively the dependence of the steady-state pH gradient on the light intensity. Even in more refined analyses of experiments, the quantitative theoretical description is in full accordance with the experimental results; this is illustrated by considering the effect of valinomycin on the dependence of the initial rate of proton uptake into bacteriorhodopsin liposomes on light intensity. It is concluded that mosaic nonequilibrium thermodynamics describes ion movement and energy transduction in the model system of bacteriorhodopsin liposomes and, therefore, may be applied to any other biological system performing such processes.