The importance of the binding-protein-dependent Mgl system to the transport of glucose in Escherichia coli growing on low sugar concentrations.

Glucose limitation in chemostats derepressed the binding-protein-dependent Mgl transport system, which is strongly repressed during growth in batch culture with high glucose levels. The limitation-induced Mgl activity was higher than that of batch cultures "fully induced" for the Mgl system after growth on glycerol plus fucose. Mgl- strains were impaired compared to Mgl+ bacteria in removing glucose from sugar-limited chemostats and were outcompeted in mixed continuous culture on limiting glucose. The influence of Mgl was not observed on growth with limiting maltose or non-carbohydrates, and thus was specific for glucose, a known substrate of the Mgl system. In the absence of the two glucose-specific membrane components of the phosphoenolpyruvate:sugar phosphotransferase system, non-PTS-dependent growth on glucose was observed in continuous culture, but only under sugar-limited conditions derepressing the Mgl system and not in glucose-rich batches or continuous culture. Hence growth of Escherichia coli on glucose at micromolar concentrations involves a significant contribution of a binding-protein-dependent transport system. The participation of multiple transporters in glucose transport can account for the complex non-hyperbolic dependence of growth-rate on glucose concentration and for discrepancies in studies attempting to describe growth on glucose purely in terms of phosphotransferase kinetics.

The mechanism of action of chlorhexidine.

Chlorhexidine did not inhibit ATPase in intact cells of Escherichia coli K12 W1317i-, even at bactericidal concentrations, and ATP hydrolysis was greatest at the highest concentration (40 mg/l), even though no net uptake of substrate occurred. Like dinitrophenol and tribrominated salicylanilide, polymyxin and chlorhexidine collapsed the membrane potential at inhibitory concentrations. Membrane disruption, and not ATPase inactivation, is considered the lethal event in chlorhexidine action.

Mutational analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli.

The phosphoenolpyruvate phosphotransferase system (PTS) component EIIIGlc is responsible for transport and phosphorylation of glucose via EIIGlc. It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP. Mutational analysis of EIIIGlc was performed in order to identify crucial residues mediating the interactions between EIIIGlc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIIIGlc gene, crr. Five mutated EIIIGlc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIIIGlc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase. A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly47-->Ser47 for the HA2 and HA5 mutations, Ala76-->Thr76 for HA4 mutation and Ser78-->Phe78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIIIGlc and its target protein(s).

Analysis of inducers of the E.coli lac repressor system in mammalian cells and whole animals.

Although the inducible prokaryotic lac repressor system has been successfully adapted for control of gene expression in mammalian cells, little information is available on the pharmacokinetics of beta-galactoside inducers in mammalian cells for optimizing this system. These studies directly measure the cell uptake and clearance in cultured cells and animal tissue cells of lac inducers. In these cells, the beta-galactosides, isopropyl beta-D-thiogalactoside (IPTG) and methyl beta-D-thiogalactoside (MTG), are rapidly taken up, exceeding extracellular levels in less than 2 hours. Greater than 5% of this inducer is found in the nuclear fraction, slightly exceeding the cytoplasmic concentration. Although similar in uptake, IPTG is cleared from the cultured cells significantly faster than MTG. In the mouse, the half-life of both inducers in the blood ranges from 15-30 minutes. HPLC analysis of tissue extracts from inducer-injected mice indicates that the inducer is metabolically stable and functionally able to bind to lac repressor. These results should permit improvement in the adaptation of the lac repressor system to mammalian cells and aid in the development of an adaptable system for gene control in transgenic animals.

Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier: substitution of tyrosine for histidine-322 and of leucine for serine-306.

The lac Y genes from two Escherichia coli mutants, MAB20 and AA22, have been cloned in a multicopy plasmid by a novel 'sucrose marker exchange' method. Characterization showed that the plasmids express a lactose carrier with poor affinity for lactose. Neither mutant carried out concentrative uptake with methyl beta-D-galactopyranoside, lactose, or melibiose as the substrate. Nor did the mutants catalyze counterflow or exchange with methyl beta-D-galactopyranoside. Both mutants did, however, retain the capacity to carry out facilitated diffusion with lactose or melibiose. DNA sequencing revealed that MAB20 (histidine-322 to tyrosine) and AA22 (serine-306 to leucine) have amino acid substitutions within the putative 'charge-relay' domain thought to be responsible for proton transport. Galactoside-dependent H+ transport was readily measured in both mutants. We conclude, therefore, that the presence of a histidine residue at position 322 of the lactose carrier is not obligatory for H+ transport per se.