150 research outputs found

    Glycine Betaine Fluxes in Lactobacillus plantarum during Osmostasis and Hyper- and Hypo-osmotic Shock

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    Bacteria respond to changes in medium osmolarity by varying the concentrations of specific solutes in order to maintain constant turgor. The primary response of Lactobacillus plantarum to an osmotic upshock involves the accumulation of compatible solutes such as glycine betaine, proline, and glutamate. We have studied the osmotic regulation of glycine betaine transport in L. plantarum by measuring the overall and unidirectional rates of glycine betaine uptake and exit at osmostasis, and under conditions of osmotic upshock and downshock. At steady state conditions, a basal flux of glycine betaine (but no net uptake or efflux) is observed that amounts to about 20% of the rate of “activated” uptake (uptake at high osmolarity). No direct exchange of 14C-labeled glycine betaine in the medium for unlabeled glycine betaine in the cytoplasm was observed in glucose metabolizing and resting cells, indicating that a separate glycine betaine efflux system is responsible for the exit of glycine betaine. Upon osmotic upshock, the uptake system for glycine betaine is rapidly activated (within seconds), whereas the basal efflux is inhibited. These two responses account for a rapid accumulation of glycine betaine until osmostasis is reached. Upon osmotic downshock, glycine betaine is rapidly released by the cells in a process that has two kinetic components, i.e. one with a half-life of less than 2 s which is unaffected by the metabolic status of the cells, the other with a half-life of 4–5 min in glucose-metabolizing cells which is dependent on internal pH or a related parameter. We speculate that the former activity corresponds to a stretch-activated channel, whereas the latter may be facilitated by a carrier protein. Glycine betaine uptake is strongly inhibited immediately after an osmotic downshock, but slowly recovers in time. These studies demonstrate that in L. plantarum osmostasis is maintained through positive and negative regulation of both glycine betaine uptake and efflux, of which activation of uptake upon osmotic upshock and activation of a “channel-like” activity upon osmotic downshock are quantitatively most important.

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    Nucleotide-Binding Sites of the Heterodimeric LmrCD ABC-Multidrug Transporter of Lactococcus lactis Are Asymmetric

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    LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[α-32P]ATP, which, at 4 °C, occurs predominantly at LmrC, while aluminiumfluoride (AlFx)-induced trapping of the hydrolyzed nucleotide at 30 °C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.

    Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of Lactobacillus plantarum

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    The accumulation of quaternary ammonium compounds in Lactobacillus plantarum is mediated via a single transport system with a high affinity for glycine betaine (apparent K(m) of 18 μM) and carnitine and a low affinity for proline (apparent K(m) of 950 μM) and other analogues. Mutants defective in the uptake of glycine betaine were generated by UV irradiation and selected on the basis of resistance to dehydroproline (DHP), a toxic proline analogue. Three independent DHP-resistant mutants showed reduced glycine betaine uptake rates and accumulation levels but behaved similarly to the wild type in terms of direct activation of uptake by high-osmolality conditions. Kinetic analysis of glycine betaine uptake and efflux in the wild-type and mutant cells is consistent with one uptake system for quaternary ammonium compounds in L. plantarum and a separate system(s) for their excretion. The mechanism of osmotic activation of the quaternary ammonium compound transport system (QacT) was studied. It was observed that the uptake rates were inhibited by the presence of internal substrate. Upon raising of the medium osmolality, the QacT system was rapidly activated (increase in maximal velocity) through a diminished inhibition by trans substrate as well as an effect that is independent of intracellular substrate. We also studied the effects of the cationic amphipath chlorpromazine, which inserts into the cytoplasmic membrane and thereby influences the uptake and efflux of glycine betaine. The results provide further evidence for the notion that the rapid efflux of glycine betaine upon osmotic downshock is mediated by a channel protein that is responding to membrane stretch or tension. The activation of QacT upon osmotic upshock seems to be brought about by a turgor-related parameter other than membrane stretch or tension
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