Journal articles

Three mutants of Lactococcus lactis subsp. lactis MG1363, termed EthR, DauR, and RhoR, were selected for resistance to high concentrations of ethidium bromide, daunomycin, and rhodamine 6G, respectively. These mutants were found to be cross resistant to a number of structurally and functionally unrelated drugs, among which were typical substrates of the mammalian multidrug transporter (P-glycoprotein) such as daunomycin, quinine, actinomycin D, gramicidin D, and rhodamine 6G.

Recent research on the process of biological phosphorus removal in lab-scale treatment systems has indicated that: (i) the development of an actively polyP-accumulating bacterial community after the introduction of an anaerobic period may take at least 4 months; (ii) up to 80% of all aerobic bacteria isolated from these communities are able to accumulate polyP; (iii) polyP synthesized by the bacterial communities of lab-scale treatment systems is probably mainly low polymeric, not exceeding 20 P-residues, and this polyP is rapidly degraded during the anaerobic period; (iv) the enzymatic hyd

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.

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.

Recent crystal structures of the multidrug ATP-binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification.

The MexAB-OprM drug efflux pump is central to multidrug resistance of Pseudomonas aeruginosa. The ability of the tripartite protein to confer drug resistance on the pathogen is crucially dependent on the presence of all three proteins of the complex. However, the role of each protein in the formation of the intact functional complex is not well understood. One of the key questions relates to the (in)ability of MexB to act independently of its cognitive partners, MexA and OprM.

The multidrug major facilitator superfamily transporter LmrP from Lactococcus lactis mediates protonmotive-force dependent efflux of amphiphilic ligands from the cell. We compared the role of membrane-embedded carboxylates in transport and binding of divalent cationic propidium and monovalent cationic ethidium. D235N, E327Q, and D142N replacements each resulted in loss of electrogenicity in the propidium efflux reaction, pointing to electrogenic 3H(+)/propidium(2+) antiport.

Multidrug ABC transporters can transport a wide range of drugs from the cell. Ongoing studies of the prototype mammalian multidrug resistance ATP-binding cassette transporter P-glycoprotein (ABCB1) have revealed many intriguing functional and biochemical features. However, a gap remains in our knowledge regarding the molecular basis of its broad specificity for structurally unrelated ligands.

BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.