A device for the high-pressure oxygenation of protein crystals

A system has been developed for subjecting protein crystals to hyperbaric pressures of oxygen gas in order to promote enzymatic reaction. Crystals of an oxygenase or oxidase enzyme are grown anaerobically by hanging drop vapor diffusion, under crystallization conditions modified to eliminate combustible materials such as plastic coverslips and grease. The crystalline enzyme:substrate complex can then be exposed to oxygen gas at pressures up to 60 bar using a custom-built device or "bomb." In this way, reaction is initiated synchronously throughout the crystal and subsequent flash freezing allows the trapping of enzyme:product complexes in high occupancy. These complexes can then be structurally characterized by conventional monochromatic X-ray crystallography. The bomb is furnished from naval brass and lubricated with Fomblin RT15 perfluorinated polyether grease in order to ensure compatibility with the highly oxidizing environment.     

Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay

We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.     

Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway

The initial step in the biosynthesis of the clinically important beta-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N2-(2-carboxyethyl)arginine, a beta-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-A resolution. The structure was solved in two space groups, P2(1)2(1)2(1) and P2(1)2(1)2. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a "V" conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2. The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative beta-amino acids.     

Bile-Mediated Aminoglycoside Sensitivity in Lactobacillus Species Likely Results from Increased Membrane Permeability Attributable to Cholic Acid

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 μg/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-³H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.     

First examination of sterols in the marine dinoflagellate genus Vulcanodinium

Vulcanodinium is an ecologically relevant dinoflagellate genus due to its production of neurotoxins known as pinnatoxins. We present here the first examination of the sterols of a Vulcanodinium rugosum isolate. Sterols are ringed lipids that assist in maintaining rigidity of cellular membranes, and the Dinophyceae are well-studied for their ability to produce a diverse array of sterols, many of which have chemotaxonomic utility. We have determined that V. rugosum produces a set of major sterols, namely cholesterol, dinosterol, 4α,24-dimethyl-5α-cholest-22E-en-3β-ol, and 4α,24-dimethyl-5α-cholestan-3β-ol, common to the Dinophyceae. However, this displayed marked differences from those studied members of the genera Scrippsiella and Peridinium, the closest phylogenetic relatives. Included in these differences is production by V. rugosum of a much lower percentage of dinostanol, a saturated form of dinosterol.     

Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant

Aims:  To isolate environmental bacteria capable of transforming fluoroquinolones to inactive molecules.
Methods and Results:  Bacteria were isolated from the aerobic liquor of a wastewater treatment plant on a medium containing norfloxacin (100 mg l⁻¹). Twenty-two isolates were highly resistant (minimal inhibitory concentration: 6·25−200 μg ml⁻¹) to five fluoroquinolones and six of them were positive by PCR amplification for the aminoglycoside resistance gene aac(6')-Ib. Of these, only Escherichia coli strain LR09 had the ciprofloxacin-acetylating variant gene aac(6')-Ib-cr ; HPLC and mass spectrometry showed that this strain transformed both ciprofloxacin and norfloxacin by N -acetylation. This bacterium also had mutations in the quinolone-resistance determining regions of the gyrA and parC genes.
Conclusions:  An E. coli isolate from wastewater, which possessed at least two distinct fluoroquinolone resistance mechanisms, inactivated ciprofloxacin and norfloxacin by N -acetylation.
Significance and Impact of the Study:  This is the first report of N -acetylation of fluoroquinolones by an aac(6')-Ib-cr -containing bacterium from an environmental source.     

Effect of Catalase on Hydrogen Peroxide Penetration into Pseudomonas aeruginosa Biofilms

The penetration of hydrogen peroxide into biofilms formed by wild-type and catalase-deficient Pseudomonas aeruginosa strains was measured using microelectrodes. A flowing stream of hydrogen peroxide (50 mM, 1 h) was unable to penetrate or kill wild-type biofilms but did penetrate and partially kill biofilms formed by an isogenic strain in which the katA gene was knocked out. Catalase protects aggregated bacteria by preventing full penetration of hydrogen peroxide into the biofilm.     

Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting

Fasciclin I is a membrane-associated glycoprotein that is regionally expressed on a subset of fasciculating axons during neuronal development in insects; it is expressed on apposing cell surfaces, suggesting a role in specific cell adhesion. In this paper we show that Drosophila fasciclin I is a novel homophilic cell adhesion molecule. When the nonadhesive Drosophila S2 cells are transfected with the fasciclin I cDNA, they form aggregates that are blocked by antisera against fasciclin I. When cells expressing fasciclin I are mixed with cells expressing fasciclin III, another Drosophila homophilic adhesion molecule, the mixture sorts into aggregates homogeneous for either fasciclin I- or fasciclin III-expressing cells. The ability of these two novel adhesion molecules to mediate cell sorting in vitro suggests that they might play a similar role during neuronal development.     

Development of Functional and Molecular Correlates of Vaccine-Induced Protection for a Model Intracellular Pathogen,F. tularensis LVS

In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes'' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rβ2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.     

Diversity of Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Inpatients of 30 Hospitals in Orange County,California

There is a need for a regional assessment of the frequency and diversity of MRSA to determine major circulating clones and the extent to which community and healthcare MRSA reservoirs have mixed. We conducted a prospective cohort study of inpatients in Orange County, California, systematically collecting clinical MRSA isolates from 30 hospitals, to assess MRSA diversity and distribution. All isolates were characterized by spa typing, with selective PFGE and MLST to relate spa types with major MRSA clones. We collected 2,246 MRSA isolates from hospital inpatients. This translated to 91/10,000 inpatients with MRSA and an Orange County population estimate of MRSA inpatient clinical cultures of 86/100,000 people. spa type genetic diversity was heterogeneous between hospitals, and relatively high overall (72%). USA300 (t008/ST8), USA100 (t002/ST5) and a previously reported USA100 variant (t242/ST5) were the dominant clones across all Orange County hospitals, representing 83% of isolates. Fifteen hospitals isolated more t008 (USA300) isolates than t002/242 (USA100) isolates, and 12 hospitals isolated more t242 isolates than t002 isolates. The majority of isolates were imported into hospitals. Community-based infection control strategies may still be helpful in stemming the influx of traditionally community-associated strains, particularly USA300, into the healthcare setting.