Expression and purification of AlgX in Pseudomonas aeruginosa

Abstract only availablePseudomonas aeruginosa is a Gram-negative bacterium that is the leading cause of hospital acquired infections. While this bacteria is present in water and soil, this bacteria only severely affects severely ill patients, such as those with cystic fibrosis. In cystic fibrosis patients, the bacteria will lodge in the lungs and form a coating, called a biofilm, around itself to protect it from the natural defenses of the body and antibiotics. This biofilm is formed by exopolysacchride sugar, known as alginate. The goal of this project is to, by isolating some of the proteins that help produce the biofilm, find the three-dimensional structure of those proteins. This will make it easier for various pharmaceutical research companies to create a drug to inhibit the specific active site in the proteins, preventing biofilm formation and allowing the infection to be treated with traditional antibiotics. The proteins encoded by two genes were picked for this study, algK and algX. Both are believed good targets because they have been shown in previously written papers to be critical to formation of the biofilm in P. aeruginosa. (1, 2) The cloned genes (in the form of a plasmid) previously obtained were used in the transformation step into E. coli. The lab protocol for general transformations was followed. To show that the bacteria were actually producing the protein we needed, the growth tubes were harvested; the cells lysed, and run on a 10% acrylamide gel to check for protein expression in the experimental versus control sample. The algK gel was inconclusive, however algX clearly overexpresses in large quantities. While most of the protein is insoluble, enough is soluble to warrant complete purification of the protein to set up crystallization trays. The current problem is how to optimize purification, because it co-elutes from a nickel column with contaminating proteins. A slow concentration bump of the buffers used to purify is being tested to see if it helps with this problem. References 1. Antonette Robles-Price, Thiang Yian Wong, Havard Sletta, Svein Valla, Neal L. Schiller. 2004. AlgX Is a Periplasmic Protein Required for Alginate Biosynthesis in Pseudomonas aeruginosa. J. of Bacteriology. 186:7369-7377. 2. Jain, Sumita, Franklin, Michael J., Ertesvag, Helga, Valla, Svein, Ohman, Dennis E. 2003. The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa. Molecular Microbiology. 47. 1123-1133NSF-REU Program in Biological Sciences & Biochemistr

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Indirect estimation of injected mortar volume in historical walls using the electrical resistivity tomography

The electrical resistivity tomography (ERT) represents one of the widely used geophysical techniques for the exploration of the subsurface. In the last few years, this method has been demonstrated to be an efficient reconnaissance tool not only for monitoring degradation status of walls and foundations of historical buildings, but also for imaging the spatial distribution of injected mortar, commonly employed for consolidation purposes. A 3D resistivity tomography survey was carried out on four selected wall portions of the historical church of Montepetriolo, Perugia, Central Italy. The obtained 3D resistivity distribution models before and after grouting provided suggestive images of the internal structure of the studied walls. Moreover, the spatial distribution of the zones being filled with mortar was determined quantitatively by scaling the post- to the preinjection resistivity values. Using a well known correlation between resistivity and porosity, established in the geophysical community, a further step towards a more quantitative assessment was attempted to indirectly determine the unit volumes of the injected mortar The obtained results were satisfactory and in some cases almost similar to the yard data. Furthermore, two flat-jack tests confirmed the local increment of the mechanical resistance of the studied left front (2) and right lateral (3) wall portions

Crystal Structure of Bacillus anthracis Phosphoglucosamine Mutase, an Enzyme in the Peptidoglycan Biosynthetic Pathway ▿†

Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme that participates in the cytoplasmic steps of peptidoglycan biosynthesis. As peptidoglycan is essential for bacterial survival and is absent in humans, enzymes in this pathway have been the focus of intensive inhibitor design efforts. Many aspects of the structural biology of the peptidoglycan pathway have been elucidated, with the exception of the PNGM structure. We present here the crystal structure of PNGM from the human pathogen and bioterrorism agent Bacillus anthracis. The structure reveals key residues in the large active site cleft of the enzyme which likely have roles in catalysis and specificity. A large conformational change of the C-terminal domain of PNGM is observed when comparing two independent molecules in the crystal, shedding light on both the apo- and ligand-bound conformers of the enzyme. Crystal packing analyses and dynamic light scattering studies suggest that the enzyme is a dimer in solution. Multiple sequence alignments show that residues in the dimer interface are conserved, suggesting that many PNGM enzymes adopt this oligomeric state. This work lays the foundation for the development of inhibitors for PNGM enzymes from human pathogens

Effects of olive pomace amendment on soil enzyme activities

Agricultural soils generally have a low content of organic matter, which affects soil fertility and hence crop yields. The use of olive pomace as an organic amendment could enhance soil properties due to its high content of organic matter and exchangeable cations as well as its extensive availability. However, little is known about the potentially detrimental effects of salts and high content of phenolic substances that might occur over time with the use of pomace amendment in agricultural soils. Similarly, its impact on microbial communities and enzyme activities is largely unknown. To ascertain these effects, we tested the response of soil enzyme activities (β-glucosidase, urease, acid phosphomonoesterase and dehydrogenase) starting from a long-term (8-year) previously amended soil and a control (non-amended) soil and exposing these soils to a short-term (12-week) laboratory experiment. We also focused on soil organic matter and mineral N, on relating enzymatic data to bacterial and fungal biomasses, and on an index for soil functional quality based on enzyme activities (AI3). Our results show that organic matter, enzyme activities and the soil quality index were improved by long-term treatment with olive pomace, contrasting with scant effects from short-term laboratory incubation. In spite of its high content in salts and phenolic substances, no detectable negative impact emerged from the use of this amendment. Overall, olive pomace clearly increased soil organic matter and improved soil biological activity and quality, without any detectable negative effects

Organic carbon pools and storage in the soil of olive groves of different age

Summary: Compared with annual crop cultivation, tree groves might represent a relevant land-use system to improve C sequestration, but few data are available to support this hypothesis. To evaluate the potential of olive tree (Olea europaea L., 1753) cultivation to store soil organic C (SOC), we assessed (i) the distribution of organic C in active (water-extractable and particulate organic C, WEOC and POC, respectively), intermediate (organic matter associated with stable sand-size aggregates and silt- and clay-size aggregates, SSAs and SCAs, respectively) and passive (organic matter resistant to oxidation, rSOM) pools, (ii) the phenol content of the C pools, (iii) the humic-C distribution of the intermediate C pool and (iv) the stocks of SOC pools in two olive groves of different age (7 years (OG7) and 30 years (OG30)) compared with a nearby site with cereal crops (arable soil, AS). In OG30 the organic C stock of the olive grove was no different from that of the AS, but the distribution of SOC pools changed with the age of the olive groves. The WEOC and POC increased in the Ap horizon of OGs, probably because of the herbaceous cover and distribution of chipped prunings on the soil. There were fewer SSAs in OG7 than AS, possibly because of pedoturbations from deep tillage before the olive trees were established, but they increased in OG30. The increase in SSAs and SCAs in the Bw and BC horizons of OG30 was associated with humic-C and unextractable-C and a smaller phenol content than AS. This suggested that the olive tree roots had a positive role through rhizodeposition and root turnover, which favoured the stabilization of organic matter into aggregates at depth. In contrast to the active and intermediate C pools, the passive C pool did not vary following the change in land use from arable to olive grove. Highlights: Effects of land-use change from arable to olive grove on soil organic C pools and stocks. Soil organic C stock increased from 7- to 30-year-old olive orchard. Olive tree cultivation affected active and intermediate C pools, but not the passive C pool. After 30 years, the olive grove stored an amount of SOC similar to that of the arable system