Jos C. Arents

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.

Bacteriorhodopsin in liposomes. II. Experimental evidence in support of a theoretical model

In the preceding article equations describing relevant ion flows in illuminated suspensions of bacteriorhodopsin liposomes have been derived. Here these equations are subjected to experimental tests. Changes in permeability characteristics of the liposomal membrane are brought about by addition of specific ionophores and change of medium composition. Using light-driven proton uptake and electrochemical potential differences for protons across the membrane as observation parameters, ridig attempts to falsify the derived equations are unsuccessful. Agreement between equations and experimental results is established on the point of: (i) the antagonistic effect of valinomycin and nigericin on the two components of the proton-motive force, (ii) the time dependence of the changes in transmembrane electrical and chemical potential differences after the onset of illumination. In three independent experimental systems evidence was obtained for the correctness of the postulated dependence of the turnover rate of the photochemical cycle on back pressure by the transmembrane electrochemical potential difference for protons.

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.

Relationship between chemiosmotic flows and thermodynamic forces in oxidative phosphorylation

A set of equations has been derived, describing quantitatively the relationships between flows and thermodynamic forces in the chemiosmotic model of oxidative phosphorylation. Experimental tests of these equations give information on the stoichiometric coupling constants between the different flows.

Investigation of in vivo cross-talk between key two-component systems of Escherichia coli.

Intracellular signal transfer in bacteria is dominated by phosphoryl transfer between conserved transmitter and receiver domains in regulatory proteins of so-called two-component systems. Escherichia coli contains 30 such systems, which allow it to modulate gene expression, enzyme activity and the direction of flagellar rotation. The authors have investigated whether, and to what extent, these separate systems form (an) interacting network(s) in vivo, focussing on interactions between four major systems, involved in the responses to the availability of phosphorylated sugars (Uhp), phosphate (Pho), nitrogen (Ntr) and oxygen (Arc). Significant cross-talk was not detectable in wild-type cells. Decreasing expression levels of succinate dehydrogenase (reporting Arc activation), upon activation of the Pho system, appeared to be independent of signalling through PhoR. Cross-talk towards NtrC did occur, however, in a ntrB deletion strain, upon joint activation of Pho, Ntr and Uhp. UhpT expression was demonstrated when cells were grown on pyruvate, through non-cognate phosphorylation of UhpA by acetyl phosphate.

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.

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 β‐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 β‐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.

Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria.

The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pKa of the uncoupler molecule. The absorption and fluorescene excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca-2+ stongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes. With mitochondrial membranes an effect of Ca-2+ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochrondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it. From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholiped moiety of the membrane, with its anionic group pointing to the aqueous solution.

H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

ABSTRACT Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii.

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 IIAGlc as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIAGlc 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‐β‐D‐thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIAGlc or were insensitive to inducer exclusion because enzyme IIAGlc could not bind to the lactose carrier. Furthermore, we showed that glucose 6‐phosphate caused dephosphorylation of enzyme IIAGlc. In a mutant insensitive to enzyme IIAGlc‐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 IIAGlc‐mediated catabolite repression which embodies repression by non‐ PTS carbon sources.