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Yop Fusions to Tightly Folded Protein Domains and Their Effects on Yersinia enterocolitica Type III Secretion. Vincent T. Lee, 2002.Yersinia enterocolitica organisms secrete Yop proteins via the type III pathway . Translational fusion of yop genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted . The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops . Pinpointing Biphenyl Dioxygenase Residues That Are Crucial for Substrate Interaction. Marco Zielinski, 2003.Three regions of the biphenyl dioxygenase (BDO) of Burkholderia sp . strain LB400 have previously been shown to significantly influence the interaction between enzyme and substrates at the active site . For a further discrimination within these regions, we investigated the effects of 23 individual amino acid exchanges . The regiospecificity of substrate dioxygenation was used as a sensitive means to monitor changes in the steric-electronic structure of the active site . Replacements of residues that, according to a model of the BDO three-dimensional structure, directly interact with substrates in most, but not all, cases (Met231, Phe378, and Phe384) very strongly altered this parameter (by factors of >7) . On the other hand, a number of amino acids (Ile243, Ile326, Phe332, Pro334, and Trp392) which have no contacts with substrates also strongly changed the site preference of dioxygenation (by factors of between 2.6 and 3.5) . This demonstrates that residues which had not been predicted to be influential can play a pivotal role in BDO specificity . Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis. Raphael Ber, 2003.Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis . We report the development of a new selective agar medium (termed BIN) that supports the growth of Y . pestis . The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain . High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth . Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y . pestis EV76 cultures in different agar compositions . The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced . It was found that BIN agar is more efficient in supporting colony formation and recovery of Y . pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar) . The advantage of BIN over other media has been also demonstrated in recovering virulent Y . pestis from the mixed bacterial populations found in decaying carcasses of infected mice . The BIN medium is suggested as a selective medium for isolation and recovery of Y . pestis from various backgrounds .
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