Galactoside Permease Research Articles

Structural analysis of genes participating in melibiose fermentation and isocitrate lyase production in Yersinia pestis strains of the main and nonmain subspecies

Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and non main subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of non main subspecies (caucasica, altaica, and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of non main subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.

Mécanismes d'efflux d'un substrat accumulé par la lactose perméase de Escherichia coli: étude théorique et expérimentale

At the steady-state of accumulation of intracellular lactose by the beta-galactoside permease of Escherichia coli, the rate of efflux of the substrate is equal to its rate of influx. An original experimental method and a mathematical processing of the experimental data are proposed to evaluate the relative involvements of the permease-mediated pathway and of the diffusion component in this efflux. The method consists of inducing the lac operon of the bacteria, and then of removing the inducer and allowing the cells to grow further. The permease content and the membrane surface of diffusion are thus varying independently in such a "de-induction" experiment, along which lactose uptake has been monitored at different times. The analysis of the experimental data show that, under conditions of maximal induction, over 95% of the efflux passes through the energized permease. The relevant parameters of the efflux of lactose have been computed and their values allow the prediction of most classical observations, as well as the prediction, never checked, that under physiological conditions, the higher the external substrate concentration, the higher the permease-mediated efflux, according to a saturation kinetics.

Anti-mRNA: specific inhibition of translation of single mRNA molecules.

A plasmid was constructed to generate RNA complementary to the beta-galactosidase mRNA under control of the phage lambda PL promoter. When this anti-mRNA was produced, synthesis of beta-galactosidase was dramatically inhibited (98%). Syntheses of galactoside permease and transacetylase, whose coding sequences are downstream of the beta-galactosidase coding region, are inhibited to a lesser degree, 80% and 55%, respectively. The generation of anti-mRNA that can be targeted to inhibit a single species of mRNA molecule within cells provides a potent mechanism by which specific transcripts can be translationally inactivated. This can be used to determine the function of proteins as well as to select cloned genes in a single rapid and convenient step.

Lactose inhibits the growth of Rhizobium meliloti cells that contain an actively expressed Escherichia coli lactose operon.

Expression of the Escherichia coli lactose operon in Rhizobium meliloti 104A14 made the cells sensitive to the addition of the beta-galactosides lactose, phenyl-beta-D-galactoside, and lactobionic acid. Growth stopped when the beta-galactoside was added and viability decreased modestly during the next few hours, but little cell lysis was observed and the cells appeared normal. Protein synthesis was not inhibited. Growth was inhibited only when beta-galactosidase expression was greater than 160 U. Lactose-resistant mutants had defects in the plasmid-carried E. coli beta-galactosidase or beta-galactoside permease and in the R. meliloti genome. We speculate that uncontrolled production of galactose by the action of the lactose operon proteins was responsible for growth inhibition.

Use of Escherichia coli operon-fusion strains for the study of glycerol 3-phosphate transport activity.

Strains of Escherichia coli K-12 deleted in the native lac operon and bearing both a wild-type glpT operon encoding for sn-glycerol 3-phosphate (G3P) transport and a hybrid operon in which glpT operator and promoter regions are fused to the lacZ gene were constructed. In strains with such a hybrid operon, beta-galactosidase and beta-galactoside permease become inducible by G3P. In these mutants the function and maturation of the glpT-coded proteins should be distinguishable from the level of gene expression, since the beta-galactosidase activity can serve as an index of the latter. With the aid of such mutants, it was shown that: (i) the expressions of the two neighboring operons, glpT and glpA (encoding anaerobic G3P dehydrogenase), are not coordinate; (ii) upon induction, the appearance of the cytoplasmic beta-galactosidase activity preceded that of methyl-beta-D-thiogalactoside transport activity (requiring only a cytoplasmic membrane protein) by about 4 min and that of G3P transport activity (requiring both a cytoplasmic membrane protein and a periplasmic protein) by about 9 min; and (iii) when cells grown at several temperatures from 24 to 42 degrees C were measured for G3P transport activity at 30 degrees C, the activity increased with the growth temperature, indicating that, within the range studied, the rate of transport increases with the fluidity of membrane phospholipids.

Properties of beta-galactosidase III: implications for entry of galactosides into Klebsiella.

Klebsiella sp. strain CT-200 lacks both its plasmid-borne lac operon, which specifies beta-galactosidase I, and its chromosomal lac operon, which specifies beta-galactosidase II, but it expresses a gene for a third beta-galactosidase, beta-galactosidase III, constitutively. CT-200 was examined to determine whether there was a beta-galactoside permease associated with the beta-galactosidase III gene. The failure of CT-200 to transport thiomethyl-beta-galactoside, o-nitrophenyl-beta-D-galactopyranoside, phenyl-beta-galactoside, lactulose, or galactosyl-arabinose was taken as evidence that beta-galactoside permease is not part of a beta-galactosidase III operon. Optimal assay conditions for beta-galactosidase II, whose activity was used as a measure of beta-galactoside transport, are reported here, as are an improved purification method and some physical and catalytic properties of the enzyme not previously reported.

Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31.

The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.

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 beta-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.

Expression and regulation of lactose genes carried by plasmids.

A number of plasmids carrying the lactose character have been studied. All of the plasmids examined so far code for proteins essential for lactose utilization, i.e., beta-galactosidase and galactoside permease. None of them carries enzymatically or immunologically detectable thiogalactoside transacetylase. The expression of the two enzymes is both negatively and positively controlled: they are inducible by different galactosides and are sensitive to catabolite repression. Since the plasmid-coded lactose systems have many features in common with the Escherichia coli lactose operon, it is suggested that the plasmids could have acquired the lactose genes from an E. coli chromosome.

Anaerobic energy-yielding reaction associated with transhydrogenation from glycerol 3-phosphate to fumarate by an Escherichia coli system.

A particulate subcellular fraction from Escherichia coli K-12 induced in anaerobic sn-glycerol 3-phosphate (G3P) dehydrogenase and fumarate reductase can catalyze under anaerobic conditions the transfer of hydrogens from G3P to fumarate, with attendant generation of high-energy phosphate. The phsophorylation process is more sensitive than the transhydrogenation process to inhibition by the detergent Triton X-100. The same is true with respect to sensitivity to sodium azide, carbonyl cyanide m-chlorophenylhydrazone and N,N'-dicyclohexylcarbodiimide. Such a preparation derived from cells with beta-galactoside permease can accumulate thiomethyl beta-D-galactoside anaerobically, and the accumulation can be stimulated twofold by adding G3P and fumarate. Mutants lacking the membrane-associated Mg2+-dependent adenosine triphosphatase cannot grow anaerobically on glycerol with fumarate as the hydrogen acceptor, although they can grow aerobically on glycerol alone.

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.

The Regulation and Properties of the Galactose Transport System in Escherichia coli K12

Abstract This study of galactose-induced transport in Escherichia coli K12 showed that in most strains two transport systems having the properties of galactose permease and galactoside permease are responsible for the observed transport. Several strains contain only the transport system but still grow normally on galactose. The regulation of this system was shown to be very similar to that of the operon. That is, it is induced by galactose, shows only a slight repression by glucose, and is under the control of the regulator gene. The only differences between the regulations of the transport system and that of the enzymes are the level of induction (4-fold for the transport system versus 15-fold for the enzymes) and the fact that the transport system is not under the control of the operator locus. The β-methyl galactoside transport system is not regulated by either regulatory gene in agreement with the work of Lengeler et al. (Lengeler, J., Hermann, K.O., Unsold, H.J. & Boos, W. (1971) Eur. J. Biochem. 19, 457–470). Galactose-binding protein is not essential for the activity of the transport system as there is no correspondence between the level of the two. In fact, the transport system is present at near normal levels under conditions where there is no measurable binding protein activity. Although the and β-methyl galactoside transport systems have different genetic loci and are controlled by different regulatory systems, they do not function independently when they are both present in the same cell. Rather, they interact in a complex way that results in the rate of transport by the β-methyl galactoside system being reduced at high concentrations and the Km of transport by the system being reduced 3-fold.

Characterization and intragenic position of mutations in the gene for galactoside permease of Escherichia coli.

To test certain models for the structure of the gene for galactoside permease and its product, appropriate mutants of E. coli were isolated after mutagenic treatment, partially characterized and mapped. The 69 mutants found to be in the permease gene were mapped by crosses with episomal deletions which divided the gene into 13 regions.

Inhibition by 3-deoxy-3-fluoro-D-glucose of the utilization of lactose and other carbon sources by Escherichia coli.

Summary: 3-Deoxy-3-fluoro-D-glucose (3FG) was converted to 3FG-6-phosphate by the phosphoenolpyruvate-dependent phosphotransferase system in frozen and thawed Escherichia coli. Up to 0.03g 3FG was taken up/g bacterial dry wt. Uptake of 3FG was not lethal, though 3FG at 0.1 to 10 mM completely prevented or severely inhibited utilization of lactose, fructose, glycerol, succinate, acetate and pyruvate. It prevented lactose utilization by inhibition of the synthesis and activity of both β-galactosidase and galactoside permease. 3FG-resistant mutants were isolated which were deficient in the Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system specific for glucose and for 3FG. Our findings support the view that the ‘glucose effect’ may depend upon glucose itself or a glucose derivative, rather than upon catabolic products.

Distribution of suboptimally induced β- d-galactosidase in Escherichia coli: The enzyme content of individual cells

Novick & Weiner (1957) proposed a model in which induction of the lac operon with suboptimal concentrations of inducer generates a population containing both uninduced and fully induced cells. The latter arise as cells acquire the galactoside transport system, thus initiating an autocatalytic cycle of induction since this permease can transport an inducer for its own synthesis. Evidence in favor of this model has been obtained from direct measurements of the enzyme content of individual cells, using a fluorogenic assay sensitive to one molecule of β- d-galactosidase. Fully induced cells, at the predicted frequency, were found in suboptimally induced populations of wild type strains, and of a strain lacking thiogalactoside transacetylase, but not of a strain lacking galactoside permease. In the wild type, the frequency of cells with an enzyme content intermediate between uninduced and fully induced levels was greater than the frequency predicted for cells within the autocatalytic cycle of induction. According to the model, then, in some of these cells, induction of β- d-galactosidase has occurred without formation of the permease necessary to initiate accumulation of inducer.

Inhibition of methylgalactoside transport in Escherichia coli upon the cessation of unsaturated fatty acid biosynthesis.

The activity of the methylgalactoside transport system of E. coli is impaired upon treatment with 3-decynoyl-N-acetylcysteamine, an inhibitor of unsaturated fatty-acid synthesis. Treated cells are unable to be induced for permease activity, while transport sites synthesized before treatment show a regular loss of activity. The inhibition of methylgalactoside transport occurs at a step after translation of the galactose-binding protein, a component of the permease, and appears to be highly specific, since drug-treated cells show normal viability, protein synthesis, and membrane integrity when transport activity is greatly reduced. A second transport system, the galactoside permease, shows significantly less sensitivity to the inhibitor. That the activity of this permease is maintained in the presence of this inhibitor suggests that the inhibitor does not impair energy coupling.

Interaction of Maltose Transport with the Transport of Glucose and Galactosides

In cells of Escherichia coli possessing both maltose and galactoside permease, fluxes via one permease are independent of the substrate for the other permease. However, both fluxes are partially inhibited by glucose or alpha-methyl glucoside at low concentrations in cells grown on glucose. Neither maltose nor galactosides have an inhibitory effect on glucose permease function. These observations are consistent with the hypothesis that the number of glucose permease systems on the cell surface of such cells is much larger than the number for maltose or galactosides.

Local and non-local interactions of fluxes mediated by the glucose and galactoside permeases of Escherichia coli

1. 1. When α-methyl glucoside is transported via the glucose permease of Escherichia coli, there are two levels of effects on the transport of galactosides via the galactoside permease. The first is direct because it is immediate and responds to the external concentration and not to the instantaneous internal concentrations and appears to result from competition for some membrane component common to the transport via the glucose and galactoside permeases. The second effect acting in addition to the first is indirect in that it requires several minutes for the full inhibition to be observed when low concentrations are used and responds to the internal concentration. Glucose gives the first response, but does not give the second as quickly as does α-methyl glucoside. Neither azide nor succinate aid or prevent this second effect. 2. 2. Under the usual growth conditions that result in high glucose permease, i.e. growth on glucose, galactosides have little inhibitory effect on α-methyl glucoside uptake although α-methyl glucoside effectively inhibits galactoside transport. However, under chemostat growth with glucose limitation, inhibitions are readily demonstrated. Having high levels of both permeases is not the sole requirement for observing interaction between fluxes because chemostat growth at fast dilution in a limiting mixture of glucose plus succinate results in bacteria with high levels of both permeases, but little interaction of either flux with the other.

Segregation of galactoside permease, a membrane marker during growth and cell division in Escherichia coli.

Incubation of Escherichia coli with chloramphenicol causes metabolic and biosynthetic disturbances, the best known of which is the synthesis of RNA and formation of incomplete ribosomes (chloramphenicol particles). As a result of the unbalanced biosynthesis the bacteria transferred in a growth medium exhibit a prolonged lag of recovery and also a lag before development of and lysis by phage λ857 occurs. If lactose is the sole carbon source during incubation with chloramphenicol, the extent of these disturbaces is strongly dependent on the relative amount of β-galactoside permease.

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.