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Structural Basis for Iron Binding and Release by a Novel Class of Periplasmic Iron-Binding Proteins Found in Gram-Negative Pathogens. Stephen R. Shouldice, 2004.We have determined the 1.35- and 1.45-Å structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A . M . haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever . The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host . The ferric (Fe3+) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion . The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion . Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm . Genetic Diversity of Escherichia coli Isolated from Urban Rivers and Beach Water. Sandra L. McLellan, 2004.Repetitive element anchored PCR was used to evaluate the genetic profiles of Escherichia coli isolated from surface water contaminated with urban stormwater, sanitary sewage, and gull feces to determine if strains found in environmental samples reflect the strain composition of E . coli obtained from host sources . Overall, there was less diversity in isolates collected from river and beach sites than with isolates obtained from human and nonhuman sources . Unique strain types comprised 28.8, 29.2, and 15.0% of the isolate data sets recovered from stormwater, river water, and beach water, respectively . In contrast, 50.4% of gull isolates and 41.2% of sewage isolates were unique strain types . River water, which is expected to contain E . coli strains from many diffuse sources of nonpoint source pollution, contained strains most closely associated with other river water isolates that were collected at different sites or on different days . However, river sites impacted by sewage discharge had approximately 20% more strains similar to sewage isolates than did sites impacted by stormwater alone . Beach sites with known gull fecal contamination contained E . coli most similar to other beach isolates rather than gull isolates collected at these same sites, indicating underrepresentation of possible gull strains . These results suggest large numbers of strains are needed to represent contributing host sources within a geographical location . Additionally, environmental survival may influence the composition of strains that can be recovered from contaminated waters . Understanding the ecology of indicator bacteria is important when interpreting fecal pollution assessments and developing source detection methodology . Identification of Peptides That Mimic the Pertussis Toxin Binding Site on Bovine Fetuin. John A. Bogdan, 2003.The introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough . Pertussis toxin (Ptx) is one component produced by Bordetella pertussis that is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids . A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B . Trollfors, J . Taranger, T . Lagergard, L . Lind, V . Sundh, G . Zackrisson, C . U . Lowe, W . Blackwelder, and J . B . Robbins, N . Engl . J . Med . 333:1045-1050, 1995) . The industrial production of Ptx can be performed through the cultivation of B . pertussis in well-defined growth media, in which the components can be well characterized and their origins can be documented . Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al . (R . D . Sekura, F . Fish, C . R . Manclark, B . Meade, and Y . L . Zhang, J . Biol . Chem . 258:14647-14651, 1983) . The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF . Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase . We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF . We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds . Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification . Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide .
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