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B Poolman

Publications and source records attributed to B Poolman.

At least 37 records · Page 2Linked to original sources
PubMed1995

The Lactococcal lmrP gene encodes a proton motive force-dependent drug transporter.

To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide. Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium. The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments. Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E. coli. Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules. Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate. These observations are indicative for a drug-proton antiport system. A lmrP deletion mutant was constructed via homologous recombinant using DNA fragments of the flanking region of the gene. The L. lactis (delta lmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of the functional drug-proton anti-porter LmrP, L. lactis is able to overexpress another, ATP-dependent, drug extrusion system. These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N. (1994) J. Bacteriol. 176, 6957-6964).

Amino Acid Sequence↗
PubMed1995

Kinetic analysis of lactose and proton coupling in Glu379 mutants of the lactose transport protein of Streptococcus thermophilus.

The role of Glu379 in the lactose-H+ symport protein (LacS) of Streptococcus thermophilus was studied by analyzing the kinetic mechanism of transport of wild-type and Ala379, Asp379, and Gln379 mutant proteins. Glu379 forms part of the sequence motif Lys-X-X-His-X-X-Glu that is present in a number of sugar transport proteins, including LacY of Escherichia coli. The E379A and E379Q mutants were defective in the uptake of lactose against a concentration gradient and lactose-dependent proton uptake, but catalyzed facilitated influx of lactose down a concentration gradient and equilibrium exchange with rates similar to that of the wild-type enzyme. The E379D mutant was partially defective in the coupled transport of lactose and protons. These results suggest that an acidic residue at position 379 is required for the coupled uptake of lactose and protons and are consistent with a mechanism in which lactose transport in the E379A and E379Q mutants occurs by uniport rather than proton symport. Lactose efflux down a concentration gradient in wild-type LacS and LacS-E379D increased with pH with apparent pK (pKa) values of > or = 8.5 and 8.0, respectively, whereas efflux in the E379Q mutant increased sigmoidally with a pKa of about 6.0. Imposition of an artificial membrane potential (inside negative) in membrane vesicles bearing wild-type LacS or LacS-E379Q not only inhibited the lactose efflux mediated by wild-type but also that of the mutant enzyme. To associate the role of Glu379 with specific step(s) in the translocation cycle of LacS, the properties of wild-type LacS and the Glu379 mutants have been evaluated by numerical analysis of simple kinetic schemes for translocation catalysis by solute H+ symport proteins. The properties of the wild-type enzyme are consistent with a mechanism in which the order of ligand binding on the inside is substrate first and proton last, whereas the order is random (or proton first, substrate last) at the outer surface of the membrane. The wild-type enzyme is asymmetric with regard to proton binding; the pK for proton binding on the outside is at least 4 units higher than the pK on the inside. The properties of the Glu379 mutants correspond with a lowering of the pK on the outside (pKOUT approximately pKIN), and the induction of a leak pathway in which the binary enzyme-substrate complex becomes mobile.

Amino Acid Sequence↗
PubMed1995

Uncoupling in secondary transport proteins. A mechanistic explanation for mutants of lac permease with an uncoupled phenotype.

The kinetic behavior of a H(+)-substrate symporter has been studied in which in addition to the unloaded (E) and fully loaded states (E.S.H) of the carrier also one of the binary complexes (E.S or E.H) may reorient its binding sites. This results in two types of uncoupled mutants, the ES leak and the EH leak type. The effects of pH and substrate concentration (pS) on the coupling of transport have been analyzed. In the enzyme with the ES leak, the proton:substrate stoichiometry (v(H+)/v(S)) and the substrate accumulation levels decrease sigmoidally from fully coupled at low pH to completely uncoupled at high pH. Importantly, the coupling inferred from initial rate measurements is higher than from steady state accumulation levels. In the enzyme with the EH leak, the coupling inferred from the accumulation levels increases from no coupling at low pH to full coupling at high pH and saturating substrate concentration. The v(H+)/v(S) increases sigmoidally with pH from < 1 to > 1 and is highly dependent on pS. At each pH value a substrate concentration can be found that results in apparent complete coupling between the two fluxes. The ES leak and the EH leak mutants provide a mechanism for substrate-induced and substrate-inhibited proton leakage, respectively. Furthermore, substrate efflux down a concentration gradient is inhibited by a membrane potential (inside negative) under uncoupled conditions in the case of an ES leak but not in the case of an EH leak. The properties of the mutants mimic those of various transport mutants that have been described, in particular mutants of the lactose transport protein of Escherichia coli. The analysis offers general means for targeted experimentation, which allows discrimination between various types of transport mutants.

Biological Transport↗
PubMed1995

Regulation of bacterial sugar-H+ symport by phosphoenolpyruvate-dependent enzyme I/HPr-mediated phosphorylation.

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein(s) domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). C-terminal truncation mutants were constructed and expressed in Escherichia coli and their properties were analyzed. Remarkably, the entire IIA domain (160 amino acids) could be deleted without significant effect on lactose-H+ symport and galactoside equilibrium exchange. Analysis of the LacS mutants in S. thermophilus cells suggested that transport is affected by PTS-mediated phosphorylation of the IIA domain. For further studies, membrane vesicles of S. thermophilus were fused with cytochrome c oxidase-containing liposomes, and, when appropriate, phosphoenolpyruvate (PEP) plus purified enzyme I and heat-stable protein HPr were incorporated into the hybrid membranes. Generation of a protonmotive force (delta p) in the hybrid membranes resulted in accumulation of lactose, whereas uptake of the PTS sugar sucrose was not observed. With PEP and the energy-coupling proteins enzyme I and HPr of the PTS on the inside, high rates of sucrose uptake were observed, whereas delta p-driven lactose uptake by wild-type LacS was inhibited. This inhibition was not observed with LacS(delta 160) and LacS(H552R), indicating that PEP-dependent enzyme I/HPr-mediated phosphorylation of the IIA domain (possibly the conserved His-552 residue) modulates lactose-H+ symport activity.

Amino Acid Sequence↗
PubMed1995

Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis.

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 P1-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.

Amino Acids↗
PubMed1995

Role of scalar protons in metabolic energy generation in lactic acid bacteria.

Lactic acid bacteria are able to generate a protonmotive force across the cytoplasmic membrane by various metabolic conversions without involvement of substrate level phosphorylation or proton pump activity. Weak acids like malate and citrate are taken up in an electrogenic process in which net negative charge is translocated into the cell thereby generating a membrane potential. The uptake is either an exchange process with a metabolic end-product (precursor/ product exchange) or a uniporter mechanism. Subsequent metabolism of the internalized substrate drives uptake and results in the generation of a pH gradient due to the consumption of scalar protons. The generation of the membrane potential and the pH gradient involve separate steps in the pathway. Here it is shown that they are nevertheless coupled. Analysis of the pH gradient that is formed during malolactic fermentation and citrate fermentation shows that a pH gradient, inside alkaline, is formed only when the uptake system forms a membrane potential, inside negative. These secondary metabolic energy generating systems form a pmf that consists of both a membrane potential and a pH gradient, just like primary proton pumps do. It is concluded that the generation of a pH gradient inside alkaline, upon the addition of a weak acid to cells is diagnostic for an electrogenic uptake mechanism translocating negative charge with the weak acid.

Cell Membrane↗
PubMed1995

Cation-selectivity of the L-glutamate transporters of Escherichia coli, Bacillus stearothermophilus and Bacillus caldotenax: dependence on the environment in which the proteins are expressed.

L-Glutamate transport by the H(+)-glutamate and Na(+)-glutamate symport proteins of Escherichia coli K-12 (GltPEc and GltSEc, respectively) and the Na(+)-H(+)-glutamate symport proteins of Bacillus stearothermophilus (GltTBs) and Bacillus caldotenax (GltTBc) was studied in membrane vesicles derived from cells in which the proteins were either homologously or heterologously expressed. Substrate and inhibitor specificity studies indicate that GltPEc, GltTBs and GltTBc fall into the same group of transporters, whereas GltSEc is distinctly different from the others. Also, the cation specificity of GltSEc is different; GltSEc transported L-glutamate with (at least) two Na+, whereas GltPEc, GltTBs and GltTBc catalysed an electrogenic symport of L-glutamate with > or = two H+, i.e. when the proteins were expressed in E. coli. Surprisingly studies in membrane vesicles of B. stearothermophilus and B. caldotenax indicated a Na(+)-H(+)-L-glutamate symport for both GltTBs and GltTBc. The Na+ dependency of the GltT transporters in the Bacillus strains increased with temperature. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, which influences the coupling ion selectivity.

Amino Acid Transport Systems, Acidic↗
PubMed1995

Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coli.

Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidation of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by beta-hydroxyaspartate and cysteic acid but not by alpha-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltPBsu) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was determined. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon. gltPBsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate. Both sodium- and proton-coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the family of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.

Amino Acid Sequence↗
PubMed1995

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.

Amino Acid Sequence↗
PubMed1995

The glutamate uptake regulatory protein (Grp) of Zymomonas mobilis and its relation to the global regulator Lrp of Escherichia coli.

After being expressed in Escherichia coli JC5412, which is defective in glutamate transport, a Zymomonas mobilis gene which enabled this strain to grow on glutamate was cloned. This gene encodes a protein with 33% amino acid identity to the leucine-responsive regulatory protein (Lrp) of E. coli. Although overall glutamate uptake in E. coli was increased, the protein encoded by the cloned fragment repressed the secondary H+/glutamate transport system GltP by interaction with the promoter region of the gltP gene. It also repressed the secondary, H(+)-coupled glutamate uptake system of Z. mobilis, indicating that at least one role of this protein in Z. mobilis is to regulate glutamate transport. Consequently, it was designated Grp (for glutamate uptake regulatory protein). When expressed in E. coli, Grp repressed the secondary H+/glutamate transport system GltP by binding to the regulatory regions of the gltP gene. An lrp mutation in E. coli was complemented in trans with respect to the positive expression regulation of ilvIH (coding for acetohydroxy acid synthase III) by a plasmid which carries the grp gene. The expression of grp is autoregulated, and in Z. mobilis, it depends on growth conditions. The putative presence of a homolog of Grp in E. coli is discussed.

Amino Acid Sequence↗
PubMed1995

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes.

For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described. Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro. A genetically well-defined S. cerevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter. The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p. The levels of expression as well as the initial uptake rates were increased 20-fold compared with those in a yeast strain carrying only one chromosomal MAL locus. Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains. To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump. Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested.

Biological Transport, Active↗
PubMed1995

Mechanisms of membrane toxicity of hydrocarbons.

Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this area. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified. The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review, we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in several instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins embedded in the membrane are affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed. Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering, change in lipid/protein ratio) that compensate for the changes in membrane structure are treated.

Bacteria↗
PubMed1995

The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes fused with proteoliposomes, as model systems for studies on the mechanism and regulation of transport is evaluated.

Amino Acid Sequence↗
PubMed1994

Uniport of monoanionic L-malate in membrane vesicles from Leuconostoc oenos.

L-malate transport was studied in membrane vesicles from Leuconostoc oenos MLE(-) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton-motive-force-generating system. In these hybrid membranes, accumulation of L-malate was observed in response to a pH gradient (delta pH), with the inside alkaline, but was strongly inhibited by a membrane potential (delta psi) of normal polarity (inside negative). Imposition of a delta psi, with the inside positive, by means of valinomycin-mediated potassium influx, resulted in a rapid accumulation of L-malate, indicating that L-malate was taken up in an anionic form. The results are consistent with a uniport mechanism facilitating the uptake of monoanionic L-malate, the dominant species at the low pH of the experiments. Kinetic analysis of delta pH-driven L-malate uptake in the pH range 3.0-5.8, yielded apparent affinity constants that varied less than twofold when calculated on the basis of the concentrations of monoanionic L-malate, whereas the values differed 2-3 orders of magnitude for the other species. At L-malate concentrations above 1 mM, a non-saturable transport component became apparent which may reflect passive influx of L-malic acid. Substrate specificity studies indicated that citrate and L-malate (and possibly D-lactate and L-lactate) compete for a single general carboxylate transport system. The carboxylate transport system catalysed homologous L-malate and heterologous L-malate/citrate exchange with rates similar to the rate of L-malate efflux. Since metabolic energy is conserved during malolactic fermentation in L. oenos, the underlying mechanism most likely involves electrogenic monoanionic L-malate uptake, in combination with H+ consumption in the cytoplasm, followed by diffusion outwards of lactic acid plus carbon dioxide.

Animals↗
PubMed1994

The di- and tripeptide transport protein of Lactococcus lactis. A new type of bacterial peptide transporter.

Lactococcus lactis takes up di- and tripeptides via a proton motive force-dependent carrier protein. The gene (dtpT) encoding the di-tripeptide transport protein of L. lactis was cloned by complementation of a dipeptide transport-deficient and proline auxotrophic Escherichia coli strain. Functional expression of the dipeptide transport gene was demonstrated by uptake studies of alanyl-[14C]glutamate and other peptides in E. coli cells. The di-tripeptide transport protein catalyzes proton motive force-driven peptide uptake and dipeptide exchange activity. The nucleotide sequence of dtpT was determined and the translated sequence corresponds with a protein of 463 amino acid residues. Hydropathy profiling indicates that the protein could form 12 membrane-spanning segments with the amino and carboxyl termini at the outer surface of the membrane. A secondary structure model is presented which is substantiated by analysis of DtpT-PhoA fusion constructs. Amino acid sequence comparisons showed no significant homology with other bacterial peptide transport systems nor with any other known protein. Flanking regions of the di-tripeptide transport gene were used to delete dtpT from the chromosome of L. lactis. Genetic and biochemical characterization of this mutant indicates that DtpT is the only transport protein in L. lactis for hydrophilic di- and tripeptides.

Amino Acid Sequence↗
PubMed1994

Interactions of cyclic hydrocarbons with biological membranes.

Many cyclic hydrocarbons, e.g. aromatics, cycloalkanes, and terpenes, are toxic to microorganisms. The primary site of the toxic action is probably the cytoplasmic membrane, but the mechanism of the toxicity is still poorly understood. The effects of cyclic hydrocarbons were studied in liposomes prepared from Escherichia coli phospholipids. The membrane-buffer partition coefficients of the cyclic hydrocarbons revealed that these lipophilic compounds preferentially reside in the membrane. The partition coefficients closely correlated with the partition coefficients of these compounds in a standard octanol-water system. The accumulation of hydrocarbon molecules resulted in swelling of the membrane bilayer, as assessed by the release of fluorescence self-quenching of fluorescent fatty acid and phospholipid analogs. Parallel to the expansion of the membrane, an increase in membrane fluidity was observed. These effects on the integrity of the membrane caused an increased passive flux of protons and carboxyfluorescein. In cytochrome c oxidase containing proteoliposomes, both components of the proton motive force, the pH gradient and the electrical potential, were dissipated with increasing concentrations of cyclic hydrocarbons. The dissipating effect was primarily the result of an increased permeability of the membrane for protons (ions). At higher concentrations, cytochrome c oxidase was also inactivated. The effective concentrations of the different cyclic hydrocarbons correlated with their partition coefficients between the membrane and aqueous phase. The impairment of microbial activity by the cyclic hydrocarbons most likely results from hydrophobic interaction with the membrane, which affects the functioning of the membrane and membrane-embedded proteins.

Benzene↗
PubMed1994

Solute transport and energy transduction in bacteria.

In bacteria two forms of metabolic energy are usually present, i.e. ATP and transmembrane ion-gradients, that can be used to drive the various endergonic reactions associated with cellular growth. ATP can be formed directly in substrate level phosphorylation reactions whereas primary transport processes can generate the ion-gradients across the cytoplasmic membrane. The two forms of metabolic energy can be interconverted by the action of ion-translocating ATPases. For fermentative organisms it has long been thought that ion-gradients could only be generated at the expense of ATP hydrolysis by the F0F1-ATPase. In the present article, an overview is given of the various secondary transport processes that form ion-gradients at the expense of precursor (substrate) and/or end-product concentration gradients. The metabolic energy formed by these chemiosmotic circuits contributes to the 'energy status' of the bacterial cell which is particularly important for anaerobic/fermentative organisms.

Adenosine Triphosphatases↗