Identification and modeling of a novel chloramphenicol resistance protein detected by functional metagenomics in a wetland of Lerma, Mexico

The exploration of novel antibiotic resistance determinants in a particular environment may be limited because of the presence of uncultured microorganisms. In this work, a culture independent approach based on functional metagenomics was applied to search for chloramphenicol resistance genes in agro-industrial wastewater in Lerma de Villada, Mexico. To this end, a metagenomic library was generated in Escherichia coli DH10B containing DNA isolated from environmental samples of the residual arsenic-enriched (10 mg/ml) effl uent. One resistant clone was detected in this library and further analyzed. An open reading frame similar to a multidrug resistance protein from Aeromonas salmonicida and responsible for chloramphenicol resistance was identifi ed, sequenced, and found to encode a member of the major facilitator superfamily (MFS). Our results also showed that the expression of this gene restored streptomycin sensitivity in E. coli DH10B cells. To gain further insight into the phenotype of this MFS family member, we developed a model of the membrane protein multiporter that, in addition, may serve as a template for developing new antibiotics. [Int Microbiol 2013; 16(2):103-111]Keywords: Escherichia coli; chloramphenicol; functional metagenomics; major facilitator superfamily; homology models; membrane proteins; arseni

Structure-Function Relationships of the Follicle-Stimulating Hormone Receptor

The follicle-stimulating hormone receptor (FSHR) plays a crucial role in reproduction. This structurally complex receptor is a member of the G-protein coupled receptor (GPCR) superfamily of membrane receptors. As with the other structurally similar glycoprotein hormone receptors (the thyroid-stimulating hormone and luteinizing hormone-chorionic gonadotropin hormone receptors), the FSHR is characterized by an extensive extracellular domain, where binding to FSH occurs, linked to the signal specificity subdomain or hinge region. This region is involved in ligand-stimulated receptor activation whereas the seven transmembrane domain is associated with receptor activation and transmission of the activation process to the intracellular loops comprised of amino acid sequences, which predicate coupling to effectors, interaction with adapter proteins, and triggering of downstream intracellular signaling. In this review, we describe the most important structural features of the FSHR intimately involved in regulation of FSHR function, including trafficking, dimerization, and oligomerization, ligand binding, agonist-stimulated activation, and signal transduction

RMSD matrix analysis (gray scale) for the TM helices of LHCGR.

(A) replicate R1, (B) replicate R2, (C) replicate R3, and (D) trajectory in POPC. Red solid lines along the diagonal were drawn for visual identification of self-similar groups. The cluster analysis is shown in the insets. Conformations in clusters include structures within 0.5 Å of RMSD among each other. The cluster number is identified in the vertical axis. Horizontal segments along the curve identify the time frames forming a given cluster.</p

Structural alignment between FSHR-AF2 (cyan structure) and the FSHR-TM (orange structure); for reference, Cα of cysteine residues are depicted as spheres in yellow and in blue for the FSHR-TM and FSHR-AF2, respectively. The open intracellular conformation suggests that the models corresponds to the activated conformation.

Structural alignment between FSHR-AF2 (cyan structure) and the FSHR-TM (orange structure); for reference, Cα of cysteine residues are depicted as spheres in yellow and in blue for the FSHR-TM and FSHR-AF2, respectively. The open intracellular conformation suggests that the models corresponds to the activated conformation.</p

Stability of the membrane bilayer in replicates R1-R3 for the FSHR and LHCGR systems.

Stable averages were calculated after 50 nanoseconds for box area (xy-dimensions) and box height (z-dimension), both parameters important for monitoring the equilibration of the membrane bilayer according to the area per lipid and hydrophobic height in SDPC lipid molecules. (TIF)</p

Principal component (PC) analysis in the FSHR.

Projections on PC1-PC2 (A-C) and PC3-PC4 (D-F) of trajectories R1-3 (blue dots). Projections for groups 3405 in R1, 1171 in R2, and 0733 in R3 are included (black dots). The groups conform subclusters of conformational states whose RMSD difference in the TM domain region is within a.5 Å cutoff.</p

Projection of the PC1 on the 3D of the P10 loop and the hinge α-helix in FSHR (orange structure).

Color code: R1-yellow, R2-white, R3-purple. The HR domain for each replicate is shown for visualization of the amplitde and direction of the motion. (MPG)</p

Mass distribution as function of the bilayer normal.

Water and SDPC functional groups were identified in both, FSHR (top panel) and LHCGR (bottom panel) systems. Calculation were performed for the last 40 ns of trajectory for replicates R1-R3. Distributions are normalized to the number of atoms/Å3. (TIF)</p

Average of kink angles at TM helix 6.

Calculation for the last 164 ns of the R1-R3 trajectories in FSHR and LHCGR. Standard deviations calculated employing the blocking average method. (DOCX)</p

Fig 12 -

Distances among LRR, HR and TM domains of the FSHR (Å). Blue dots, group 3406 of R1; magenta dots, group 1171 of R2; and black dots, group 0733 of R3.</p