Hw van Veen

Multidrug resistance in Lactococcus lactis : Evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane

Lactococcus lactis possesses an ATP‐dependent drug extrusion system which shares functional properties with the mammalian multidrug resistance (MDR) transporter P‐glycoprotein. One of the intriguing aspects of both transporters is their ability to interact with a broad range of structurally unrelated amphiphilic compounds. It has been suggested that P‐glycoprotein removes drugs directly from the membrane. Evidence is presented that this model is correct for the lactococcal multidrug transporter through studies of the extrusion mechanism of BCECF‐AM and cationic diphenylhexatriene (DPH) derivatives from the membrane. The non‐fluorescent probe BCECF‐AM can be converted intracellularly into its fluorescent derivative, BCECF, by non‐specific esterase activities. The development of fluorescence was decreased upon energization of the cells. These and kinetic studies showed that BCECF‐AM is actively extruded from the membrane before it can be hydrolysed intracellularly. The increase in fluorescence intensity due to the distribution of TMA‐DPH into the phospholipid bilayer is a biphasic process. This behaviour reflects the fast entry of TMA‐DPH into the outer leaflet followed by a slower transbilayer movement to the inner leaflet of the membrane. The initial rate of TMA‐DPH extrusion correlates with the amount of probe associated with the inner leaflet. Taken together, these results demonstrate that the lactococcal MDR transporter functions as a ‘hydrophobic vacuum cleaner’, expelling drugs from the inner leaflet of the lipid bilayer. Thus, the ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters.

The ABC family of multidrug transporters in microorganisms

Multidrug transporters are membrane proteins that are able to expel a broad range of toxic molecules from the cell. In humans, the overexpression of the multidrug resistance P-glycoprotein (Pgp) and the multidrug resistance-associated protein MRP1 (MRP) is a principal cause of resistance of cancers to chemotherapy. These multidrug transporters belong to the ATP-binding cassette (ABC) family of transport proteins that utilize the energy of ATP hydrolysis for activity. In microorganisms, multidrug transporters play an important role in conferring antibiotic resistance on pathogens. In the last decade, homologs of human Pgp and MRP have been found in microorganisms such as Plasmodium falciparum, Candida albicans, Saccharomyces cerevisiae and, more recently, in Lactococcus lactis. In this review, we will summarize the current state of knowledge on three major aspects of microbial ABC-type multidrug transporters: (i) the functional and structural similarities among these proteins in prokaryotic and eukaryotic cells, (ii) the molecular mechanism of these transporters, and (iii) their potential physiological role.

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 recombination using DNA fragments of the flanking region of the gene. The L. lactis (ΔlmrP) 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 antiporter 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).

Phosphate transport in prokaryotes: molecules, mediators and mechanisms

Bacteria have evolved sophisticated P>i transport systems which combine high affinity with coupling to metabolic energy. This review discusses the current evidence concerning the physiological, biochemical, and molecular properties of these P>i transport systems in prokaryotes. Major developments of the past years will be presented with emphasis on three kinds of issues. First, work on P>i transport in Escherichia coli and the polyphosphate-accumulating Acinetobacter johnsonii has assigned a novel biochemical mechanism and provided additional descriptive information for the transport of P>i and divalent cations. It is therefore appropriate to summarize these new facts and emphasize their general relevance for pro- and eukaryotic cells. Second, recent work on the bioenergetics of P>i transport in A. johnsonii has demonstrated the profound role of the transmembrane P>i gradient in energy transducing processes such as the accumulation of solutes, and the generation of a proton motive force. These findings and their significance for the survival of the cell during metabolic stress conditions will be discussed. Finally, polyphosphate-accumulating microorganisms play a valuable role in biotechnological applications, such as in wastewater treatment. As such organisms are still underrepresented in current molecular microbiological studies, the investigations in A. johnsonii described here may serve as a useful precedent for those to come.Bacteria have evolved sophisticated P>i transport systems which combine high affinity with coupling to metabolic energy. This review discusses the current evidence concerning the physiological, biochemical, and molecular properties of these P>i transport systems in prokaryotes. Major developments of the past years will be presented with emphasis on three kinds of issues. First, work on P>i transport in Escherichia coli and the polyphosphate-accumulating Acinetobacter johnsonii has assigned a novel biochemical mechanism and provided additional descriptive information for the transport of P>i and divalent cations. It is therefore appropriate to summarize these new facts and emphasize their general relevance for pro- and eukaryotic cells. Second, recent work on the bioenergetics of P>i transport in A. johnsonii has demonstrated the profound role of the transmembrane P>i gradient in energy transducing processes such as the accumulation of solutes, and the generation of a proton motive force. These findings and their significance for the survival of the cell during metabolic stress conditions will be discussed. Finally, polyphosphate-accumulating microorganisms play a valuable role in biotechnological applications, such as in wastewater treatment. As such organisms are still underrepresented in current molecular microbiological studies, the investigations in A. johnsonii described here may serve as a useful precedent for those to come.

The role of transport processes in survival of lactic acid bacteria. Energy transduction and multidrug resistance

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.

RESTRICTIVE USE OF DETERGENTS IN THE FUNCTIONAL RECONSTITUTION OF THE SECONDARY MULTIDRUG TRANSPORTER LMRP

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.

Activation by Gene Amplification of pitB, Encoding a Third Phosphate Transporter of Escherichia coli K-12

Two systems for the uptake of inorganic phosphate (P(i)) in Escherichia coli, PitA and Pst, have been described. A revertant of a pitA pstS double mutant that could grow on P(i) was isolated. We demonstrate that the expression of a new P(i) transporter, PitB, is activated in this strain by a gene amplification event.

Structure-function analysis of multidrug transporters in Lactococcus lactis

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Multidrug resistance in lactic acid bacteria : molecular mechanisms and clinical relevance

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type multidrug transporter LmrA in Lactococcus lactis are representatives of the two major classes of multidrug transporters found in pro- and eukaryotic organisms. Therefore, knowledge of the molecular properties of LmrP and LmrA will have a wide significance for multidrug transporters in all living cells, and may enable the development of specific inhibitors and of new drugs which circumvent the action of multidrug transporters. Interestingly, LmrP and LmrA are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by t he same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA will represent an intriguing new area of research.

Polyphosphate-accumulating bacteria and enhanced biological phosphorus removal

More than 50 years ago, Jeener and Brachet (1944) observed that addition of inorganic phosphate (Pi) to a suspension of yeast cells previously subjected to phosphate starvation induced massive accumulation of a basophilic substance within the cells. Soon afterwards this substance was isolated and identified as inorganic polyphosphate (polyP) (Schmidt et al.1946; Wiame 1948). This was by no means the first isolation of polyP from a microorganism. As early as 1888 Liebermann had obtained polyP from yeast, probably metaphosphate. Over the next 50 years hardly any paper on this polyP from yeast appeared. The work of Wiame and others marks the beginning of biological research on inorganic polyP.