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Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7. G. O'Flynn, 2004.Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura . This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E . coli O157:H7 . Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E . coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro . Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C . However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge . All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology . The frequency of BIM formation (10–6 CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10–4 CFU) . In addition, BIMs commonly reverted to phage sensitivity within 50 generations . In an initial meat trial experiment, the phage cocktail completely eliminated E . coli O157:H7 from the beef meat surface in seven of nine cases . Given that the frequency of BIM formation is low (10–6 CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment . The Crystal Structure of Zn(II)-Free Treponema pallidum TroA, a Periplasmic Metal-Binding Protein, Reveals a Closed Conformation. Yong-Hwan Lee, 2002.We previously demonstrated that Treponema pallidum TroA is a periplasmic metal-binding protein (MBP) with a distinctive alpha-helical backbone . To better understand the mechanisms of metal binding and release by TroA, we determined the crystal structure of the apoprotein at a resolution of 2.5 Å and compared it to that of the Zn(II)-bound form (Protein Data Bank accession code 1toa) . apo-TroA shows a conformation even more closed than that of its Zn(II)-bound counterpart due to a 4° tilt of the C-terminal domain (residues 190 through 308) about an axis parallel to the poorly flexible backbone helix . This domain tilting pushes two loops (residues 248 through 253 and 277 through 286) towards the metal-binding site by more than 1 Å, resulting in an unfavorable interaction of I251 with D66 . To avoid this contact, D66 shifts towards H68, one of the four Zn(II)-coordinating residues . The approach of this negative charge coincides with the flipping of the imidazole side chain of H68, resulting in the formation of a new hydrogen bond . The conformational change of H68, along with a slight rearrangement of D279, a C-terminal domain Zn(II)-coordinating residue, distorts the metal-binding site geometry, presumably causing the release of the bound metal ion . Ligand binding and release by TroA, and presumably by other members of the MBP cluster, differs from the "Venus flytrap" mechanism utilized by bacterial nonmetal solute-binding receptors . Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core. Peter P. Sheridan, 2003.The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates . We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9°C for over 100,000 years . Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 107 cells/ml . Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2°C . Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries . Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically . Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found . Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species . Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification . One sequence was over 12% distant from its closest relative and may represent a novel order or family . These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years . Description of a "Phoenix" Phenomenon in the Growth of Campylobacter jejuni at Temperatures Close to the Minimum for Growth. A. F. Kelly, 2003.When Campylobacter jejuni cultures that had been grown in broth at 39°C were subcultured into fresh medium at 30°C, there was a transient period of growth followed by a decline in viable-cell numbers before growth resumed once more . We propose that this complex behavior is the net effect of the growth of inoculum cells followed by a loss of viability due to oxidative stress and the subsequent emergence of a spontaneously arising mutant population that takes over the culture .
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