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Novel, Rapid, and Inexpensive Cell-Based Quantification of Antimalarial Drug Efficacy. Tyler N. Bennett, 2004.We report on the development of a new SYBR Green I-based plate assay for analyzing the activities of antimalarial drugs against intraerythrocytic Plasmodium falciparum . This assay is considerably faster, less labor-intensive, and less expensive than conventional radiotracer (e.g., [3H]hypoxanthine and [3H]ethanolamine)-based assays or P . falciparum lactate dehydrogenase activity-based assays . The assay significantly improves the pace at which antimalarial drug discovery efforts may proceed . Localization and Characterization of Two Novel Genes Encoding Stereospecific Dioxygenases Catalyzing 2(2,4-Dichlorophenoxy)propionate Cleavage in Delftia acidovorans MC1. Kathleen M. Schleinitz, 2004.Two novel genes, rdpA and sdpA, encoding the enantiospecific  -ketoglutarate dependent dioxygenases catalyzing R,S-dichlorprop cleavage in Delftia acidovorans MC1 were identified . Significant similarities to other known genes were not detected, but their deduced amino acid sequences were similar to those of other
-ketoglutarate dioxygenases . RdpA showed 35% identity with TauD of Pseudomonas aeruginosa, and SdpA showed 37% identity with TfdA of Ralstonia eutropha JMP134 . The functionally important amino acid sequence motif HX(D/E)X23-26(T/S)X114-183HX10-13R/K, which is highly conserved in group II
-ketoglutarate-dependent dioxygenases, was present in both dichlorprop-cleaving enzymes . Transposon mutagenesis of rdpA inactivated R-dichlorprop cleavage, indicating that it was a single-copy gene . Both rdpA and sdpA were located on the plasmid pMC1 that also carries the lower pathway genes . Sequencing of a 25.8-kb fragment showed that the dioxygenase genes were separated by a 13.6-kb region mainly comprising a Tn501-like transposon . Furthermore, two copies of a sequence similar to IS91-like elements were identified . Hybridization studies comparing the wild-type plasmid and that of the mutant unable to cleave dichlorprop showed that rdpA and sdpA were deleted, whereas the lower pathway genes were unaffected, and that deletion may be caused by genetic rearrangements of the IS91-like elements . Two other dichlorprop-degrading bacterial strains, Rhodoferax sp . strain P230 and Sphingobium herbicidovorans MH, were shown to carry rdpA genes of high similarity to rdpA from strain MC1, but sdpA was not detected . This suggested that rdpA gene products are involved in the degradation of R-dichlorprop in these strains .
The Spectrum of Spontaneous Rifampin Resistance Mutations in the rpoB Gene of Bacillus subtilis 168 Spores Differs from That of Vegetative Cells and Resembles That of Mycobacterium tuberculosis. Wayne L. Nicholson, 2002.Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the ß subunit of RNA polymerase . In this study, we demonstrated that spores of B . subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells . In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB, H482R, from vegetative cells . Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L . The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis. The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells . Synthesis of a Klebsiella pneumoniae O-Antigen Heteropolysaccharide (O12) Requires an ABC 2 Transporter. Luis Izquierdo, 2003.A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K . pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5  . A total of eight open reading frames (ORFs) (wbO12 gene cluster) were necessary to produce K . pneumoniae O12-antigen LPS in E . coli K-12 . A complete analysis of the K . pneumoniae wbO12 cluster revealed an interesting coincidence with the wbO4 cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA) . This prompted us to generate mutants of K . pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S . marcescens N28b (O4) obtained previously . Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides . The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity . However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen . The K . pneumoniae O12 WbbL protein performs the same function as WbbL from S . marcescens O4 in either the S . marcescens O4 or E . coli K-12 genetic background .
Computer Simulation of Clostridium botulinum Strain 56A Behavior at Low Spore Concentrations. L. Zhao, 2003.It is generally assumed that spore behavior is independent of spore concentration, but recently published mathematical models indicate that this is not the case . A Monte Carlo simulation was employed in this study to further examine the independence assumption by evaluating the inherent variance in spore germination data . All simulations were carried out with @Risk software . A total of 500 to 4,000 iterations were needed for each simulation to reach convergence . Lag time and doubling time from a higher inoculum concentration were used to simulate the time to detection (TTD) at a lower inoculum concentration under otherwise identical environmental conditions . The point summaries of the simulated and observed TTDs were recorded for the 26 simulations, with kinetic data at the target inoculum concentration . The ratios of the median (Rm = medianobs/mediansim) and 90% range (Rr = 90% rangeobs/90% rangesim) were calculated . Most Rm and Rr values were greater than one, indicating that the simulated TTDs were smaller and more homogeneous than the observed ones . Rr values departed farther from one than Rm values . Ratios obtained when simulating 1 spore with 10,000 spores deviated the farthest from one . Neither ratio was significantly different from the other when simulating 1 spore with 100 spores or simulating 100 spores with 10,000 spores . When kinetic data were not available, the percent positive observed at the 95th percentile of the simulated TTDs was obtained . These simulation results confirmed that the assumption of independence between spores is not valid .
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