Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers

The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose-inducible promoters P(tac) and P(trc) by the natural inducer lactose and the synthetic inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) was investigated. Lactose requires active transport by LacY, whereas IPTG can freely penetrate the cell wall. In E. coli strains lacking a functional LacY, IPTG is required for induction of P(tac) and P(trc). In E. coli strains carrying a functional LacY, induction of P(trc) and P(tac) with intermediate concentrations of lactose gave rise to two subpopulations, one fully induced and one uninduced, whereas a single, fully induced population resulted when high inducer concentrations were used. In contrast, induction with IPTG gave rise to a single population of cells at all inducer concentrations in both lacY and lacY(+) strains.     

Investigating autocatalytic gene expression systems through mechanistic modeling.

A structured model of gene expression, which incorporates the stochastic behavior of cellular processes, was developed to examine the "all-or-none" phenomenon observed in autocatalytic systems (e.g. the lac operon). Autocatalytic expression systems typically have the genes encoding the inducer transport proteins controlled by internal inducer levels, so that transport of the inducer increases production of the transport protein. The model was able to predict the unique behaviors of autocatalytic expression systems that have been experimentally observed and provided valuable insight into the role of population heterogeneity in these systems. The simulations substantiate the importance of stochastic processes on induction of gene expression in autocatalytic systems. The simulation results show that the all-or-none phenomenon is governed largely by random cellular events, and that population-averaged variations in gene expression are due to changes in the frequency of full gene induction in individual cells rather than to uniform variations in gene expression across the entire population. In addition, the model shows how concentrations of inducer too low to induce expression in uninduced cells can maintain induction in pre-induced cultures. A comparison of induction behaviors from an autocatalytic system and a system having constitutive synthesis of the transport protein showed that transport protein levels must be decoupled from inducer control to achieve homogeneous expression of a gene of interest in all cells of a culture.     

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.

Escherichia coli forms three permeases that can transport the amino acid tryptophan: Mtr, AroP, and TnaB. The structural genes for these permeases reside in separate operons that are subject to different mechanisms of regulation. We have exploited the fact that the tryptophanase (tna) operon is induced by tryptophan to infer how tryptophan transport is influenced by the growth medium and by mutations that inactivate each of the permease proteins. In an acid-hydrolyzed casein medium, high levels of tryptophan are ordinarily required to obtain maximum tna operon induction. High levels are necessary because much of the added tryptophan is degraded by tryptophanase. An alternate inducer that is poorly cleaved by tryptophanase, 1-methyltryptophan, induces efficiently at low concentrations in both tna+ strains and tna mutants. In an acid-hydrolyzed casein medium, the TnaB permease is most critical for tryptophan uptake; i.e., only mutations in tnaB reduce tryptophanase induction. However, when 1-methyltryptophan replaces tryptophan as the inducer in this medium, mutations in both mtr and tnaB are required to prevent maximum induction. In this medium, AroP does not contribute to tryptophan uptake. However, in a medium lacking phenylalanine and tyrosine the AroP permease is active in tryptophan transport; under these conditions it is necessary to inactivate the three permeases to eliminate tna operon induction. The Mtr permease is principally responsible for transporting indole, the degradation product of tryptophan produced by tryptophanase action. The TnaB permease is essential for growth on tryptophan as the sole carbon source. When cells with high levels of tryptophanase are transferred to tryptophan-free growth medium, the expression of the tryptophan (trp) operon is elevated. This observation suggests that the tryptophanase present in these cells degrades some of the synthesized tryptophan, thereby creating a mild tryptophan deficiency. Our studies assign roles to the three permeases in tryptophan transport under different physiological conditions.     

Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport

Adhya, Sankar (University of Wisconsin, Madison), and Harrison Echols. Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport. J. Bacteriol. 92:601-608. 1966.-The inhibitory effect of glucose on the induction of the enzymes required for galactose utilization ("glucose effect") was studied in Escherichia coli. Experiments on the uptake into the cell of labeled inducers (d-galactose-C(14) and d-fucose-H(3)) pointed to inhibition at the level of inducer transport as the possible primary mechanism of the glucose effect in the case of the gal enzymes. This interpretation was supported by the finding that a mutant constitutive for the lac enzymes was resistant to glucose inhibition of galactose induction of the gal enzymes; the mutant had acquired a glucose-resistant alternative transport mechanism for galactose via the constitutively synthesized galactoside permease. Further support for the transport inhibition model was provided by the finding that glucose did not substantially inhibit induction of the gal enzymes when glucose and galactose were produced intracellularly by beta-galactosidase hydrolysis of lactose, even if excess glucose was added. The inducer uptake experiments also showed that d-galactose and d-fucose probably enter the cell via different transport systems, although uptake of both compounds was inhibited by glucose.     

Light requirement for induction and continuous accumulation of an ammonium-inducible NADP-specific glutamate dehydrogenase in chlorella

The ammonium-inducible NADP-specific glutamate dehydrogenase of Chlorella sorokiniana was shown to require light for both its induction by ammonia in uninduced cells, and its continuous accumulation in fully induced cells. Addition of ammonia to uninduced cells in the light resulted in a 35-minute induction lag followed by linear and coincident increases in enzyme activity and antigen. Enzyme activity was not induced in the dark; however, transfer of these cells to the light resulted in an immediate increase in enzyme activity and antigen. The absence of an induction lag suggested that mRNA sequences and/or an enzyme precursor with different antigenic properties than the active holoenzyme accumulated in cells in the dark in ammonium medium. When fully induced cells were transferred to the dark, the activity of the enzyme quickly ceased to accumulate. In contrast to the NADP-specific isozyme, the cells also contain a constitutive NAD-specific isozyme which was shown to accumulate in cells in the dark in either ammonium or nitrate medium.     

Biochemical studies of melibiose metabolism in wild type and mel mutant strains of Salmonella typhimurium

I identified two enzyme activities, alpha-galactosidase and a galactoside permease, required for melibiose metabolism by Salmonella typhimurium. These activities are very low under normal growth conditions, but their production can be induced by melibiose and gratuitously by melibiitol. Melibiose induction is severely inhibited by glucose, but the glucose effect can be countered by 3', 5' cyclic adenosine monophosphate. I isolated two phenotypic classes of mutants not able to utilize melibiose as a carbon source. One class, Car(-), is deficient in the phosphotransferase system. The other, Mel, lacks either alpha-galactosidase, galactoside permease, or both functions.     

Inducible System for the Utilization of β-Glucosides in Escherichia coli I. Active Transport and Utilization of β-Glucosides

Wild-type Escherichia coli strains (beta-gl(-)) do not split beta-glucosides, but inducible mutants (beta-gl(+)) can be isolated which do so. This inducible system consists of a beta-glucoside permease and an aryl beta-glucoside splitting enzyme. Both can be induced by aryl and alkyl beta-glucosides. In beta-gl(-) and noninduced beta-gl(+) cells, C(14)-labeled thioethyl beta-glucoside (TEG) is taken up by a constitutive permease, apparently identical with a glucose permease (GP). This permease has a high affinity for alpha-methyl glucoside and a low affinity for aryl beta-glucosides. No accumulation of TEG occurs in a beta-gl(-) strain lacking glucose permease (GP(-)). In induced beta-gl(+) strains, there appears a second beta-glucoside permease with low affinity for alpha-methyl glucoside and high affinity for aryl beta-glucosides. Autoradiography shows that TEG is accumulated by the beta-glucoside permease and glucose permease in two different forms (one being identical with TEG, the other probably phosphorylated TEG). In GP(+) beta-gl(+) strains with high GP activity, alkyl beta-glucosides induce the enzyme and the beta-glucoside permease after a prolonged induction lag, and they competitively inhibit the induction by aryl beta-glucosides. The induction lag and competition do not exist in GP(-) beta-gl(+) strains. It is assumed that phosphorylated alkyl and thioalkyl beta-glucosides inhibit the induction, and that this inhibition is responsible for the induction lag.     

Energetics of galactose,proline, and glutamine transport in a cytochrome-deficient mutant of salmonella typhimurium

The effect of inhibitors and uncouplers on the osmotic shock-sensitive transport systems for glutamine and galactose (by the β-methyl galactoside permease) was compared to their effect on the osmotic shock-resistant proline and galactose permease systems in cytochrome-deficient cells of Salmonella typhimurium SASY28. Both osmotic shock-sensitive and -resistant systems were sensitive to uncouplers and to inhibitors of the membrane-bound Ca²⁺, Mg²⁺-activated adenosine triphosphatase. This suggests that uptake by both types of systems is energized in these cells by an electrochemical gradient of protons formed by ATP hydrolysis through the ATPase.     

Product induction in Pseudomonas acidovorans of a permease system which transports L-tryptophan

Growth of Pseudomonas acidovorans in the presence of l-tryptophan resulted in the appearance of a tryptophan transport system which was extremely sensitive to sodium azide or 2,4-dinitrophenol. Asparagine-grown cells possessed no detectable tryptophan "permease" activity. Substitution of l-kynurenine for l-tryptophan in the growth medium also induced the tryptophan permease activity, along with tryptophan oxygenase and kynurenine formamidase. This is the first reported example of the product induction of a permease activity. Irrespective of whether Pseudomonas cells are grown in the presence of d- or l-tryptophan, the resulting induced tryptophan permease activity is specific for the l-isomer. In addition, the radioactive compounds l-leucine, l-phenylalanine, or dl-5-hydroxytryptophan are not transported. When dl-5-fluorotryptophan is a component of the inducing medium (with l-tryptophan), induction of tryptophan permease activity, as well as tryptophan oxygenase, is inhibited. In the permease assay system, using normally induced cells, the fluoroanalogue inhibited strikingly tryptophan transport. Therefore, this analogue may inhibit induction by blocking inducer transport into the cell. When added to the l-tryptophan-inducing medium, dl-7-azatryptophan markedly enhanced induction of tryptophan oxygenase, but the level of tryptophan permease activity was not further elevated. The mechanism of this analogue is unclear at present. Invariant tryptophan permease activity levels are found in cells grown with 5 or 15 mml-tryptophan or 5 mml-kynurenine, whereas the respective tryptophan oxygenase levels are greatly different. Together with other results, these results indicate that the synthesis of tryptophan permease activity is not coordinate with that of tryptophan oxygenase. Tryptophan transport is strongly inhibited by l-formylkynurenine and by l-kynurenine. These two metabolites were prepared in radioactive form, and they are actively transported following bacterial growth on l-tryptophan or l-kynurenine. Preliminary results suggest the tryptophan permease activity may be distinct from the permease(s) activity for l-formylkynurenine and l-kynurenine. Kynurenine, then, is capable of inducing tryptophan permease and kynurenine permease activities.     

Membrane Translocation of Mannitol in Escherichia coli Without Phosphorylation

Galactosyl-mannitol can be transported into cells of Escherichia coli by beta-galactoside permease and can be hydrolyzed rapidly to mannitol and galactose by beta-galactosidase. When a mutant strain lacking enzyme I of the phosphoenolpyruvate phosphotransferase system and constitutive in the lactose system was presented with galactosyl-mannitol in which the mannitol moiety was labeled with (3)H, the liberated mannitol remained inside the cell if the Enzyme II complex of the phosphoenolpyruvate phosphotransferase system for mannitol was uninduced. It is postualted that one of the enzyme II proteins can still catalyze translocation of mannitol across the cell membrane even when phsophorylation is not possible.     

Role of lac genes in induction of beta-galactosidase synthesis by galactose

Strain BL1003, a lacO mutant, synthesizes beta-galactosidase constitutively at a low rate. The enzyme is further inducible by d-galactose to the same differential rate as is seen in the presence of an optimal concentration of thiomethylgalactoside. lacY Mutants derived from strain BL1003 are not inducible by galactose, although they synthesize beta-galactosidase at the low constitutive rate characteristic of the parent. Galactose is a weak inducer of beta-galactosidase synthesis in wild-type Escherichia coli K-12, but it is more effective when the wild type has been preinduced with isopropyl-beta-d-thiogalactoside. Nevertheless, the rise in the differential rate of synthesis in response to galactose in a preinduced wild-type culture is much lower than in strain BL1003. Thus, two factors are involved in the induction of strain BL1003 by galactose: the mutant operator and the constitutive permease. The operator has an altered sensitivity to the i product-galactose complex. The low constitutive level of permease enabled the cells, at the high concentrations of galactose used (5 x 10(-2)m), to maintain a sufficient internal concentration for further induction.     

Fast measurement of galactoside transport by lactose permease

Lactose permease of Escherichia coli was reconstituted into vesicles of dimyristoylphosphatidylcholine, and the rate of galactoside counterflow was measured in the millisecond time range. The turnover number and the half-saturation constant for transport agree with the values known for cells. This result demonstrates that lactose permease is the sole protein necessary for galactoside transport. Furthermore, lactose permease seems not to require a high level of negatively charged lipids or a certain degree of unsaturation of the lipid hydrocarbon chains. However, the lipids must be in the fluid state, because the transport rate drastically decreases below the lipid ordered fluid phase transition.     

Lag in adaptation to lactose as a probe to the timing of permease incorporation into the cell membrane.

If bacteria are incapable of forming and incorporating proteins into the cytoplasmic membranes in all phases of the cell cycle, then not all cells from an asynchronous culture should be capable of growth when switched to a new carbon and energy source whose metabolism requires new membrane function. The transfer of an inducible culture to low lactose provides such a situation since the cells cannot grow unless galactoside permease can function to concentrate the lactose internally. From such experiments, it was concluded that the Y gene product of the lac operon is synthesized, incorporated, and can start functioning in active transport, at any time throughout the bulk of the cell cycle. Not only were the lags before growth re-ensued much shorter than would be expected if the membrane transport capability could only be developed in a small portion of the cycle, but brief pulses of a gratuitous inducer shortened the lags much further. Three types of Escherichia coli ML 30 culture were studied: cells that had exhausted the limiting glucose; cells taken directly from glucose-limited chemostats; and a washed suspension of highly catabolite repressed cells from cultures grown in high levels of glucose and gluconate. The growth studies reported here were performed on-line with a minicomputer. They represent at least an order of magnitude increase in accuracy in estimating growth parameters over previous instrumentation.     

Malate Utilization by a Group D Streptococcus: Regulation of Malic Enzyme Synthesis by an Inducible Malate Permease

Induction of an nicotinamide adenine dinucleotide-specific malic enzyme and a malate entry system permits Streptococcus faecalis to grow at the expense of malate. Evidence is presented which shows that biosynthesis of the permease, but not of the malic enzyme, is subject to catabolite repression by glucose. In contrast to the malic enzyme, the catalytic function of the entry system does not appear to be inhibited by intermediate products of glycolysis. Although the induction of the entry system does not appear to be coordinated with the induction of the malic enzyme, the latter process is dependent upon the permease for the transport and accumulation of inducer.     

Inhibitor studies of dissimilative Fe(III) reduction by Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans")

Aerobic respiration and dissimilative iron reduction were studied in pure, batch cultures of Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans"). Specific respiratory inhibitors were used to identify elements of electron transport chains involved in the reduction of molecular oxygen and Fe(III). When cells were grown at a high oxygen concentration, dissimilative iron reduction occurred via an abbreviated electron transport chain. The induction of alternative respiratory pathways resulted from growth at low oxygen tension (less than 0.01 atm [1 atm = 101.29 kPa]). Induced cells were capable of O2 utilization at moderately increased rates; dissimilative iron reduction was accelerated by a factor of 6 to 8. In cells grown at low oxygen tension, dissimilative iron reduction appeared to be uncoupled from oxidative phosphorylation. Models of induced and uninduced electron transport chains, including a mathematical treatment of chemical inhibition within the uninduced, aerobic electron transport system, are presented. In uninduced cells respiring anaerobically, electron transport was limited by ferrireductase activity. This limitation may disappear among induced cells.     

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.