Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport

Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant "uncoupled" for beta-galactoside/H+ symport [Wong, P. T. S., Kashket, E. R., & Wilson, T. H. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 63], are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-D-galactopyranoside (TMG) in the presence of an H+ electrochemical gradient (interior negative and alkaline). Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H+ influx is only about 15% of that observed in vesicles containing wild-type lac permease (ML 308-225). Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 308-225 vesicles. When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide [Viitanen, P., Garcia, M. L., Foster, D. L., Kaczorowski, G. J., & Kaback, H. R. (1983) Biochemistry 22, 2531]. That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent Km. Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sucrose transport by the Escherichia coli lactose carrier.

Several lines of evidence suggest that sucrose is transported by the lactose carrier of Escherichia coli. Entry of sucrose was monitored by an osmotic method which involves exposure of cells to a hyperosmotic solution of disaccharide (250 mM). Such cells shrink (optical density rises), and if the solute enters the cell, there is a return toward initial values (optical density falls). By this technique sucrose was found to enter cells at a rate approximately one third that of lactose. In addition, the entry of [14C]sucrose was followed by direct analysis of cell contents after separation of cells from the medium by centrifugation. Sucrose accumulated within the cell to a concentration 160% of that in the external medium. The addition of sucrose to an anaerobic suspension of cells resulted in a small alkalinization of the external medium. These data are consistent with the view that the lactose carrier can accumulate sucrose by a proton cotransport system. The carrier exhibits a very low affinity for the disaccharide (150 mM) but a moderately rapid Vmax.     

Characterization of lactose carrier mutants which transport maltose

Brooker and Wilson (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963) previously isolated lactose carrier mutants which were able to transport maltose. All of the mutants were found to be single amino acid substitutions for alanine 177 or for tyrosine 236. In the present study, we have examined the ability of these mutants to transport maltose, lactose, o-nitrophenyl-beta-D-galactopyranoside, methyl-beta-D-thiogalactopyranoside, and H+. Both the position 177 and 236 mutants have enhanced rates of maltose transport and exhibit apparent Km values for maltose which are substantially less than that of the wild-type strain. The position 177 mutants transport lactose and other galactosides at a normal rate and with normal affinity during downhill transport and show counterflow transport rates which are faster than the wild-type strain. Interestingly, these mutants are markedly defective in accumulating substrates against a concentration gradient, yet retain a normal H+:galactoside stoichiometry. The position 236 mutants appear to be defective in the downhill, uphill, and counterflow transport of galactosides but exhibit a normal H+:galactoside stoichiometry.     

Towards an understanding of the structural basis of ''forbidden'' transport pathways in the Escherichia coli lactose carrier: mutations probing the energy barriers to uncoupled transport

Recent progress in the analysis of mutants of the Escherichia coli lactose carrier function is reviewed, with special emphasis on the structural basis for energy barriers which prevent 'forbidden' conformational changes. Mutations which break down the barriers to forbidden isomerizations involving the binary carrier:sugar (CS) and carrier:proton (CH) complexes have been obtained in several laboratories. These mutants allow uncoupled transport of H+ or galactoside in the lactose carrier which normally couples cation and sugar movement in a 1:1 stoichiometry. These uncoupled mutants appear to be associated with changes in both sugar and cation recognition, suggesting that the physical interactions forming the basis for co-substrate recognition and uncoupling are not independently variable. By postulating that translocation involves transformation of the stable intermediate of the co-transport cycle to unstable transition state conformations of the carrier, it is possible to consider the consequences of mutagenesis in terms of transition state theory. Consistent with several experimental observations, the analysis predicts in each mutant the occurrence of more than one abnormality in the transport cycle (such as changes in sugar recognition, cation recognition or the coupling reaction). We have called the general phenomenon a 'mutational double-effect' because any mutation which alters the Gibbs free energy change of one reaction in the transport cycle must affect the free energy change of at least one other reaction in this cycle.     

Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier. Replacement of histidine 322 by tyrosine or phenylalanine

Mutations have been introduced into the Escherichia coli lac Y gene by oligonucleotide-directed mutagenesis such that the lactose carrier contains either tyrosine or phenylalanine in place of histidine 322. These mutants did not carry out active accumulation of lactose, melibiose, or methyl-beta-D-galactopyranoside, but facilitated diffusion was still catalyzed. Galactoside-dependent H+ transport, measured with the pH electrode, was retained in both mutants. We conclude that although histidine 322 is important for energy transduction, neither an electronegative atom nor a dissociable proton is essential for proton cotransport with lactose or melibiose.     

Lysis of Escherichia coli mutants by lactose.

Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which as a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K+ content no lysis occurred. Lysis required high levels of beta-galctosidase and lactose transport activity. MM6, the parental strain of MM6-13, has lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, beta-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of MG2+, lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher slat concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2+, lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulafte, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and beta-galactosidase activity, are important in lysis.     

Sensitivity of efflux-driven carrier turnover to external pH in mutants of the Escherichia coli lactose carrier that have tyrosine or phenylalanine substituted for histidine-322. A comparison of lactose and melibiose

Two Escherichia coli lactose carrier mutants (tyrosine or phenylalanine substituted for histidine 322) were studied under conditions of net efflux or equilibrium exchange. Net lactose efflux by either mutant was 10-20-fold slower than by the parent and was sensitive to extracellular pH (5.6-8.0). The presence of extracellular lactose (equilibrium exchange) failed to accelerate loss of [14C]lactose, indicating that the step(s) rate limiting for exchange were also rate limiting for net lactose efflux. Net melibiose efflux by the Phe-322 mutant was comparable to the normal carrier, while that by the Tyr-322 mutant was 5-fold faster (pH 7.0). Melibiose efflux by either mutant was sensitive to pH (5.6-8.0). Melibiose in the extracellular medium significantly accelerated loss of [3H]melibiose from either mutant, showing that slow exchange is a sugar-specific phenomenon and not an intrinsic property of these mutants. The sugar-specific effect of these mutations could mean that the defect in these mutants is not on the path of the proton, although alternative explanations cannot as yet be eliminated. The modest effect of these mutations on the transport rate indicates that His-322 contributes a far smaller free energy increment to catalyzing of H+/galactoside cotransport than active site histidines contribute to catalyzing peptide bond hydrolysis in serine proteases. We interpret this to mean that in chemical terms the function of these catalytic histidine residues differ considerably.     

Transport of alpha-p-nitrophenylgalactoside by the lactose carrier of Escherichia coli.

alpha-p-Nitrophenylgalactoside was found to be accumulated by the lactose transport-system of Escherichia coli. This fact may help to resolve the differences in the reported number of sugar binding sites of the lactose transport protein in nonenergized and energized membrane vesicles.     

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.     

The role of protons in the mechanism of galactoside transport via the lactose permease of Escherichia coli

The kinetic mechanism of lactose transport across the cytoplasmic membrane has been investigated and the results related to standard models for the lactose-H+ symport reaction using computer simulation. It is shown that the biphasic kinetics reported for lactose uptake (Kaczorowski, G.J. and Kaback, H.R. (1979) Biochemistry 18, 3691-3697) are consistent with random binding of lactose and protons and rapid subsequent translocation of the ternary lactose-H+-permease complex. Such a model is also shown to explain the observed dependence of the kinetic parameters on the magnitude of the protonmotive force. Both sugar and protons are shown to cause product inhibition of lactose flux and the ability of standard models to account for the pattern of inhibition is discussed. Three apparent dissociation constants have been determined for the protonation reactions in the external medium: two (pKa 6.3 and 9.6) control the activity of the permease, whilst the third (pKa 8.3) controls the affinity of the permease for galactosides. A similar set of dissociation constants has been determined for the internal reactions. Again two (pKa 6 and 9.8) control activity and a third (pKa 8.8) controls the affinity for galactosides. The dissociation reactions characterised by pKa 8.3, 8.8, 9.6 and 9.8 are attributed to the dissociation of the substrate (symported) proton from the binary proton-permease complexes (pKa 8.3 and 8.8) and the ternary proton-galactoside-permease complexes (pKa 9.6 and 9.8). The third pair (pKa 6.3 and 6.0) must be interpreted as describing a separate protonation reaction which may have a regulatory or auxiliary role in transport.     

Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.     

Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector.

Summary The isolation and properties of a hybrid plasmid carrying the Y gene of the lac operon of Escherichia coli are described. The lactose carrier protein, coded for by the Y gene, is readily identified upon lac operon induction in strains carrying the plasmid. The protein comprises about 15% of the cytoplasmic membrane protein synthesized in the first generation after induction, compared with a wild type strain induced under the same conditions where lactose carrier protein comprises 1.4% of the cytoplasmic membrane protein.     

Characterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli

lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.     

Isolation of mutants of Escherichia coli uncoupled in oxidative phosphorylation using hypersensitivity to streptomycin.

Mutants of Escherichia coli, harbouring the uncA401 or uncB402 alleles, were found to take up streptomycin more rapidly than the coupled parent strains. The increased rate of uptake results in greater sensitivity of the uncoupled strains, compared to the parent strains, to low concentrations of streptomycin. Studies with unc+ revertants showed that hypersensitivity to streptomycin is attributable to the mutation causing uncoupling. The uptake of streptomycin in an unc- strain is abolished by addition of the chemical uncoupler carbonylcyanide m-chlorophenylhydrazone. The phenotype of hypersensitivity to streptomycin can be used as a selection procedure for the isolation of uncoupled strains. In an experiment reported here, nine out of 12 strains isolated as being sensitive to streptomycin (at 2.5 micrograms/ml), were found to be unable to grow on succinate as a sole source of carbon. Five of the nine Suc- strains were found to be uncoupled in oxidative phosphorylation, and two of the five uncoupled strains lacked Mg2+-ATPase activity. The mutations causing uncoupling were cotransducible with the ilv genes.     

The phospholipid requirement for activity of the lactose carrier of Escherichia coli

The transport activity of the lactose carrier of Escherichia coli has been reconstituted in proteoliposomes composed of different phospholipids. The maximal activity was observed with the natural E. coli lipid as well as mixtures containing phosphatidylethanolamine or phosphatidylserine. Phosphatidylcholine or mixtures of phosphatidylcholine with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activity. The lactose carrier reconstituted with amino phospholipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine, and dioleoylphosphatidylcholine) revealed a progressive decrease in both counterflow and proton motive force-driven lactose uptake activities. Trinitrophenylation of phosphatidylethanolamine in the E. coli proteoliposomes resulted in a marked reduction in lactose carrier activity. Partial restitution of transport activity was obtained by detergent extraction of the carrier from these inactive proteoliposomes and reconstitution of the carrier into proteoliposomes containing normal E. coli lipid. These results suggest that the amino group of the amino phospholipids (e.g. phosphatidylethanolamine and phosphatidylserine) is required for the full function of the lactose carrier from E. coli.     

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.     

Characterization of Escherichia coli null mutants for glutaredoxin 2.

Three Escherichia coli glutaredoxins catalyze GSH-disulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB(-)) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB(-) cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.