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LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump. This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher. The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding. Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change. Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding. Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher. This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.

Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of structurally unrelated compounds. Similar to its eucaryotic homologue P-gp, this protein is a member of the ATP-binding cassette (ABC) superfamily. Different predictive models, based on hydropathy profiles, have been proposed to describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectroscopy, combined with limited proteolysis, to investigate the secondary structure and the orientation of the transmembrane segments of LmrA. We bring the first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented a-helices. Furthermore, a new approach was developed in order to provide information about membrane domain dynamics. Monitoring the infrared linear dichroism spectra in the course of H-1/H-2 exchange allowed to focus the recording of exchange rates on the membrane-embedded region of the protein only. This approach revealed an unusual structural dynamics, indicating high flexibility in this antibiotic binding and transport region.

Solute transport in Saccharomyces cerevisiae can be regulated through mechanisms such as trans-inhibition and/or catabolite inactivation by nitrogen or carbon sources. Studies in hybrid membranes of S. cerevisiae suggested that the maltose transport system Mal61p is fully reversible and capable of catalyzing both influx and efflux transport. This conclusion has now been confirmed by studies in a S. cerevisiae strain lacking the maltase enzyme. Whole cells of this strain, wherein the orientation of the maltose transporter is fully preserved, catalyze fully reversible maltose transport. Catabolite inactivation of the maltose transporter Mal61p was studied in the presence and absence of maltose metabolism and by the use of different glucose analogues. Catabolite inactivation of Mal61p could be triggered by maltose, provided the sugar was metabolized, and the rate of inactivation correlated with the rate of maltose influx. We also show that 2-deoxyglucose, unlike 6-deoxyglucose, can trigger catabolite inactivation of the maltose transporter. This suggests a role for early glycolytic intermediates in catabolite inactivation of the Mal61 protein. However, there was no correlation between intracellular glucose-6-phosphate or ATP levels and the rate of catabolite inactivation of Mal61p. On the basis of their identification in cell extracts, we speculate that (dideoxy)-trehalose and/or (deoxy)-trehalose-6-phosphate trigger catabolite inactivation of the maltose transporter.

Bacillus subtilis was grown at its growth temperature limits and at various temperatures in between the lower and upper growth temperature boundary. Liposomes were made of the extracted membrane lipids derived from these cells. The headgroup composition of the cytoplasmic membrane lipids did not differ significantly at the lower (13°C) and upper (50°C) temperature boundary. The averaged lipid acyl chain length, degree of saturation, and ratio of iso- and anteiso-branched fatty acids increased with the temperature. At the temperature of growth, the membranes were in a liquid-crystalline phase, but liposomes derived from cells grown at 13°C were almost threefold more viscous than those derived from 50°C grown cells. The temperature dependence of the proton permeability of the liposomes was determined using the acid-pulse method with monitoring of the outside pH with the fluorescent probe pyranine. The proton permeability of each liposome preparation increased with the temperature. However, the proton permeability of the liposomes at the growth temperature of the cells from which the lipids were derived was almost constant. These data indicate that the growth temperature dependent variation in lipid acyl chain composition permits maintenance of the proton permeability of the cytoplasmic membrane. This [`]homeo-proton permeability adaptation' precludes futile cycling of protons at higher growth temperatures and allows cells to sustain the proton motive force as a driving force for essential energy transducing processes. http://www.sciencedirect.com/science/article/B6T1T-47YY9YH-C/1/94023ddec523ae7d12d336f6972e3351

In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K + (or Na + ) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.

In extreme environments, mainly Archaea are encountered. The archaeal cytoplasmic membrane contains unique ether lipids that cannot easily be degraded, are temperature- and mechanically resistant, and highly salt tolerant. Moreover, thermophilic and extreme acidophilic Archaea possess membrane-spanning tetraether lipids that form a rigid monolayer membrane which is nearly impermeable to ions and protons. These properties make the archaeal lipid membranes more suitable for life and survival in extreme environments than the ester-type bilayer lipids of Bacteria or Eukarya.

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from beta-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of beta-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo; (iii) use of oligopeptides of 5-10 residues becomes only possible after uptake via Opp; (iv) only a few (10-14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.

Transport of hydrophilic di- and tripeptides into Lactococcus lactis is mediated by a proton motive force-driven peptide transport protein (DtpT) that shares similarity with eukaryotic peptide transporters, e.g., from kidney and small intestine of rabbit, man, and rat. Hydropathy profiling in combination with the ''positive inside rule'' predicts for most of the homologous proteins an a-helical bundle of 12 transmembrane segments, but the positions of these transmembrane segments and the location of the amino and carboxyl termini are by no means conclusive. The secondary structure of DtpT was investigated by analysing 42 DtpT-alkaline phosphatase fusion proteins, generated by random or directed fusions of the corresponding genes. These studies confirm the presence of 12 transmembrane segments but refute several other predictions made of the secondary structure. Data obtained from the fusion proteins were substantiated by studying the accessibility of single cysteine mutants in putative cytoplasmic or extracellular loops by membrane (im)permeant sulfhydryl reagents. The deduced topology model of DtpT consists of a bundle of 12 alpha-helixes with a short amino and a large carboxyl terminus, both located at the cytoplasmic site of the membrane. On the basis of sequence comparisons with DtpT, it seems likely that the structure model of the amino-terminal half of DtpT also holds for the eukaryotic peptide transporters, whereas the carboxyl-terminal half is largely different.

Energy transduction in the anaerobic, thermophilic bacterium Clostridium fervidus relies exclusively on Na+ as the coupling ion. The Na+ ion gradient across the membrane is generated by a membrane-bound ATPase (G. Speelmans, B. Poolman, T. Abee, and W. N. Konings, J. Bacteriol. 176:5160-5162, 1994). The Na+-ATPase complex was purified to homogeneity. It migrates as a single band in native polyacrylamide gel electrophoresis and catalyzes Na+-stimulated ATPase activity. Denaturing gel electrophoresis showed that the complex consists of at least six different polypeptides with apparent molecular sizes of 66, 61, 51, 37, 26, and 17 kDa. The N-terminal sequences of the 66- and 51-kDa subunits were found to be significantly homologous to subunits A and B, respectively, of the Na+-translocating V-type ATPase of Enterococcus hirae. The purified V1V0 protein complex was reconstituted in a mixture of Escherichia coli phosphatidylethanolamine and egg yolk phosphatidylcholine and shown to catalyze the uptake of Na+ ions upon hydrolysis of ATP. Na+ transport was completely abolished by monensin, whereas valinomycin stimulated the uptake rate. This is indicative of electrogenic sodium transport. The presence of the protonophore SF6847 had no significant effect on the uptake, indicating that Na+ translocation is a primary event and in the cell is not accomplished by an H+-translocating pump in combination with an Na+-H+ antiporter.

Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50&C. Since a high concentration of Na 1 ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na 1 was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na 1 ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na 1 and Li 1 ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed. The newly isolated strain LBS3 is a gram-positive, sporeforming, anaerobic thermoalkaliphile that grows optimally at 508C and an alkaline pH of 9.5. The strain was isolated from a hot-lake inlet at Lake Bogoria in Kenya and represents a new group within the gram-positive Clostridium-Bacillus subphyllum(24).Onlyafewthermoalkaliphilesareknowntodate,like

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy-coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli, chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides, chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein-dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue gamma-glutamyl-hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H+-linked glutamate carrier of E. coli, was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides.

Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize Liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent dyes from: PC Liposomes in;a Delta psi-stimulated manner, and not that of neutral and cationic fluorescent dyes. This activity is blocked in PG liposomes. Nisin, however, efficiently dissipates the Delta psi in cytochrome c oxidase proteoliposomes reconstituted with PG, with a threshold Delta psi, requirement of about -100 mV. Nisin associates with the anionic surface of PG liposomes and disturbs the lipid dynamics near the phospholipid polar head group-water interface. Further studies with a novel cationic lantibiotic, epilancin K7, indicate that this molecule penetrates into the hydrophobic carbon region of the Lipid bilayer upon the imposition of a Delta psi. It is concluded that nisin acts as an anion-selective carrier in the absence of anionic phospholipids. In vivo, however, this activity is likely to be prevented by electrostatic interactions with anionic lipids of the target membrane. It is suggested that pore formation by cationic (type A) lantibiotics involves the local perturbation of the bilayer structure and a Delta psi-dependent reorientation of these molecules from a surface-bound into a membrane-inserted configuration.

In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P-1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT(-) mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp(-) and DtpT(-) Opp(-) mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.

In natural waters and domestic waste waters in which divalent metal ions are present in excess of Pi, H2PO4-, HPO4(2-), and MeHPO4 prevail at pH values physiological for Acinetobacter johnsonii 210A (pH 5.5-8.0). In view of the ability of this organism to extensively accumulate Pi and divalent cations in cytoplasmic polyphosphate granules, the substrate specificity of its two Pi transport systems was studied. The constitutive, proton motive force-driven Pi carrier, previously shown to be dependent on divalent cations, plays a major role in the divalent cation and Pi flux by translocating MeHPO4 rather than Pi. This notion is confirmed by the observation that divalent cations are cotransported with Pi in a 1:1 stoichiometry in proteoliposomes containing reconstituted Pi carrier protein. In contrast, the Pi repressible, periplasmic binding protein-dependent Pi transport system mediates the uptake of H2PO4- and HPO4(2-). Pi uptake, but not MeHPO4 uptake, was stimulated in cells under Pi limitation, and the periplasmic Pi-binding protein has affinity for H2PO4- and HPO4(2-), but not for MeHPO4. When operating in concert, both systems enable A. johnsonii 210A to efficiently acquire Pi from its habitat through uptake of the predominant Pi species.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.

In extreme environments varying from hot to cold, acidic to alkaline, and highly saline, mainly Archaea are found. Thermophilic and extremely acidophilic Archaea have a membrane that contains membrane spanning tetraether lipids. These tetra-ether membranes have a limited permeability for protons even at the high temperatures of growth and this property makes it possible for thermophilic archaea to maintain a viable proton motive force under the extreme conditions. -Ether lipids cannot be degraded easily and are highly stable which is also a requirement for life under extreme conditions. Psychrophilic and mesophilic Bacteria, and all Archaea adjust the lipid composition of their membranes so that the proton permeability of their membranes remains within a narrow range. This phenomenon is termed 'homeoproton permeability adaptation'. Thermophilic Bacteria are the only prokaryotes that are unable to control the proton permeability of their membranes. These organisms have to rely on the less permeable sodium ions in energy transducing processes in their membrane.

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho -vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis , one for the artificial pH probe 2′,7′-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N -(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L. lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.

In all organisms, adenosine triphosphate (ATP) provides metabolic energy for driving energy-dependent processes. It is synthesized and/or utilized by enzymes known as F-type and V-type ATPases, which are small rotary motors1,2. Both types consist of a headpiece, F1 or V1, respectively, which is connected by a stalk region to the membrane-bound part, FO or VO. Electron microscopic analysis of negatively stained particles has revealed a peripheral stalk, or stator, between V1 and VO of the V-type (Na+)ATPase of the thermophilic bacterium Clostridium fervidus3,4, like that in F-type ATPases5,6. We have analysed many more particles and now present a more complete structure of the V-type ATPase stator moiety.

The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force. The inhibition of the activity of LmrP by detergents restricted the range of compounds that could be used for the solubilization and reconstitution of the protein because low concentrations of detergent are retained in proteoliposomes. Surprisingly, dodecyl maltoside did not modulate the activity of LmrP. Therefore, LmrP was solubilized with dodecyl maltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecyl maltoside-destabilized, preformed liposomes prepared from Escherichia coli phospholipids and egg phosphatidylcholine. Reconstituted LmrP mediated the transport of multiple drugs in response to an artificially imposed pH gradient, demonstrating that the protein functions as a proton motive force-dependent multidrug transporter, independent of accessory proteins. These observations are relevant for the effective solubilization and reconstitution of multidrug transporters belonging to the major facilitator superfamily, which, in view of their broad drug specificity, may strongly interact with detergents.

The influence of pH and the salt concentration on the proton and sodium ion permeability of liposomes formed from lipids of the halophile Halobacterium salinarum and the haloalkaliphile Halorubrum vacuolatum were studied. In contrast with liposomes formed from Escherichia coli lipids, liposomes formed from halophilic lipids remained stable up to 4 M of NaCl and KCl. The proton permeability of the liposomes from lipids of halophiles was independent of the salt concentration and was essentially constant between pH 7 and pH 9. The sodium ion permeability increased with the salt concentration but was 10- to 100 fold lower than the proton permeability. It is concluded that the membranes of halophiles are stable over a wide range of salt concentrations and at elevated pH values and are well adapted to the halophilic conditions.

The proton-coupled Pho84 phosphate permease of Saccharomyces cerevisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes. Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria. Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease. The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate.

The maltose transporter of Saccharomyces cerevisiae is subject to rapid, irreversible inactivation in the presence of glucose. Loss of transport function was paralleled by a decrease in amount of transporter protein and most likely involves endocytosis and degradation of the protein in the vacuole. This (catabolite) inactivation of Mal61p was triggered not only by glucose but also by 2-deoxy-d-glucose, which cannot be metabolized beyond 2-deoxy-d-glucose phosphate. The signal that targets membrane proteins specifically for catabolite inactivation is unknown. To investigate whether or not specific modification of Mal61p triggers the inactivation, putative protein kinase A and C phosphorylation sites were removed, and the transport activities and levels of the mutant proteins upon addition of glucose were followed in time. Three Mal61p mutants, i.e. S295A, T363A, and S487A, exhibited significantly reduced rates of inactivation in the presence of glucose. Likewise, in wild-type Mal61p the rate of inactivation and degradation of the protein paralleled each other in the case of T363A. On the contrary, for the S295A and S487A mutants the rates of protein degradation were slowed down more profoundly than was the loss of transport activity. These observations indicate that (i) some form of modification (e.g. phosphorylation) of the protein precedes breakdown, (ii) the modification inactivates Mal61p, and (iii) the inactivation of Mal61p is not necessarily followed by proteolytic degradation.

F- and V-type ATPases are central enzymes in energy metabolism that couple synthesis or hydrolysis of ATP to the translocation of H + or Na + across biological membranes. They consist of a soluble headpiece that contains the catalytic sites and an integral membrane-bound part that conducts the ion flow. Energy coupling is thought to occur through the physical rotation of a stalk that connects the two parts of the enzyme complex. This mechanism implies that a stator-like structure prevents the rotation of the headpiece relative to the membrane-bound part. Such a structure has not been observed to date. Here, we report the projected structure of the V-type Na + -ATPase of Clostridium fervidus as determined by electron microscopy. Besides the central stalk, a second stalk of 130 Å in length is observed that connects the headpiece and membrane-bound part in the periphery of the complex. This additional stalk is likely to be the stator.

Publisher Summary This chapter discusses the cell biological mechanisms through which prokaryotes develop multidrug resistance. The majority of bacterial multidrug transporters characterized thus far, operates via a secondary transport mechanism. A number of genes encoding TEXANs and Mini TEXANs are cloned and sequenced. Analysis of these primary sequences reveals a striking similarity in the overall structure, suggesting that the proteins may function via a similar mechanism. Transport studies in membrane vesicles of Lactococcus lactis ( L. lactis )and Escherichia coli ( E. coli )are demonstrated the drug/proton antiport mechanism of the TEXAN LmrP. Smr and EmrE are purified and characterized upon reconstitution into proteoliposomes. These studies exhibit that a single multidrug resistance protein is able to function as a drug pump, and show the Δp-dependence of drug transport via the Mini TEXANs. The discovery of the ABC-type multidrug transporter LmrA in L. lactis shows overlap with that of the human multidrug resistance P-glycoprotein, the TEXANs LmrP and Bmr, and Mini TEXANs Smr, EmrE and QacC. On the other hand, the substrate specificity of these multidrug transporters is very different from that of the TetA(B) and other transporters dedicated to the efflux of a specific drug or class of drugs.

Penicillium chrysogenum utilizes phenylacetic acid as a side chain precursor in penicillin G biosynthesis. During industrial production of penicillin G, phenylacetic acid is fed in small amounts to the medium to avoid toxic side effects. Phenylacetic acid is taken up from the medium and intracellularly coupled to 6-aminopenicillanic acid. To enter the fungal cell, phenylacetic acid has to pass the plasma membrane. The process via which phenylacetic acid crosses the plasma membrane was studied in mycelia and liposomes. Uptake of phenylacetic acid by mycelium was nonsaturable, and the initial velocity increased logarithmically with decreasing external pH. Studies with liposomes demonstrated a rapid passive flux of the protonated species through liposomal membranes. These results indicate that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species. The rate of phenylacetic acid uptake at an external concentration of 3 mM is at least 200-fold higher than the penicillin production rate in the Panlabs P2 strain. In this strain, uptake of phenylacetic acid is not the rate-limiting step in penicillin G biosynthesis.

The strictly aerobic, polyphosphate-accumulating Acinetobacter johnsonii strain 210A degrades its polyphosphate when oxidative phosphorylation is impaired. The endproducts of this degradation, divalent metal ions and inorganic phosphate, are excreted as a neutral metal-phosphate (MeHPO4) chelate via the electrogenic MeHPO4/H+ symport system of the organism. The coupled excretion of MeHPO4 and H+ in A. johnsonii 210A can generate a proton motive force. In membrane vesicles and deenergized cells, a membrane potential of about -70 mV and transmembrane pH gradient of about -8 mV were formed in response to an imposed outwardly directed MeHPO4 concentration gradient of 120 mV (initial value). The MeHPO4 efflux-induced proton motive force could drive energy-requiring processes, such as the accumulation of L-proline and L-lysine and the synthesis of ATP via the membrane-bound F0F1 H(+)-ATPase. In vivo 31P NMR studies of polyphosphate degradation in anaerobic cell suspensions revealed the presence of a considerable outwardly directed phosphate gradient across the cytoplasmic membrane corresponding to a MgHPO4 concentration gradient of at least 100 mV. This MgHPO4 concentration gradient was maintained for several hours. Thus, energy recycling by MeHPO4/H+ efflux will contribute significantly to the overall production of metabolic energy from the degradation of polyphosphate in A. johnsonii 210A.

The mechanism and energetics of the secondary Pi transport system of A. johnsonii were studied in membrane vesicles and proteoliposomes in which the transport protein was functionally reconstituted. Pi uptake is strictly dependent on the presence of divalent cations, like Mg2+, Ca2+, Mn2+, or Co2+. These cations form a MeHPO4 complex with up to 87% of the Pi present in the incubation mixture, suggesting that divalent cations and Pi are co-transported via a metal-phosphate chelate. Metal-phosphate uptake is driven by the proton motive force (interior negative and alkaline). The metal-phosphate/proton stoichiometry was close to unity. The transport system mediates efflux and homologous exchange of metal-phosphate, but not heterologous exchange of metal-phosphate and glycerol-3-P or glucose-6-P. Exchange and counterflow were essentially pH-independent while efflux and uptake increased with increasing pH. Efflux was inhibited by the proton motive force, whereas exchange was inhibited by the membrane potential only. These observations are consistent with an ordered mechanism for binding and dissociation of metal-phosphate and proton to and from the carrier protein and point to the recycling of a positively charged, protonated carrier protein during exchange.

Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K(+)-stimulated and Mg(2+)-stimulated ATPase was purified from membranes of these cells by solubilization with decyl-beta-D-maltoside in the presence of Escherichia coli phospholipids followed by triazine-dye affinity chromatography. This primary transport system has a substrate specificity and an inhibitor sensitivity closely similar to those of the Kdp ATPase from E. coli and is composed of three subunits with molecular masses of 70.0, 43.5, and 23.5 kDa.

The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers. A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity. Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition. These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide. Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline. The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate. The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+). These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.

The activity of the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTS) in Escherichia coli is coupled to the oxidation-reduction potential. It is inhibited when the redox potential is increased above -300 mV either via substrate oxidation or via direct addition of oxidizing agents. Depending on the point of addition, dithiothreitol either blocks or reverses these effects. Inhibition occurs at the level of sugar binding to EII. A sulfhydryl group associated with EII activity undergoes reversible oxidation to, presumably, a disulfide, resulting in the conversion of EII from a reduced, high-affinity form to an oxidized, low-affinity form which has a 10(2)-10(3) times lower affinity for the sugar. An identical change in affinity occurs as the result of the generation of a delta mu H+ during the oxidation of reduced N-methylphenazonium methosulfate or nicotinamide adenine dinucleotide. In this case, uncouplers and ionophores reverse the change. A mechanism is proposed in which the electrical potential difference across the membrane regulates the glucose PTS by shifting the midpoint potential of the EII-associated redox transition to more negative values. As a result, EII is converted to the oxidized, low-affinity state in the presence of a delta mu H+.

The anthraquinones emodin (1,3,delta-trihydroxy-6-methylanthraquinone) and emodinanthrone (1,3,8-trihydroxy-6-methylanthrone) inhibited respiration-driven solute transport at micromolar concentrations in membrane vesicles of Escherichia coli. This inhibition was enhanced by Ca ions. The inhibitory action on solute transport is caused by inhibition of electron flow in the respiratory chain, most likely at the level between ubiquinone and cytochrome b, and by dissipation of the proton motive force. The uncoupling action was confirmed by studies on the proton motive force in beef heart cytochrome oxidase proteoliposomes. These two effects on energy transduction in cytoplasmic membranes explain the antibiotic properties of emodin and emodinanthrone.

In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB , whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB . Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum .

In this thesis, the action spectrum and mechanism of microbial growth inhibition by sodium lactate were examined, with special emphasis on its use in meat products.The concentrations (mM) of lactate needed to prevent growth of various spoilage organisms and pathogens in a broth were determined and compared to those of NaCl. The sensitivity towards lactate differed between microorganisms, as did the effect of pH on the minimum inhibitory concentration (MIC) of lactate. Especially, bacterial strains that were able to grow at low water activity (≤0.95) in the presence of NaCl were inhibited by sodium lactate (Staphylococcus aureus, Listeria monocytogenes, Brochothrix thermosphacta), indicating that the microbial quality of food products could be increased when part of the NaCl content would be exchanged by sodium lactate. This is also true when the influence of lactate on toxin production, spore germination and heat resistance of spores from Clostridium botulinum is considered.The effects on growth characteristics of pH, temperature and sodium lactate or NaCl concentrations below the MIC, as examined with Listeria innocua in a broth, were successfully translated into a relatively simple mathematical model that was subsequently validated in a Bologna-type sausage. Microbial growth in the sausage was much slower than in broth, but this might be related to the growth conditions being less favourable. The parameter value for the maximum specific growth rate in the absence of lactate was re-estimated using the data obtained in sausage and as a result, the model predictions were statistically acceptable.Listeria innocua and Lactococcus lactis which had been grown in the presence of sodium lactate were better capable of regulating their intracellular pH (pH in ) than control cells (grown without lactate). This is probably related to an increase in the amount and/or activity of the proton ATPase (F 1 F 0 -ATPase) in response to a decrease in the pH in . The mechanism of microbial growth inhibition by sodium lactate remains to be further elucidated, since the decrease in pH in was not enough to explain why microbial growth in the presence of lactate is prevented at low pH.

The biological removal of phosphorus from waste water is an attractive method to control eutrophication of surface waters. The process is currently perceived to depend on the provision of alternate stages in which the activated sludge is subjected to anaerobic and aerobic conditions. A characteristic feature of such plant is that P i , after being released from biomass in the anaerobic stage, is reincorporated into biomass during aeration, together with part or all of the influent P i . Analysis of the population structure of activated sludge has focussed attention on the strictly aerobic, gram-negative genus Acinetobacter as being one of the important genera in enhanced biological phosphorus removal. However, due to the lack of insight into the relevant physiological processes in these microorganisms our understanding of the mechanisms of enhanced biological phosphorus removal is only superficial.The project of this thesis was initiated to study the mechanisms and regulation of P i uptake and efflux in the polyphosphate-accumulating Acinetobacter johnsonii 210A. The nature of polyphosphates and the enzymology of their metabolism have been a subject of previous studies with A.johnsonii 210A and other Acinetobacter spp. Chapter 1 presents a review of these investigations and those concerning the molecular mechanisms of P i transport in prokaryotes. The results described in this thesis show that A.johnsonii 210A is well adapted to the environmental conditions encountered in activated sludge plants through (i) the efficient acquisition of the predominant P i species from its habitat, and (ii) the ability to survive prolonged periods of anaerobiosis, by using polyphosphate as a source of metabolic energy when oxidative phosphorylation is impaired.P i is taken up in A.johnsonii 210A against a concentration gradient by energydependent, carrier-mediated processes (Chapter 2). Kinetic analysis of P i uptake in cells grown under P, limitation, revealed the presence of two P i transport systems with an apparent Kt for P i of 0.7 and 9μM. The high-affinity permease could be classified as an ATP- and periplasmic binding protein-dependent P i uptake system. Induction of this system under P i limitation, and the ability to maintain a low internal P i by the synthesis of polyphosphate enable the organism to reduce the P i concentration in the environment to micromolar levels. The low-affinity system is a constitutive secondary P i transport system involved in P i uptake and efflux.P i transport via the secondary transport system was studied in membrane vesicles and proteoliposomes in which the carrier protein was successfully reconstituted (Chapter 3). These model systems allow detailed studies on the mechanism of P i transport without the interference of polyphosphate metabolism or other cellular processes. P i uptake is strongly dependent on the presence of divalent metal ions, such as Mg 2+, Ca 2+, Mn 2+, or Co 2+. These cations form a MeHPO 4 complex with up to 87% of the P i present in the incubation mixtures, suggesting that divalent cations and P i are cotransported via aMeHPO 4 complex. MeHPO 4 uptake is driven by the proton motive force with an mechanistic MeHPO 4 /H +stoichiometry of one. The pH dependence of various modes of facilitated diffusion processes, such as efflux, exchange, and counterflow catalyzed by the MeHPO 4 carrier suggests that H +and MeHPO4 binding and release to and from the carrier protein occur via an ordered mechanism.In view of the similarities between P i transport in cells of A.johnsonii 210A and Escherichia coli, a more extensively studied organism (Chapter 2), the mechanism and energetics of the phosphate inorganic transport (Pit) system of E. coli were investigated (Chapter 4). P i and metal transport studies in proteoliposomes containing reconstituted Pit protein identified Pit as a MeHPO 4 /H +symport system. The effects of pH and the proton motive force on the different modes of MeHPO 4 transport are consistent with the ordered binding model proposed for the MeHPO 4 transporter in A. johnsonii 210A.Chapter 5 describes the substrate specificity of the two P i transport systems in A. johnsonii 210A in relation to P i speciation in the aquatic environment. In natural waters and domestic waste water in which divalent metal ions are present in excess of P i , the species H 2 PO4-, HPO42-and MeHPO 4 prevail at physiological pH values for Acinetobacter (pH 5.5 to 8.0). The transport of MeHPO 4 by the secondary P i transport system is demonstrated in proteoliposomes by the (i) divalent cation dependent uptake and efflux of P i , (ii) P i -dependent uptake of Ca 2+and Mg 2+, (iii) equimolar transport of P i and Ca 2+, and (iv) inhibition by Mg 2+of Ca 2+uptake in the presence of P i , but not of P i uptake in the presence of Ca 2+.The transport of MeHPO 4 is closely related to the metabolism of cytoplasmic polyphosphate granules in which P i and divalent cations are accumulated. H 2 PO4-and HPO42-are translocated by the primary P i uptake system. P i uptake, but not MeHPO 4 uptake, was stimulated in cells under P i limitation. The periplasmic P i -binding protein showed affinity for H 2 PO4-and HPO42-, but not for MeHPO 4 .Chapter 6 demonstrates the presence of high-affinity secondary transport systems for L-lysine, L-alanine and L-proline in A. johnsonii 210A. The lysine and alanine carriers translocate their solute in symport with one proton. In contrast, the proline carrier is strictly dependent on the presence of Na +ions and mediates Na +/proline symport. The low internal Na +concentration, necessary for optimal proline uptake, is achieved by a sodium/proton antiporter. High-affinity systems will enable the organism to scavenge the environment for traces of metabolizable substrates and to recapture endogenous compounds leaking out of the cell.Retention of metabolites will become particularly important for survival when oxidative phosphorylation is impaired in A.johnsonii 210A. In Chapter 7, evidence is presented for the ability of the organism (i) to use polyphosphate as a source of metabolic energy during anaerobiosis, (ii) to maintain a considerable, outwardly directed MeHPO4 gradient across the cytoplasmic membrane during the degradation of polyphosphate, and (iii) to generate a proton motive force by the excretion of MeHPO 4 and H +via the MeHPO 4 carrier. This MeHPO 4 efflux-induced proton motive force can drive energy- requiring processes such as the accumulation of lysine and proline, and the synthesis of ATP. Conservation of metabolic energy from polyphosphate degradation may enable A. johnsonii 210A to survive alternating aerobic/anaerobic conditions as encountered in natural habitats and wastewater treatment plants.The significance of the here described findings for the cotransport of P i and divalent metal ions across biomembranes and the recycling of metabolic energy in microorganisms by the excretion of inorganic endproducts is discussed in Chapter 8.

A prerequisite for the stabilization of important features, such as aroma production, in starter strains used in dairy fermentations, is an extensive knowledge of the genetic basis of these properties. In this thesis the genetic basis of citrate metabolism in Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp. are studied and genetic aspects of citrate-fermenting Leuconostoc strains are analyzed.- Chapter II describes the development of a cloning system for Leuconostoc which was necessary due to the lack of natural transformation systems.- Chapter III describes the use of polymerase chain reaction to classify Leuconostoc strains.- Since lactose is the most important carbohydrate used by lactic acid bacteria, knowledge of the genetic basis of the genes involved in lactose metabolism can offer important data concerning gene regulation and expression. Chapters IV and V describe the cloning and expression of the lactose genes of Leuconostoc ssp.- Cloning and functional expression in Escherichia coli of the the citrate permease gene of Lactococcus lactis subsp. lactis var. diacetylactis are described in Chapter VI.- A comparison of the lactococcal citrate permease with the Leuconostoc lactis citrate permease gene is described in Chapter VII.- A summary together with concluding remarks are given in Chapter VIII.