Temperature-sensitive mutants of Escherichia coli affecting beta-galactoside transport

Six different temperature-sensitive (ts) mutants have been isolated which have parental beta-galactoside permease levels at low temperatures but have decreased permease levels when grown at high temperatures. These mutants were derived from Escherichia coli ML308 (lacI(-)Y(+)Z(+)A(+)). After N-methyl-N'-nitro-N'-nitro-soguanidine mutagenesis, ampicillin was used to select for cells unable to grow on low lactose concentrations at 42 C. Temperature-sensitive mutants were assayed for galactoside permease activity after growth in casein hydrolysate medium at 25 or 42 C by measuring both radioactive methylthio-beta-d-galactoside uptake and in vivo o-nitrophenyl-beta-d-galactoside hydrolysis. The six conditional isolates have decreased levels of galactoside permease which are correlated with decreased growth rates at elevated temperatures. The low permease levels are not due to a temperature labile lacY gene product but rather to a temperature labile synthesis rate of functional permease. Some of the mutants exhibit a ts increase in permeability as shown by the increased leakage of intracellular beta-galactosidase and by the increased rate of in vivo o-nitrophenyl-beta-d-galactoside hydrolysis via the nonpermease mediated entry mechanism. Preliminary evidence indicates that transport in general is decreased in these mutants, yet there is some specificity in the mutational lesion since glucoside transport is unaffected. All these observations suggest that these mutants have ts alterations in membrane synthesis which results in pleiotropic effects on various membrane functions.     

(Study on the beta-galactoside permease of Escherichia coli). Solubilization of a thiodigalactoside-binding protein from E.coli membrane containing beta-galactoside permease.

A thiodigalactoside binding protein is solubilized from membrane vesicles of $\text{Escherichia}\text{Coli}$ containing the M protein by use of the detergents Triton X-100 or Emulfogen BC 720. Thiodigalactoside binding affinity of the soluble protein is the same as the membrane embedded β-galactoside permease whereas the residual particulate fraction is free of affinity for this substrate.     

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 1. Functional studies

The effects of various monoclonal antibodies against purified lac carrier protein on carrier-mediated lactose transport were studied in right-side-out membrane vesicles and in proteoliposomes reconstituted with purified lac carrier protein. Out of more than 60 monoclonal antibodies tested, only one antibody, designated 4B1, inhibits transport. Furthermore, the nature of the inhibition is highly specific in that the antibody inhibits only those transport reactions that involve net proton translocation (i.e., active transport, carrier-mediated influx and efflux under nonenergized conditions, and lactone-induced proton influx). In contrast, the antibody has little effect on equilibrium exchange and no effect on generation of the proton electrochemical gradient or on the ability of the carrier to bind a high-affinity ligand. Clearly, therefore, the antibody alters the relationship between lactose and proton translocation at the level of the lac carrier protein. When entrance counterflow is studied with external [1-14C]lactose at saturating and subsaturating concentrations, it is apparent that antibody 4B1 mimics 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 antibody has no effect on the rate or extent of counterflow when external lactose is saturating but stimulates the efficiency of counterflow when external lactose is below the apparent Km. It seems likely, therefore, that the antibody either inhibits the rate of deprotonation or alters the equilibrium between protonated and deprotonated forms of the carrier. Monovalent Fab fragments prepared from antibody 4B1 inhibit transport in a manner that is similar qualitatively to that of the intact antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactose permease of Escherichia coli catalyzes active beta-galactoside transport in a gram-positive bacterium.

The following several lines of evidence demonstrate that lactose permease (LacY) of Escherichia coli is assembled into the cytoplasmic membrane of gram-positive Corynebacterium glutamicum, expressing the lacY gene, as a functional carrier protein. (i) LacY was detected immunologically in the cytoplasmic membrane fraction of the heterologous host. (ii) Recombinant C. glutamicum cells bearing the lacY gene displayed an increased influx of o-nitrophenyl-beta-D-galactopyranoside, which was inhibited by N-ethylmaleimide. (iii) Washed cells were capable of accumulating methyl-beta-D-thiogalactoside about 60-fold. (iv) The uptake of methyl-beta-D-thiogalactoside was energy dependent and could be inhibited by the addition of 10 microM carbonyl cyanide-m-chlorophenylhydrazone. LacY of E. coli was active in the recombinant C. glutamicum cells despite the different membrane lipid compositions of these organisms.     

Localization of acyl carrier protein in Escherichia coli.

Acyl carrier protein was localized by immunoelectron microscopy in the cytoplasm of Escherichia coli. These data are inconsistent with the previous report of an association between acyl carrier protein and the inner membrane (H. Van den Bosch, J.R. Williamson, and P.R. Vagelos, Nature [London] 228:338-341, 1970). Moreover, bacterial membranes did not bind a significant amount of acyl carrier protein or its thioesters in vitro. A thioesterase activity specific for long-chain acyl-acyl carrier protein was associated with the inner membrane.     

Alternative-substrate inhibition and the kinetic mechanism of the beta-galactoside/proton symport of Escherichia coli.

The effects of competing alternative substrates on the rate of uptake by galactoside/proton symport were investigated. These experiments produced a decrease in apparent maximum velocity with increased alternative-substrate concentration that cannot be accounted for by a simple ordered mechanism. This, together with non-linearities in the variation of the apparent kinetic constants with alternative-substrate concentration, can be accounted for by a random mechanism for galactoside and proton binding.     

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.     

Membrane topology of the melibiose carrier of Escherichia coli.

The minimum structural information necessary to formulate and assess mechanistic models of integral membrane protein function is that of membrane topology. This paper characterizes the topological structure of the melibiose carrier of Escherichia coli based on constraints provided by genetic fusions to the compartment-specific reporter protein alkaline phosphatase. Twenty-eight unique chimeras exhibiting either low alkaline phosphatase activity (cytoplasmic location of the fusion joint) or high alkaline phosphatase activity (periplasmic location of the fusion joint) were characterized and used in conjunction with Goldman-Engelman-Steitz hydropathy analysis to model topological structure. The melibiose carrier is predicted to have a cytoplasmic amino terminus, two sets of six transmembrane domains separated by an unusually large cytoplasmic loop ("six-loop-six" arrangement), and a 45-residue cytoplasmic carboxyl tail. Remarkably, the identical six-loop-six arrangement is predicted from the hydrophobicity plots of the H(+)-coupled lactose, arabinose, xylose, and citrate cotransporters of E. coli, the glucose transporter from rat brain, the family of glucose transporters isolated from various human tissues and cell lines, and the human, mouse, and hamster multidrug resistance transporters (Henderson, P.J.F. (1990) Res. Microbiol. 141, 316-328; Maloney, P.C. (1990) Res. Microbiol. 141, 374-383). Such a broad degree of conservation (or convergence) suggests a distinct structural and/or mechanistic advantage associated with the six-loop-six motif. The nature of this advantage is as yet unknown.     

The glucose-specific carrier of the Escherichia coli phosphotransferase system.

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS). 2',3'-Epoxypropyl beta-d-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates. They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter. 3a is the only analogue that inhibits EIIGlc by binding to the high-affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7-anhydro-d,l-glycero-alpha-d-gluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl alpha-d-glucopyranoside (1a) and methyl alpha-d-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-alpha-d-glucopyranoside (1e), beta-d-glucopyranosyl isothiocyanate (3c) and beta-d-glucopyranosyl phenyl isothiocyanate (3d). Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter. Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.     

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.     

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.     

Reconstitution of the melibiose carrier of Escherichia coli

Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.     

Multiple forms of beta-ketoacyl-acyl carrier protein synthetase in Escherichia coli.

Two forms of beta-ketoacyl-acyl carrier protein (ACP) synthetase (designated I and II) have been identified in extracts of Escherichia coli. Synthetase I corresponds to the condensing enzyme that was studied earlier (GREENSPAN, M.D., ALBERTS, A.W., and VAGELOS, P.R. (1969) J. Biol. Chem. 244, 6477-6485); synthetase II represents a new form of the enzyme. Synthetase II was isolated as a homogeneous protein. It differs from synthetase I in having a higher molecular weight (76,999 versus 66,000), a lower pH optimum (5.5 to 6.1 versus 7.2), and a greater resistance to denaturation by heat. Synthetase II is similar to synthetase I in that both are inactivated by iodoacetamide, and prior incubation of the enzymes with fatty acyl thioesters prevents the inhibitory effect of iodoacetamide. Both also react with a fatty acyl thioester to form an acyl-enzyme intermediate, and the latter reacts with malonyl-ACP to form a beta-ketoacyl thioester. Specificity studies indicated that synthetase II, like synthetase I, has similar affinities with saturated and cis unsaturated fatty acyl thioesters of ACP that are intermediates in the synthesis of saturated and unsaturated fatty acids, respectively. The two synthetases differ only with respect to reactivity with palmitoleyl thioesters: synthetase II has a lower Km and higher Vmax than synthetase I with palmitoleyl-ACP. This finding suggests that synthetase II functions specifically in the elongation of palmitoleyl-ACP to form cis-vaccenyl-ACP. An investigation of synthetases I and II in two classes of unsaturated fatty acid auxotrophs revealed that synthetase I is absent in one class, fabB. Addition of wild type synthetase I to fabB fatty acid synthetase, which synthesizes only saturated fatty acids, permitted this fatty acid synthetase to synthesize unsaturated fatty acids. These experiments indicate that synthetase I plays a critical role in the synthesis of unsaturated fatty acids.     

On the structure-function relationship of acyl carrier protein of Escherichia coli.

The conformations of Escherichia coli acyl carrier protein (ACP) and acetylated ACP have been studied as a function of pH and salt concentration by circular dichroism measurements. The results show that the amino groups of ACP in their protonated form are important for maintaining the native conformation of the protein at physiological pH. However, externally added cations (divalent more effectively than monovalent ones) can substitute for the ammonium groups in maintaining the ordered structure pf ACP. It is suggested that both the ammonium groups of ACP and externally added cations reduce the repulsion between carboxylate groups of ACP and thereby prevent the unfolding of the protein. A reduction of the number of negatively charged carboxylate groups by either protonation or chemical modification abolished the requirement for either ammonium groups or other cations. A qualitative agreement between the effect of salt on the conformation and on the biological activity of acetylated ACP has been observed. The single arginine residue of acetylated ACP has been modified by treatment with a trimer of 2,3-butanedione with the resulting derivative of ACP retaining most of its biological activity.