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Use of Semiconductor Quantum Dots for Photostable Immunofluorescence Labeling of Cryptosporidium parvum. Lai Yoke Lee, 2004.Cryptosporidium parvum is a waterborne pathogen that poses potential risk to drinking water consumers . The detection of Cryptosporidium oocysts, its transmissive stage, is used in the latest U.S . Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-Cryptosporidium sp . monoclonal antibody (MAb) . However, FITC exhibits low resistance to photodegradation . This property will inevitably limit the detection accuracy after a short period of continuous illumination . In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection . In this study, QD605-streptavidin together with biotinylated MAb was used for C . parvum oocyst detection . The C . parvum oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above . The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 ± 501 oocysts (mean ± standard deviation) was determined to be 20 nM . The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (P > 0.126) when various C . parvum oocyst concentrations were used . The QD605 was highly photostable while the FITC intensity decreased to 19.5% ± 5.6% of its initial intensity after 5 min of continuous illumination . The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water . This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting C . parvum oocysts with the advantage of having a high photobleaching threshold . Novel Type of ADP-Forming Acetyl Coenzyme A Synthetase in Hyperthermophilic Archaea: Heterologous Expression and Characterization of Isoenzymes from the Sulfate Reducer Archaeoglobus fulgidus and the Methanogen Methanococcus jannaschii. Meike Musfeldt, 2002.Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + Pi  acetate + ATP + CoA) in Archaea and eukaryotic protists . The only characterized ACD in archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145-kDa heterotetramers ( 2, ß2) . The coding genes for the
and ß subunits are located at different sites in the P . furiosus chromosome . Based on significant sequence similarity of the P . furiosus genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus and one ORF was identified in the hyperthermophilic methanogen Methanococcus jannaschii . The ORFs constitute fusions of the homologous P . furiosus genes encoding the
and ß subunits . Two ORFs, AF1211 and AF1938, of A . fulgidus and ORF MJ0590 of M . jannaschii were cloned and functionally overexpressed in Escherichia coli . The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities . In contrast to the Pyrococcus ACD, the ACDs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous P . furiosus
and ß subunits in an
ß (AF1211 and MJ0590) or ß (AF1938) orientation . The data indicate that A . fulgidus and M . jannaschii contains a novel type of ADP-forming acetyl-CoA synthetase in Archaea, in which the subunit polypeptides and their coding genes are fused .
The Phosphate Starvation Stimulon of Corynebacterium glutamicum Determined by DNA Microarray Analyses. Takeru Ishige, 2003.The phosphate (Pi) starvation stimulon of Corynebacterium glutamicum was characterized by global gene expression analysis by using DNA microarrays . Hierarchical cluster analysis of the genes showing altered expression 10 to 180 min after a shift from Pi-sufficient to Pi-limiting conditions led to identification of five groups comprising 92 genes . Four of these groups included genes which are not directly involved in P metabolism and changed expression presumably due to the reduced growth rate observed after the shift or to the exchange of medium . One group, however, comprised 25 genes, most of which are obviously related to phosphorus (P) uptake and metabolism and exhibited 4- to >30-fold-greater expression after the shift to Pi limitation . Among these genes, the RNA levels of the pstSCAB (ABC-type Pi uptake system), glpQ (glycerophosphoryldiester phosphodiesterase), ugpAEBC (ABC-type sn-glycerol 3-phosphate uptake system), phoH (unknown function), nucH (extracellular nuclease), and Cgl0328 (5'-nucleotidase or related esterase) genes were increased, and pstSCAB exhibited a faster response than the other genes . Transcriptional fusion analyses revealed that elevated expression of pstSCAB and ugpAEBC was primarily due to transcriptional regulation . Several genes also involved in P uptake and metabolism were not affected by Pi starvation; these included the genes encoding a PitA-like Pi uptake system and a putative Na+-dependent Pi transporter and the genes involved in the metabolism of pyrophosphate and polyphosphate . In summary, a global, time-resolved picture of the response of C . glutamicum to Pi starvation was obtained . Bacteria Associated with Cysts of the Soybean Cyst Nematode (Heterodera glycines). Sarah M. Nour, 2003.The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world . The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops . To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means . Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst . Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 ± 0.5) x 105 bacteria (mean ± standard deviation) with various cellular morphologies . Filamentous fungi were also observed . Live-dead staining indicated that the majority of cyst bacteria were viable . The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature . Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition . Bands shared by all eight samples included the actinobacterium genera Actinomadura and Streptomyces . A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium . These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence . Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp . This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents . Single-Cell Enumeration of an Uncultivated TM7 Subgroup in the Human Subgingival Crevice. Cleber C. Ouverney, 2003.Specific oligonucleotide hybridization conditions were established for single-cell enumeration of uncultivated TM7 and IO25 bacteria by using clones expressing heterologous 16S rRNA . In situ analysis of human subgingival crevice specimens revealed that a greater proportion of samples from sites of chronic periodontitis than from healthy sites contained TM7 subgroup IO25 . In addition, IO25 bacterial cells from periodontitis site samples were more abundant and fourfold longer than IO25 cells from healthy site samples .
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