Unliganded maltose-binding protein triggers lactose transport in an Escherichia coli mutant with an alteration in the maltose transport system.

Escherichia coli accumulates malto-oligosaccharides by the maltose transport system, which is a member of the ATP-binding-cassette (ABC) superfamily of transport systems. The proteins of this system are LamB in the outer membrane, maltose-binding protein (MBP) in the periplasm, and the proteins of the inner membrane complex (MalFGK2), composed of one MalF, one MalG, and two MalK subunits. Substrate specificity is determined primarily by the periplasmic component, MBP. However, several studies of the maltose transport system as well as other members of the ABC transporter superfamily have suggested that the integral inner membrane components MalF and MalG may play an important role in determining the specificity of the system. We show here that residue L334 in the fifth transmembrane helix of MalF plays an important role in determining the substrate specificity of the system. A leucine-to-tryptophan alteration at this position (L334W) results in the ability to transport lactose in a saturable manner. This mutant requires functional MalK-ATPase activity and the presence of MBP, even though MBP is incapable of binding lactose. The requirement for MBP confirms that unliganded MBP interacts with the inner membrane MalFGK2 complex and that MBP plays a crucial role in triggering the transport process.     

Induction of lactose transport in Escherichia coli during the absence of phospholipid synthesis.

Induction of lactose transport and of beta-galactosidase synthesis was examined in two Escherichia coli strains that require exogenous glycerol for phospholipid synthesis and growth. No preferential inhibition of lactose transport induction was observed when phospholipid synthesis was restricted to 5 to 10% of the normal rate. We conclude that the lactose transport system does not require concurrent phospholipid synthesis for its functional assembly.     

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.     

A novel mutant of the lac transport system of Escherichia coli.

A new type of lactose permease mutant, called lacY^f, does not actively transport the usual substrates; but it does facilitate the entry of β-galactosides into Escherichia coli K-12. The kinetics of facilitated entry, as assayed by hydrolysis of o-nitrophenyl-β-d-galactopyranoside by intact cells are identical to those observed with wild-type permease. However, the mutant permease activity is not affected by SH reagents or the substrate analog β-d-galactosyl-1-thio-β-d-galactopyranoside which strongly inhibit wild-type activity. Furthermore, the kinetics of formation of permease in the mutant following addition of inducer and the kinetics subsequent to removal of inducer differ strikingly from those observed in wild-type strains. The results are consistent with a block in the maturation of permease in the mutant resulting in the accumulation of a large amount of permease precursor. Studies of the $\text{lacY}{}^{\mathrm{+}}\text{lacY}{}^{\mathrm{f}}$ heterogenotes provide evidence for a subunit structure for the lactose permease.     

Evidence for a protonmotive force related regulatory system in Escherichia coli and its effects on lactose transport

Strains of Escherichia coli with mutations in the eup (energy-uncoupled phenotype) locus do not grow on nonfermentable carbon sources, have reduced growth yields on limiting glucose, are insensitive to colicins A and K, exhibit resistance to aminoglycoside antibiotics, and are defective in protonmotive force coupled active transport. eup mutations do not result in lowered protonmotive force. Here we show that deenergization of a eup+ strain results in the appearance of a new low KT, low Vmax form of the lactose carrier; in a strain deleted of the eup locus, deenergization does not evoke the low KT, low Vmax form of the lactose carrier. Cells bearing a eup point mutation and exhibiting the Eup- phenotype possess the low KT, low Vmax form of the lactose carrier even when energized. In addition to affecting the kinetic parameters of the lactose carrier, the eup point mutation also reduces the KT and Vmax of the proline carrier. On the basis of these findings, we suggest that the normal eup gene product mediates a novel regulation of lactose carrier function following deenergization. The defect in proline and lactose transport caused by eup point mutations may stem from an altered eup product aberrantly mediating the regulation under energized conditions. Finally, the pleiotropy associated with eup point mutations may be indicative of those protonmotive force driven functions that are subject to eup regulation.     

Energetic studies of lactose active transport in Escherichia coli membrane vesicles

The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method. Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively. The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane. The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles. Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate. The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35%. The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose. No heat of reaction is contributed by lactose movement across the membrane without symport with H+.     

lacY mutant of Escherichia coli with altered physiology of lactose induction.

A mutant of Escherichia coli is described that grew on lactose only in the presence of isopropylthiogalactoside. This cell contained a defect in the lacY gene that resulted in the formation of a transport system with a poor affinity for lactose. The inability to grow on lactose alone was due to the failure of induction by this disaccharide. This failure of inducation was presumably due to a defect in lactose accumulation which resulted in significant reduction in the formation of allo-lactose, the true inducer of lac operon. These results are consistent with the view that the capacity to accumulate lactose plays an important physiological role in the induction of the enzymes necessary for its utilization.     

Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution

The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.