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Genetics of Metabolic Variations between Yersinia pestis Biovars and the Proposal of a New Biovar, microtus. Dongsheng Zhou, 2004.Yersinia pestis has been historically divided into three biovars: antiqua, mediaevalis, and orientalis . On the basis of this study, strains from Microtus-related plague foci are proposed to constitute a new biovar, microtus . Based on the ability to ferment glycerol and arabinose and to reduce nitrate, Y . pestis strains can be assigned to one of four biovars: antiqua (glycerol positive, arabinose positive, and nitrate positive), mediaevalis (glycerol positive, arabinose positive, and nitrate negative), orientalis (glycerol negative, arabinose positive, and nitrate positive), and microtus (glycerol positive, arabinose negative, and nitrate negative) . A 93-bp in-frame deletion in glpD gene results in the glycerol-negative characteristic of biovar orientalis strains . Two kinds of point mutations in the napA gene may cause the nitrate reduction-negative characteristic in biovars mediaevalis and microtus, respectively . A 122-bp frameshift deletion in the araC gene may lead to the arabinose-negative phenotype of biovar microtus strains . Biovar microtus strains have a unique genomic profile of gene loss and pseudogene distribution, which most likely accounts for the human attenuation of this new biovar . Focused, hypothesis-based investigations on these specific genes will help delineate the determinants that enable this deadly pathogen to be virulent to humans and give insight into the evolution of Y . pestis and plague pathogenesis . Moreover, there may be the implications for development of biovar microtus strains as a potential vaccine . Patterns of Amphotericin B Killing Kinetics against Seven Candida Species. Emilia Cantón, 2004.In a previous study tolerance to amphotericin B (AMB) was found among Candida parapsilosis and C . dubliniensis strains by seeding the whole volumes of wells used for MIC determinations, and minimum fungicidal concentrations (MFC) for non-C . albicans Candida strains were demonstrated to be above the levels safely achievable in serum . As an extension of that study, we performed time-kill assays with 26 blood culture isolates (6 C . albicans, 5 C . parapsilosis, 5 C . krusei, 4 C . glabrata, 3 C . lusitaniae, and 3 C . tropicalis isolates), 3 oropharyngeal C . dubliniensis isolates, 3 AMB-susceptible isolates (ATCC 90028, ATCC 22019, ATCC 6254), and 6 AMB-resistant isolates (ATCC 200955, ATCC 200956, ATCC 200950, ATCC 200951, ATCC 200952, ATCC 200953) using RPMI 1640 medium and 0.12 to 32 µg of AMB per ml and determined the numbers of CFU per milliliter at 0, 2, 4, 8, 12, 24, and 48 h . MFCs and time-kill patterns were species specific (MFCs,  1 µg/ml for all C . dubliniensis and C . albicans isolates except AMB-resistant strain ATCC 200955; MFCs, 2 to >16 µg/ml for the other isolates) . The times required to reach the fungicidal endpoint (99.9% killing) at four times the MIC were 2 h for C . albicans and C . dubliniensis, 16 h for C . glabrata, 24 h for C . parapsilosis and C . lusitaniae, and
 40 h for C . tropicalis and C . krusei . The killing rate increased as the AMB concentration was increased up to 2 µg/ml . The highest killing rates were achieved for C . albicans, C . dubliniensis, and C . lusitaniae, while viable C . tropicalis, C . krusei, and C . parapsilosis cells were present after 48 h (MICs,
2 µg/ml) when AMB was used at 2 µg/ml . Time-kill curves and MFCs can detect viable cells after 48 h when AMB is used at
2 µg/ml . The failure of AMB treatment could be due to its poor killing activity against some species at the concentrations reached in patients' serum .
Identification of Two prpDBC Gene Clusters in Corynebacterium glutamicum and Their Involvement in Propionate Degradation via the 2-Methylcitrate Cycle. Wilfried A. Claes, 2002.Genome sequencing revealed that the Corynebacterium glutamicum genome contained, besides gltA, two additional citrate synthase homologous genes (prpC) located in two different prpDBC gene clusters, which were designated prpD1B1C1 and prpD2B2C2 . The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80% . Significant sequence similarities were found also to the prpBCDE operons of Escherichia coli and Salmonella enterica, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway . Homologous and heterologous overexpression of the C . glutamicum prpC1 and prpC2 genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases . Growth tests showed that C . glutamicum used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days . Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in C . glutamicum the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway . Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the C . glutamicum prpD2B2C2 genes by propionate as an additional carbon source . Mutational analyses revealed that only the prpD2B2C2 genes were essential for the growth of C . glutamicum on propionate as a sole carbon source, while the function of the prpD1B1C1 genes remains obscure . Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism. Jeff J. Engebretson, 2003.An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences . T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability . The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated . In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences . Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100) . Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs . Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs . It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range . The latter constraint was found to be more significant for estimating restriction endonuclease fidelity . Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities . All restriction endonucleases used in this study detected  70% of the OTUs at richness values greater than 50 OTUs per modeled community . Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation) .
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