The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (K(d)) for ligand binding, number of binding-sites, transport affinities (K(m)), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4-5 d.

The ATP-binding cassette (ABC) transporter GlnPQ is an essential uptake system for amino acids in gram-positive pathogens and related nonpathogenic bacteria. The transporter has tandem substrate-binding domains (SBDs) fused to each transmembrane domain, giving rise to four SBDs per functional transporter complex. We have determined the crystal structures and ligand-binding properties of the SBDs of GlnPQ from Enterococcus faecalis, Streptococcus pneumoniae, and Lactococcus lactis. The tandem SBDs differ in substrate specificity and affinity, allowing cells to efficiently accumulate different amino acids via a single ABC transporter. The combined structural, functional, and thermodynamic analysis revealed the roles of individual residues in determining the substrate affinity. We succeeded in converting a low-affinity SBD into a high-affinity receptor and vice versa. Our data indicate that a small number of residues that reside in the binding pocket constitute the major affinity determinants of the SBDs.

Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3,3-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (⌬). Wild-type cells, grown aerobically in the presence of heme, generated a ⌬ even in the presence of the F 1 -F o ATPase inhibitor N,N-dicyclohexylcarbodiimide, while a cytochrome bd-negative mutant strain (CydA⌬) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydA⌬ cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.Lactococcus lactis has a long history of use in the production of fermented dairy products, such as cheese and buttermilk, under mainly anaerobic conditions. Studies on the aerobic growth of L. lactis have therefore been focused mainly on the effect of oxygen on fermentation patterns (25) or cell damage due to the formation of reactive oxygen species (3,8,32).These damaging effects of oxygen on L. lactis cells are not observed when cells are grown in the presence of both oxygen and a heme source (9,30,45). Aerated, heme-grown L. lactis cells display new characteristics such as increased growth yield, resistance to oxidative and acid stress, and improved long-term survival when stored at low temperatures (40). These traits are important for industrial applications, and the use of heme to increase the efficiency of biomass production of starter cultures has been described previously (10,13,37). The increased growth efficiency of aerated heme-grown L. lactis cells is due to a shift from homolactic to mixed-acid fermentation, more complete glucose utilization in non-pH-controlled batch cultures, and possibly energy generation by NADH oxidation via the electron transfer chain (ETC) (9). The ability to generate metabolic energy via NADH oxidation by the ETC will be the subject of this work. Increased growth efficiency will make L. lactis more useful as a cell factory for the production of biomass-related compounds such as proteins and vitamins.Heme is an essential cofactor of cytochrome complexes in the electron transport chains of respiring cells (14, 52). Furthermore, the genomes of several L. lactis strains contain genes which, when expressed, could form a simple ETC if supplied with heme (13). Genes encoding menaquinone biosynthesis enzymes and a bd-type cytochrome (mena)quinoloxidase have, for example, been identified in the genomes of strains IL-1403 and SK11 (http://genome.ornl.gov/microbial/lcre/) (6). The (me...

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine ␤-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mah