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.     

Counter-transport mediated by the lactose permease of Escherichia coli

When the two main energy yielding pathways, respiration and the membrane ATPase of Escherichia coli are poisoned, the lactose permease is unable to accomplish accumulative transport of thiogalactosides, but the efflux of pre-loaded substrate can be coupled to a transiently uphill transport of exogenous substrate. This transient uphill transport, called overshoot has been reexamined with the possibility of an obligate H⁺ cotransport in mind. Overshoot can be diminished but not suppressed by a proton-conducting uncoupler, carbonyl cyanide $\text{m}$ chlorophenylhydrazone, (CCCP) and by a liposoluble cation, triphenyl-methyl phosphonium (TPMP⁺). The effect of other factors, such as temperature, amount of permease and pH were also explored. The overshoot was found to decrease with increasing pH, until at pH 8 it became negligible. This is in sharp contrast with the relatively flat pH dependence of uphill and downhill transport in unpoisoned cells. CCCP and TPMP⁺ had no inhibitory effect on the overshoot at pH 6 and below.     

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.     

Lactose transport in Escherichia coli cells Dependence of kinetic parameters on the transmembrane electrical potential difference

We determine the kinetic parameters $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of lactose transport in Escherichia coli cells as a function of the electrical potential difference (Δψ) at pH 7.3 and $\text{Δ}\text{pH}\text{= 0}$. We report that transport occurs simultaneously via two components: a component which exhibits a high $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ (larger than 10 mM) and whose contribution is independent of Δψ, a component which exhibits a low $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ independent of Δψ (0.5 mM) but whose $\text{V}$ increases drastically with increasing Δψ. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon Δψ of $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ and $\text{V}$ of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49–63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H⁺/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s)) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H⁺/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H⁺/lactose stoichiometry different for active transport and facilitated diffusion.     

Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ($\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H⁺ symport, to an observable decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$, and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H⁺ symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.     

Co-transport of Na+ and methul-beta-D-thiogalactopyranoside mediated by the melibiose transport system of Escherichia coli.

Na⁺-dependent transport of methyl-β-D-thiogalactopyranoside (TMG) mediated by the melibiose transport system was investigated in $\text{Escherichia coli}$ mutants lacking the lactose transport system. When an inwardly-directed electro-chemical potential difference of Na⁺ was imposed across the membrane of energy depleted cells, transient uptake of TMG was observed. Addition of TMG to cell suspensions under anaerobic conditions caused a transient acidification of the medium. This acidification was observed only in the presence of Na⁺ or Li⁺. These results support the idea that TMG is taken up by a mechanism of Na⁺-TMG co-transport via the melibiose transport system in $\text{Escherichia coli}$.     

Proton efflux associated with melibiose permease activity in Salmonella typhimurium.

Transport of thiomethyl-β-D-galactoside (TMG) via the melibiose permease system (TMG permease II) in $\text{Salmonella typhimurium}$ is known to be a sodium-dependent co-transport system. We have shown that this co-transport of sodium and TMG is associated with extrusion of protons from the cells. The rate and extent of proton extrusion during TMG uptake were measured in wild-type cells and mutants containing internal and extended deletions in the $\text{pts}$ locus. No differences between these various strains were noted.     

Alteration of Mycobacterium phlei membrane structure by freezing and thawing: reversal by heating.

Membrane vesicles from Mycobacterium phlei which contain the electron transport chain, when subjected to freezing to ?70 °C followed by slow thawing, exhibited a decreased level of phosphorylation coupled to the oxidation of substrates. This loss in oxidative phosphorylation was restored following heat treatment (50 °C for 10 min) of the membranes. Freeze treatment (?70 °C for 10 min) of membrane vesicles also resulted in a decrease in membrane bound coupling factor-latent ATPase activity. The soluble coupling factor(s) or cryoprotective agents (i.e., glycerol or dimethyl sulfoxide) were found to protect the membrane vesicles from the effects of freezing. Membrane vesicles depleted of particulate bound coupling factor were sensitive to exposure to low temperatures; however, complete protection was afforded by the addition of coupling factor. In addition, prolonged sonication of electron transport particles resulted in lowered $\text{P}\text{O}$ ratios, and heat treatment of these sonicated particles restored $\text{P}\text{O}$ ratio. Therefore, it appears that the effects of heat treatment and freeze treatment on membrane vesicles are reversible. The steady state level of reduced cytochrome b was considerably higher (40%) in heat-treated electron transport particles as compared to untreated particles (28.5%); electron transport particles subjected to freeze treatment showed a lower steady state level of cytochrome b (16.6%) as compared to electron transport particles. The steady state level of cytochrome b in freeze-treated particles returned to the original level (27.5%) for electron transport particles when subjected to heat treatment. Nevertheless, the total amount of enzymatically reducible cytochrome b was the same for all membranes after subjection to the various types of treatment. In contrast to cytochrome b, the reduced steady state levels of cytochrome c, and a + a₃ were not altered by heat or freeze treatment.     

Quantitative aspects of active transport by the lactose transport system of Escherichia coli

Accumulation by the lactose transport system of Escherichia coli has been measured in cells induced so as to contain increasing numbers of membrane carriers. Carrier activity was assayed both by the rate of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ entry and the initial rate of accumulation of $\text{methyl-1-thio-gb-}\text{d}\text{-galactopyranoside}$. At the steady-state cells with a low number of carriers accumulated considerable amounts of substrate when compared to the fully induced control. This is consistent with the hypothesis that there are two distinct routes of both entry and of exit: a carrier mediated pathway and a diffusion component. When these two factors are evaluated quantitatively they account for the observed relationship between the number of carriers and the steady-state accumulation achieved.     

Reconstitution of LAC carrier function in cholate-extracted membranes from Escherichia coli.

Extraction of $\text{Escherichia}\text{coli}$ ML 308-225 membrane vesicles with cholate yields a particulate fraction containing 10 to 15% of the phospholipid and about 70% of the protein of intact vesicles. Addition of phospholipid to the particulate fraction in the presence of cholate, followed by sonication and removal of detergent by gel filtration yields a vesicular preparation that exhibits $\text{lac}$ carrier function as judged by transient increases in 6′-(N-dansyl)aminohexyl-1-thio-β-D-galactopyranoside fluorescence in the presence of either a lactose diffusion gradient or an artificially-generated membrane potential (interior negative). Activity is not observed in the absence of phospholipid, in the presence of N-ethylmaleimide or in analogous preparations from ML 30 vesicles that do not contain the β-galactoside transport system.     

Thermal analysis of bacteriophage T4.

The process of bacteriophage T4 morphogenesis was studied using a heat leakage scanning calorimeter. Thermograms of defective mutant 49 (am NG727) in permissive and non-permissive cells of $\text{Escherichia coli}$ showed a difference in thermal properties between packaged and non-packaged DNA molecules. $\text{In vivo}$, non-packaged DNA carried out their thermal transition at 85°C, the same temperature as that of T4 DNA melting measured in the standard saline citrate buffer, while the packaged DNA gave a sharper peak at 87°C due to some interaction with the head shell structure. Empty head shells showed a sharp heat absorption peak at 89°C both $\text{in vivo}$ and $\text{in vitro}$, indicating the high degree of cooperativity in their conformational changes.     

Imidazole acetic acid as a substitute for cAMP.

The ability of imidazole acetic acid (IA) to substitute for cAMP was demonstrated by use of a series of strains carrying a lesion in the $\text{cya}$ structural gene. The substitution of IA for cAMP was specific for the L-arabinose operon in that this compound was ineffective in substituting for cAMP in the lactose or maltose catabolic systems. The cAMP receptor protein (CRP) and the $\text{araC}$ gene product were necessary for the IA mediated induction of the L-arabinose system.     

A major role for chloride in (3H)- noradrenaline transport by rat heart adrenergic nerves

The effect of Cl^? and other anions on (³H)-noradrenaline line (NA) transport by bisected rat heart atrial appendages $\text{in}$$\text{vitro}$ has been studied. It was found that NA active transport, at the plasma membrane level, shows an absolute dependency on Cl^?, with a half-maximal activation of transport occurring at 6 mM Cl^? and complete saturation at 50 mM. Cl^? effects are due to changes in transport Km, while Vmax is not changed. Only one class of sites for Cl^? seem to be present in the transport system. Br^? can substitute for Cl^? with 90% effectiveness, nitrate and iodide are less effective, while larger anions are very poor substitutes. In addition, heart atrial hemi-appendages have been characterized as a suitable preparation for studies of this type.     

Use of lac operon fusions to isolate Escherichia coli mutants with altered expression of the fumarate reductase system in response to substrate and respiratory controls.

Strains of $\text{E}\text{.}\text{coli}$ with fusions between the $\text{lac}$ structural genes and the promoter region of the fumarate reductase system were constructed from a parental strain deleted in the native $\text{lac}$ operon. Like fumarate reductase in wild-type cells, β-galactosidase in these fusion strains is inducible by fumarate, but only under anaerobic conditions. From one of these strains, three classes of mutants altered in the expression of the hybrid operon were isolated. By anaerobic selection for growth on lactose in the absence of fumarate, mutants that synthesize β-galactosidase constitutively both aerobically and anaerobically were obtained. By aerobic selection for growth on lactose in the presence of fumarate, mutants that are inducible in the enzyme both aerobically and anaerobically and mutants that are inducible in the enzyme only aerobically were obtained. The regulatory behaviors of the mutants studied suggest that substrate and respiratory control of the expression of the fumarate reductase complex are mechanistically connected.     

Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins.

A novel iron uptake system was observed in pseudorevertants of $\text{Escherichia}$,$\text{coli}$ strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.     

The in vivo equivalence of a cell-free system for RNA processing and transport

The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm $\text{in vivo}$, has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA $\text{in vivo}$. Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the $\text{in vivo}$ norm. The results emphasize the importance of establishing the $\text{in vivo}$ equivalence in cell-free systems designed to study RNA synthesis, processing and transport.     

Inhibitory effect of unconjugated bilirubin on p-aminohippurate transport in rat kidney cortex slices

(1) The effects of unconjugated bilirubin on the accumulation of $\text{p-}\text{aminohippurate}$, kinetics of $\text{p-}\text{aminohippurate}$ uptake, the efflux of pre-accumulated $\text{p-}\text{aminohippurate}$ and water and electrolyte distribution were investigated in the rat kidney cortical slice. (2) The addition of unconjugated bilirubin to the incubation medium decreased the 60 min slice-to-medium concentration ratio of $\text{p-}\text{aminohippurate}$. (3) The decrease in $\text{p-}\text{aminohippurate}$ accumulation by unconjugated bilirubin was found to be more pronounced by increasing the concentration of pigment in the medium. (4) The rate of uptake of $\text{p-}\text{aminohippurate}$ as a function of $\text{p-}\text{aminohippurate}$ concentration differed in aerobiosis and anaerobiosis, and unconjugated bilirubin decreased only the uptake of $\text{p-}\text{aminohippurate}$ in aerobic conditions. (5) The efflux of pre-accumulated $\text{p-}\text{aminohippurate}$ decreased when unconjugated bilirubin concentration in the medium was low (10–20 μM) but the efflux increased when the concentration of pigment was much higher (100 μM). (6) The addition of unconjugated bilirubin to the medium (40–100 μM) increased intracellular sodium and total tissue water content, and decreased intracellular potassium and oxygen consumption of tissue. However the slices incubated with low concentration of pigment (20 μM) did not exhibit significative changes in cellular functional parameters. (7) These findings suggest that unconjugated bilirubin impairs $\text{p-}\text{aminohippurate}$ transport by a complex mechanism that might involve binding of pigment to sites necessary for anion transport, although effects related to pigment toxicity or to its oxidative decomposition are not excluded.     

Prolonged production of hydrogen gas by a chloroplast biocatalytic system.

The evolution of H₂ gas in an $\text{in vitro}$ illuminated chloroplast plus hydrogenase system was shown to function for six and a half hours at a continuous rate of about 10 μmoles H₂/mg. chlorophyll/hour. Chloroplasts from various plant species, using different isolation media, and storage in a fixed state (glutaraldehyde) at 4°, ?5° and ?196° were shown to catalyze H₂ production. Both $\text{Clostridium}$ and $\text{E. coli}$ hydrogenase were used. From the use of the photosystem II inhibitors DCMU and DBMIB and heat inactivation of photosystem II, it was concluded that H₂O was the source of electrons for H₂ gas production.     

Evidence that ATP exerts control of the rate of glucose utilization in the intact Escherichia coli cell by altering the cellular level of glucose-6-P, an intermediate known to inhibit glucose transport in vitro

The effects of treating nitrogen-starved cultures of $\text{Escherichia coli}$ W4597 (K) with various doses of 2,4-dinitrophenol include increases in the rates of glucose utilization, decreases in ATP and glucose-6-P and maintenance of the level of fructose-1, 6-P₂. A quantitative correlation was observed between the increases in the rates of glucose utilization and decreases in glucose-6-P in agreement with the observation made $\text{in vitro}$ that glucose-6-P inhibits glucose transport in $\text{E. coli}$. A quantitative correlation was also observed between glucose-6-P and ATP indicating that the fall in glucose-6-P is effected by the fall in ATP which indirectly signals increased glucose utilization and increased ATP production.