Methanogenesis and the K+ transport system are activated by divalent cations in ammonia-treated cells of Methanospirillum hungatei

We describe a K+ transport system in Methanospirillum hungatei cells depleted of cytoplasmic K+ via an ammonia/K+ exchange reaction (Sprott, G. D., Shaw, K. M., and Jarrell, K. F. (1984) J. Biol. Chem. 259, 12602-12608). Ammonia-treated cells contained low concentrations of ATP and were unable to make CH4 or to transport 86Rb+. All of these properties were restored by CaCl2, MgCl2, or MnCl2, and not by CoCl2 or NiCl2. The Rb+ transport system had a Km of 0.42 and Vmax of 29 nmol/min X mg; K+ inhibited competitively. Both H2 and CO2 were required for appreciable transport, whereas air, valinomycin, or nigericin were potent inhibitors. The influx of Rb+ was electrogenic and associated with proton efflux, producing a delta pH (alkaline inside) in acidic media. In the absence of K+ (or Rb+), the activation of CH4 synthesis by Mg2+ produced little change in the cytoplasmic pH, showing that methanogenesis did not elicit a net efflux of protons. The pH optimum for transport was in the range 6.0-7.3 where the transmembrane pH gradient would contribute minimally to the proton motive force. Protonophores at pH 6.3 caused a partial decline in CH4 synthesis and the ATP content and dramatically collapsed Rb+ transport. These and other inhibitor experiments, coupled with the fact that the Rb+ gradient was too large to be in equilibrium with the proton motive force alone, suggest a role for both ATP and the proton motive force in Rb+ transport. Also, a role for K+ in osmoregulation is indicated.     

ATP-driven calcium transport in membrane vesicles of Streptococcus sanguis.

Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulating 45Ca2+. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with 45Ca2+ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells.     

Energy coupling to net K+ transport in Escherichia coli K-12.

Energy coupling for three K+ transport systems of Escherichia coli K-12 was studied by examining effects of selected energy sources and inhibitors in strains with either a wild type or a defective (Ca2+, Mg2+)-stimulated ATPase. This approach allows discrimination between transport systems coupled to the proton motive force from those coupled to the hydrolysis of a high energy phosphate compound (ATP-driven). The three K+ transport systems here studied are: (a) the Kdp system, a repressible high affinity (Km=2 muM) system probably coded for by four linked Kdp genes; (b) the Trka system, a constitutive system with high rate and modest affinity (Km=1.5 mM) defined by mutations in the single trkA gene; and (c) the TrkF system, a nonsaturable system with a low rate of uptake (Rhoads, D.B., Waters, F.B., and Epstein, W. (1976) J. Gen. Physiol. 67, 325-341). Each of these systems has a different mode of energy coupling: (a) the Kdp system is ATP-driven and has a periplasmic protein component; (b) the TrkF system is proton motive force-driven; and (c) the TrkA system is unique among bacterial transport systems described to date in requiring both the proton motive force and ATP for activity. We suggest that this dual requirement represents energy fueling by ATP and regulation by the proton motive force. Absence of ATP-driven systems in membrane vesicles is usually attributed to the requirement of such systems for a periplasmic protein. This cannot explain the failure to demonstrate the TrkA system in vesicles, since this system does not require a periplasmic protein. Our findings indicate that membrane vesicles cannot couple energy to ATP-driven transport systems. Since vesicles can generate a proton motive force, the inability of vesicles to generate ATP or couple ATP to transport (or both) must be invoked to explain the absence of TrkA in vesicles. The TrkF system should function in vesicles, but its very low rate may make it difficult to identify.     

Interconversion of components of the bacterial proton motive force by electrogenic potassium transport.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.     

Calcium transport in membrane vesicles of Streptococcus cremoris

Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A.J.M., Hellingwerf, K.J. & Konings, W.N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing delta psi with a concomitant increase in delta pH stimulates Ca2+ uptake while dissipation of the delta pH results in a reduced rate of Ca2+ uptake. Also an artificially generated delta pH, interior acid, can drive Ca2+ uptake in S. cremoris membrane vesicles. Ca2+ uptake depends strongly on the presence of external phosphate while Ca2+-efflux-induced proton flux is independent of the presence of external phosphate. Ca2+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,N'-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.     

ATP-driven exchange of Na+ and K+ ions by Streptococcus faecalis

We describe the characterization of KtrII, a novel potassium transport system of Streptococcus faecalis, first discovered by H. Kobayashi [1982) J. Bacteriol. 150, 506-511). KtrII requires sodium ions and mediates the stoichiometric exchange of internal Na+ for external K+. Potassium accumulation is not energized by the electrochemical potentials of either H+ or Na+; the energy source is probably ATP. Two lines of evidence indicate that KtrII is a manifestation of the sodium-stimulated ATPase reported earlier (Heefner, D. L., and Harold, F. M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2798-2802). (i) Mutants that lack the ATPase also lack KtrII, and revertants recover both in parallel. (ii) KtrII and the Na+-ATPase are induced in parallel when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential. KtrII is not induced in response to K+ deprivation. We propose that the Na+-ATPase exchanges Na+ for K+ ions.     

Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane.

The bacteriocin lactacin F is bactericidal against Lactobacillus delbrueckii, Lactobacillus helveticus, and Enterococcus faecalis. Activity against L. delbrueckii was recently shown to be dependent on two peptides, LafA and LafX, which are encoded within the lactacin F operon (T. R. Klaenhammer, FEMS Microbiol. Rev. 12:39-87, 1993). It has been proposed that two peptides form an active lactacin F complex. In this study, the action of lactacin F against E. faecalis ATCC 19443 and the effects of various environmental parameters were investigated in detail. Addition of lactacin F induced the loss of K+ from cells of L. delbrueckii, Lactobacillus johnsonii 88-4, and E. faecalis, while the lactacin F producer L. johnsonii VPI 11088 was not affected by the bacteriocin. Lactacin F caused an immediate loss of cellular K+, depolarization of the cytoplasmic membrane, and hydrolysis of internal ATP in E. faecalis. Lactacin F induced loss of K+ in 3,3',4',5-tetrachlorosalicylanilide-treated cells, indicating that pores are formed in the absence of a proton motive force. ATP hydrolysis was not due to dissipation of the proton motive force but was most likely caused by efflux of inorganic phosphate, resulting in a shift of the ATP hydrolysis equilibrium. Action of lactacin F was optimal at acidic pH values and was reduced in the presence of di- and trivalent cations. The lanthanide gadolinium (Gd3+) prevented action of lactacin F completely at a concentration of 0.2 mM. Lactacin F-induced loss of cell K+ was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-affinity potassium uptake system in Bacillus acidocaldarius.

Cells of Bacillus acidocaldarius that were grown with 2.7 mM K+ expressed a low-affinity K+ uptake system. The following observations indicate that its properties closely resemble those of the Escherichia coli Trk and Streptococcus faecalis KtrI systems: (i) the B. acidocaldarius system took up K+ with a Km of 1 mM; (ii) it accepted Rb+ (Km of 6 mM; same Vmax as for K+); (iii) it was still active in the presence of low concentrations of sodium; (iv) the observed accumulation ratio of K+ maintained by metabolizing cells was consistent with K+ being taken up via a K+-H+ symporter; and (v) K+ uptake did not occur in cells in which the ATP level was low. Under the latter conditions, the cells still took up methylammonium ions via a system that was derepressed by growth with low levels of ammonium ions, indicating that in the acidophile ammonium (methylammonium) uptake requires a high transmembrane proton motive force rather than ATP.     

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of -200 to -250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.     

Accumulation of arsenate, phosphate, and aspartate by Sreptococcus faecalis.

Uptake of arsenate and phosphate by Streptococcus faecalis 9790 is strictly dependent on concurrent energy metabolism and essentially unidirectional. targinine supports uptake only in presence of glycerol or related substances; glycerol is not directly involved in transport but depletes the cellular orthophosphate pool and thus relieves feedback inhibition of transport. Uptake of phosphate and arsenate is stimulated by K+ and by other permeant cations. The results suggest that electroneutrality is preserved by compensatory movement of either H+ or OH minus. Ionophores and N,N'-dicyclohexylcarbodiimide, which prevent establishment of a proton motive force, block the accumulation of thiomethylgalactoside and of threonine but not that of arsenate or phosphate. We conclude that arsenate accumulation requires adenosine 5'-triphosphate but is not driven by the proton-motive force. However, conditions and reagents that lower the cytoplasmic pH do inhibit accumulation of arsenate and phosphate, suggesting that uptake depends on the capacity of the cells to maintain a neutral or alkaline cytoplasm. We therefore propose that phosphate accumulation is an electroneutral exchange for OH driven by adenosine 5'-triphosphate or by a metabolite thereof. Accumulation of aspartate and glutamate also requires adenosine 5'-triphosphate but not the proton-motive force and may involve a similar mechanism.     

Sodium-stimulated ATPase in Streptococcus faecalis.

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.     

Sodium/proton antiporter in Streptococcus faecalis.

Streptococcus faecalis, like other bacteria, accumulates potassium ions and expels sodium ions. This paper is concerned with the pathway of sodium extrusion. Earlier studies (D.L. Heefner and F.M. Harold, Proc. Natl. Acad. Sci. USA 79:2798-2802, 1982) showed that sodium extrusion is effected by a primary, ATP-linked sodium pump. I report here that cells grown under conditions in which sodium ATPase is not induced can still expel sodium ions. This finding suggested the existence of an alternate pathway. Sodium extrusion by the alternate pathway requires the cells to generate a proton motive force. This conclusion rests on the following observations. (i) Sodium extrusion required glucose. (ii) Sodium extrusion was observed at neutral pH, which allows the cells to generate a proton motive force, but not at alkaline pH, which reduces the proton motive force to zero. (iii) Sodium extrusion was inhibited by the addition of dicyclohexylcarbodiimide and of proton-conducting ionophores. (iv) In response to an artificial pH gradient (with the exterior acid), energy-depleted cells exhibited a transient sodium extrusion which was unaffected by treatments that dissipated the membrane potential and which was blocked by proton conductors. I propose that streptococci have two independent systems for sodium extrusion: an inducible sodium ATPase and a constitutive sodium/proton antiporter.     

Mechanism of glutamate uptake in Zymomonas mobilis.

The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.     

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.     

Solubilization and functional reconstitution of the proline transport system of Escherichia coli

The membrane carrier for L-proline (product of the putP gene) of Escherichia coli K12 was solubilized and functionally reconstituted with E. coli phospholipid by the cholate dilution method. The counterflow activity of the reconstituted system was studied by preloading the proteoliposomes with either L-proline or the proline analogues: L-azetidine-2-carboxylate or 3,4-dehydro-L-proline. The dilution of such preloaded proteoliposomes into a buffer containing [3H]proline resulted in the accumulation of this amino acid against a considerable concentration gradient. A second driving force for proline accumulation was an electrochemical potential difference for Na+ across the membrane. More than a 10-fold accumulation was seen with a sodium electrochemical gradient while no accumulation was found with proton motive force alone. The optimal pH for the L-proline carrier activities for both counterflow and sodium gradient-driven uptake was between pH 6.0 and 7.0. The stoichiometry of the co-transport system was approximately one Na+ for one proline. The effect of different phospholipids on the proline transport activity of the reconstituted carrier was also studied. Both phosphatidylethanolamine and phosphatidylglycerol stimulate the carrier activity while phosphatidylcholine and cardiolipin were almost inactive.     

Light-driven amino acid uptake in Streptococcus cremoris or Clostridium acetobutylicum membrane vesicles fused with liposomes containing bacterial reaction centers.

Reaction centers of the phototrophic bacterium Rhodopseudomonas palustris were introduced as proton motive force-generating systems in membrane vesicles of two anaerobic bacteria. Liposomes containing reaction center-light-harvesting complex I pigment protein complexes were fused with membrane vesicles of Streptococcus cremoris or Clostridium acetobutylicum by freeze-thawing and sonication. Illumination of these fused membranes resulted in the generation of a proton motive force of approximately -110 mV. The magnitude of the proton motive force in these membranes could be varied by changing the light intensity. As a result of this proton motive force, amino acid transport into the fused membranes could be observed. The initial rate of leucine transport by membrane vesicles of S. cremoris increased exponentially with the proton motive force. An H+/leucine stoichiometry of 0.8 was determined from the steady-state level of leucine accumulation and the proton motive force, and this stoichiometry was found to be independent of the magnitude of the proton motive force. These results indicate that the introduction of bacterial reaction centers in membrane vesicles by the fusion procedure yields very attractive model systems for the study of proton motive force-consuming processes in membrane vesicles of (strict) anaerobic bacteria.     

Energy transduction in the methanogen Methanococcus voltae is based on a sodium current.

We provide experimental support for the proposal that ATP production in Methanococcus voltae, a methanogenic member of the archaea, is based on an energetic system in which sodium ions, not protons, are the coupling ions. We show that when grown at a pH of 6.0, 7.1, or 8.2, M. voltae cells maintain a membrane potential of approximately -150 mV. The cells maintain a transmembrane pH gradient (pH(in) - pH(out)) of -0.1, -0.2, and -0.2, respectively, values not favorable to the inward movement of protons. The cells maintain a transmembrane sodium concentration gradient (sodium(out)/sodium(in)) of 1.2, 3.4, and 11.6, respectively. While the protonophore 3,3',4',5-tetrachlorosalicylanilide inhibits ATP formation in cells grown at pH 6.5, neither ATP formation nor growth is inhibited in cells grown in medium at pH 8.2. We show that when grown at pH 8.2, cells synthesize ATP in the absence of a favorably oriented proton motive force. Whether grown at pH 6.5 or pH 8.2, M. voltae extrudes Na+ via a primary pump whose activity does not depend on a proton motive force. The addition of protons to the cells leads to a harmaline-sensitive efflux of Na+ and vice versa, indicating the presence of Na+/H+ antiporter activity and, thus, a second mechanism for the translocation of Na+ across the cell membrane. M. voltae contains a membrane component that is immunologically related to the H(+)-translocating ATP synthase of the archaeabacterium Sulfolobus acidocaldarius. Since we demonstrated that ATP production can be driven by an artificially imposed membrane potential only in the presence of sodium ions, we propose that ATP production in M. voltae is mediated by an Na+-translocating ATP synthase whose function is coupled to a sodium motive force that is generated through a primary Na+ pump.     

Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris.

In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force.     

Stoichiometry of the H+-ATPase of Escherichia coli cells during anaerobic growth

The H+/ATP stoichiometry of the H+-ATPase was investigated in Escherichia coli cells growing under anaerobic conditions at pH 6 and 7. The protonmotive force was determined from the intracellular accumulation of benzoate and tetraphenylphosphonium ions, as well as the accumulation of lactose in this lac operon inducible, but beta-galactosidase negative strain. The phosphorylation potential was calculated from the cellular concentrations of ATP, ADP and inorganic phosphate. By comparing the phosphorylation potential and the proton motive force under these steady state conditions, the H+/ATP stoichiometry was determined to be 3, similar to the value previously found in the same cells growing under aerobic conditions.     

Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.

Wild-type Streptomyces clavuligerus NRRL 3585 is unable to utilize glucose. A glucose-utilizing (gut-1) mutant of S. clavuligerus NRRL 3585 has been obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The gut-1 mutant is able to grow on glucose or galactose, while the wild type is unable to catabolize these hexoses. Similar binding affinities of glucose by cells of the wild type and the gut-1 mutant were found, but the wild type was unable to complete glucose transport. A soluble intracellular ATP-dependent (but not phosphoenolpyruvate-dependent) glucokinase activity was found both in the wild type and the gut-1 mutant. The gut-1 mutant has acquired a functional transport system that allows transport of glucose, 2-deoxyglucose, and galactose, as shown by hexose competition experiments. The gut-1 transport system concentrates glucose inside the cell at least 10- to 20-fold and is strongly inhibited by respiratory inhibitors, which prevent the establishment of a proton motive force, and by proton-conducting ionophores, suggesting that it is energized by a proton motive force. The new transport system is not completely sugar specific (transporting galactose and glucose through the same system), as opposed to the hexose-specific system reported in wild-type Streptomyces griseus.