Browsing by Author "Xu, Bu"
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- Detection of Salmonella spp. Using a Generic and Differential FRET-PCRZhang, Jilei; Wei, Lanjing; Kelly, Patrick; Freeman, Mark; Jaegerson, Kirsten; Gong, Jiansen; Xu, Bu; Pan, Zhiming; Xu, Chuanling; Wang, Chengming (PLOS, 2013-10-16)To facilitate the detection of Salmonella and to be able to rapidly and conveniently determine the species/subspecies present, we developed and tested a generic and differential FRET-PCR targeting their tetrathionate reductase response regulator gene. The differential pan-Salmonella FRET-PCR we developed successfully detected seven plasmids that contained partial sequences of S. bongori and the six S. enterica subspecies. The detection limit varied from ∼5 copies of target gene/per PCR reaction for S. enterica enterica to ∼200 for S. bongori. Melting curve analysis demonstrated a Tm of ∼68°C for S. enterica enterica, ∼62.5°C for S. enterica houtenae and S. enterica diarizonae, ∼57°C for S. enterica indica, and ∼54°C for S. bongori, S. enterica salamae and S. enterica arizonae. The differential pan-Salmonella FRET-PCR also detected and determined the subspecies of 4 reference strains and 47 Salmonella isolated from clinically ill birds or pigs. Finally, we found it could directly detect and differentiate Salmonella in feline (5/50 positive; 10%; one S. enterica salamae and 4 S. enterica enterica) and canine feces (15/114 positive; 13.2%; all S. enterica enterica). The differential pan-Salmonella FRET-PCR failed to react with 96 non-Salmonella bacterial strains. Our experiments show the differential pan-Salmonella FRET-PCR we developed is a rapid, sensitive and specific method to detect and differentiate Salmonella.
- Structural and Mutational Analyses of Aspergillus fumigatus SidA: A Flavin-Dependent N-hydroxylating EnzymeFedkenheuer, Michael Gerald (Virginia Tech, 2012-07-27)SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector.