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

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.

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

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.

Kinetic Equations for Longwavelength Excitations of the Quark-Gluon Plasma

We show that longwavelength excitations of the quark-gluon plasma are described by simple kinetic equations which represent the exact equations of motion at leading order in $g$. Properties of the so-called ``hard thermal loops'', i.e. the dominant contributions to amplitudes with soft external lines, find in this approach a natural explanation. In particular, their generating functional appears here as the effective action describing long wavelength excitations of the plasma.Comment: January 8, 1993; 8 pages; SPhT/93-

A transition research perspective on governance for sustainability

In this paper we present the transition approach as an integrated perspective to understand and possibly orient our society towards sustainable development. Transition management is based upon complex adaptive system thinking and seeks to deal with ongoing changes in society in an evolutionary manner so as to influence these ongoing changes in terms of speed and direction: towards sustainability. Since the concept of sustainability is inherently normative, subjective and ambiguous, we argue that (unlike some more traditional approaches to sustainable development) we should focus on an open facilitation and stimulation of social processes towards sustainability. Transition perspective poses novel challenges for research: there are no unequivocal answers, nor is it clear how these processes should be governed. We thus end this paper by formulating the basic research questions central to the search for governance for sustainability

Improving police integrity in Uganda: Impact assessment of the police accountability and reform project

Uganda and in particular the Ugandan police are perceived as highly corrupt. To address the integrity of police officers, an intervention called the Police Accountability and Reform Project (PARP) was implemented in selected police districts between 2010 and early 2013. This paper studies the impact of PARP for a sample of 600 police officers who were interviewed about police integrity by means of 12 hypothetical vignette cases depicting context-specific, undesirable behavior of varying degrees of severity. The assessments of the cases by the police officers are analyzed using propensity score matching, inverse probability weighting, and seemingly unrelated regression techniques. We show that the self-selection of police officers into the program is unlikely to drive the results. The results suggest that officers participating in PARP activities (1) judge the presented cases of misconduct more severely, (2) are more inclined to report misconduct, and (3) also expect their colleagues to judge misbehavior at the police level more critically although the latter two coefficient estimates are smaller in size. This suggests that PARP activities have affected the perception of police officers but only encouraged them moderately to actually take action against bad practices

Energetics and Mechanism of Drug Transport Mediated by the Lactococcal Multidrug Transporter LmrP

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli. LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Δψ) and the transmembrane proton gradient (ΔpH), pointing to an electrogenic drug/proton antiport mechanism. Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-sideout membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a “hydrophobic vacuum cleaner” mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA.