Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system

Abstract Background Increasing our understanding of antibiotic resistance mechanisms is critical. To enable progress in this area, methods to rapidly identify and characterize antibiotic resistance conferring enzymes are required. Results We have constructed a sensitive reporter system in Escherichia coli that can be used to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, resulted in luminescence production as a result of expression of the lux genes carried by the reporter plasmid. Introduction of another plasmid into this system expressing TetX, a tetracycline-inactivating enzyme, caused a marked loss in luminescence due to enzyme-mediated reduction in the intracellular Tc concentration. Data generated for the TetX enzyme using the reporter system could be effectively fit with the known Km and kcat values, demonstrating the usefulness of this system for quantitative analyses. Conclusion Since members of the TetR family of repressors regulate enzymes and pumps acting upon almost every known antibiotic and a wide range of other small molecules, reporter systems with the same design as presented here, but employing heterologous TetR-related proteins, could be developed to measure enzymatic activities against a wide range of antibiotics and other compounds. Thus, the assay described here has far-reaching applicability and could be adapted for high-throughput applications

The CRISPR/Cas Adaptive Immune System of Pseudomonas aeruginosa Mediates Resistance to Naturally Occurring and Engineered Phages

Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function

The X-ray structure of 4-aminobenzyl alcohol (4-aminophenylmethanol)

A second polymorph of 4-aminobenzyl alcohol [orthorhombic, a = 8.95051(15), b = 5.8248(1), c = 12.1645(2) Å, space group Pna21] shows a "herringbone" structure with stacks of hydrogen-bonded molecules when viewed down the b-axis.Publisher PDFPeer reviewe

Revised Status of Rare and Endangered Unionaea (Mollusca: Margaritiferidae, Unionidae) in Arkansas

Harris and Gordon (1987) reviewed the distribution and status of 18 rare and /or endangered unionacean bivalve species (commonly referred to as clams, mussels, freshwater mussels, naiads) that occur or have occurred in Arkansas. They discussed four species that were federally listed as endangered, four species that were considered endangered or extirpated within Arkansas, four species considered threatened within Arkansas, four species of special concern within Arkansas, and two species for which the conservation status was considered uncertain due to questions regarding taxonomic validity. Numerous unionacean field surveys have been performed during 1986 1996, and a substantial database of new distributional and relative abundance information has been accumulated. Two additional unionacean species have been listed as federally endangered, one additional species has been listed as federally threatened, and one endangered species has been newly discovered within Arkansas bringing the total number of federally protected unionacean species occurring within Arkansas to eight. The conservation status of 16 additional unionacean species occurring in Arkansas is discussed also

Overexpression of Protein Kinase C βII Induces Colonic Hyperproliferation and Increased Sensitivity to Colon Carcinogenesis

Protein kinase C βII (PKC βII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC βII function in vivo, we generated transgenic mice that overexpress PKC βII in the intestinal epithelium. Transgenic PKC βII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC βII mice exhibit elevated colonic β-catenin levels and decreased glycogen synthase kinase 3β activity, indicating that PKC βII stimulates the Wnt/adenomatous polyposis coli (APC)/β-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC βII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/β-catenin signaling pathway

Growth Curves of Four Species of Commercially Valuable Freshwater Mussels (Bivalva: Unionidae)

North American freshwater mussels (Bivalvia: Unionidae) have been exploited commercially for over 100 years and have been regulated using shell size limits and/or harvest seasons. Presently, freshwater mussels are considered a threatened faunal group in North America due to the large numbers of endangered, threatened or special concern species. Therefore, management of this fauna should emphasize their long-term sustainability. The objectives of this study were 1) to construct von Bertalanffy growth curves for selected commercially-most-valuable species, Fusconaia ebena, Megalonaias nervosa, Amblema plicata and Quadrula quadrula, from five rivers and two reservoirs, 2) to compare species-specific von Bertalanffy growth curves from different rivers and reservoirs, and 3) to provide information on size at onset of sexual maturity in F. ebena and A. plicata. Von Bertalanffy growth curves of four commercially valuable Ambleminae species were used in this study to compare drainage-specific growth. Growth curves for all four species investigated were significantly different between pairs of drainages. Approximate size at onset of sexual maturity was determined for Arkansas F. ebena and A. plicata. Von Bertalanffy growth curves, coupled with life history and population dynamics information, could be useful in assessing and determining national/state harvest sizes and/or drainage specific harvest sizes once annual growth line formation is confirmed