Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump

The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump-component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled

The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 Å resolution

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 Å resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane -barrel domain in a fashion that may mimic proteinlipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export

Anomalous diffusion with absorption: Exact time-dependent solutions

Recently, analytical solutions of a nonlinear Fokker-Planck equation describing anomalous diffusion with an external linear force were found using a non extensive thermostatistical Ansatz. We have extended these solutions to the case when an homogeneous absorption process is also present. Some peculiar aspects of the interrelation between the deterministic force, the nonlinear diffusion and the absorption process are discussed.Comment: RevTex, 16 pgs, 4 figures. Accepted in Physical Review

Nonextensivity of the cyclic Lattice Lotka Volterra model

We numerically show that the Lattice Lotka-Volterra model, when realized on a square lattice support, gives rise to a {\it finite} production, per unit time, of the nonextensive entropy $S_q= \frac{1- \sum_ip_i^q}{q-1}$ $(S_1=-\sum_i p_i \ln p_i)$. This finiteness only occurs for $q=0.5$ for the $d=2$ growth mode (growing droplet), and for $q=0$ for the $d=1$ one (growing stripe). This strong evidence of nonextensivity is consistent with the spontaneous emergence of local domains of identical particles with fractal boundaries and competing interactions. Such direct evidence is for the first time exhibited for a many-body system which, at the mean field level, is conservative.Comment: Latex, 6 pages, 5 figure

Magneto-transport and magnetic susceptibility of SmFeAsO1-xFx (x = 0.0 and 0.20)

Bulk polycrystalline samples, SmFeAsO and the iso-structural superconducting SmFeAsO0.80F0.20 are explored through resistivity with temperature under magnetic field {\rho}(T, H), AC and DC magnetization (M-T), and Specific heat (Cp) measurements. The Resistivity measurement shows superconductivity for x = 0.20 sample with Tc(onset) ~ 51.7K. The upper critical field, [Hc2(0)] is estimated ~3770kOe by Ginzburg-Landau (GL) theory. Broadening of superconducting transition in magneto transport is studied through thermally activated flux flow in applied field up to 130 kOe. The flux flow activation energy (U/kB) is estimated ~1215K for 1kOe field. Magnetic measurements exhibited bulk superconductivity with lower critical field (Hc1) of ~1.2kOe at 2K. In normal state, the paramagnetic nature of compound confirms no trace of magnetic impurity which orders ferromagnetically. AC susceptibility measurements have been carried out for SmFeAsO0.80F0.20 sample at various amplitude and frequencies of applied AC drive field. The inter-granular critical current density (Jc) is estimated. Specific heat [Cp(T)] measurement showed an anomaly at around 140K due to the SDW ordering of Fe, followed by another peak at 5K corresponding to the antiferromagnetic (AFM) ordering of Sm+3 ions in SmFeAsO compound. Interestingly the change in entropy (marked by the Cp transition height) at 5K for Sm+3 AFM ordering is heavily reduced in case of superconducting SmFeAsO0.80F0.20 sample.Comment: 18 pages text + Figs: comments/suggestions welcome ([email protected]