Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-β,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.     

The α-galactosidase from Escherichia coli K12

Growth of Escherichia coli on melibiose requires the induced synthesis of α-galactoside permease and α-galactosidase. Hydrolysis of the chromogenic substrate p-nitrophenyl-σ-galactoside by whole bacteria is dependent on intact oxidative metabolism. The α-galactosidase from E. coli was isolated for the first time as a soluble enzyme. In cell-free extracts p-nitrophenyl-α-galactoside hydrolisis was observed only at high protein concentrations and the activity decreased exponentially with the square of the dilution. The reason for this behaviour was shown to be that, unlike other known α-galactosidases, the enzyme of E. coli requires NAD. For optimal activity the enzyme also requires Mn²⁺, a high concentration of 2-mercaptoethanol, and a pH of 8.1. The approximate molecular weight of the active from of α-galactosidase as determined by sedimentation in a sucrose gradient is 200 000. Due to the instability of the enzyme, its purification has not been achieved.     

Differential effects of visible light on active transport in E. coli

Light of wavelengths above 400 nm inactivated several active transport systems in E. coli ML 308. Rates of inactivation for uptake of threonine, glycine, leucine and methionine were similar and differed from those for methyl thio-β-D-galactoside and phenylalanine. These differential effects indicate that inactivation of the threonine, glycine, leucine and methionine systems is linked to a common photochemical lesion differing from that involved in the inactivation of the methyl thio-β-D-galactoside and phenylalanine systems. These lesions may serve as labels to identify molecules involved in transport or energy coupling processes.     

Half-of-the sites reactivity in the catalytic mechanism of yeast glyceraldehyde 3-phosphate dehydrogenase

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.     

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H⁺/sugar cotransport. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ is less dependent on the need for proton reextrusion, probably because the stoichiometry of H⁺/substrate cotransport is greater for lactose than for ONPG.     

Determining discharges from the Table Mountain Group (TMG) aquifer to wetlands in the Southern Cape, South Africa

The focus of this study was to determine whether coastal wetlands in lowland settings could be dependent on groundwater from the deep circulating confined Table Mountain Group (TMG) aquifer. Groundwater interactions with wetlands are normally perceived to be limited to primary aquifers. A comparative study was done between two endorheic coastal wetlands in the southern Cape. Earlier reports stated that these groundwater dependent wetlands were fed by discharges from the fixed dunes surrounding them. On the basis of a three-dimensional electrical conductivity (EC) interpolation for Groenvlei, a hydrological link between the TMG aquifer and Groenvlei and Van Kervelsvlei was investigated by measuring water level and quality of groundwater and surface water. Water quality parameters used were EC, pH, Na⁺, Fe²⁺ and Cl⁻. The results from this, and an accompanying study, on the basis of water quality and plant nutrient cycling assessments, indicated direct groundwater discharges from the TMG to at least Van Kervelsvlei, with Groenvlei receiving secondary discharges from the TMG via Van Kervelslvlei. These findings significantly affect the current knowledge on which water balance models are based for the determination of groundwater availability for the area. Handling editor: D. Hamilton The first author did the research as part of his PhD in the Department of Earth Sciences of the University of the Western Cape, Bellville, South Africa. Funding is quoted under acknowledgements.