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Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus. Trygve Brautaset, 2004.Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of50°C . With the exception of mannitol, no sugars have beenreported to support rapid growth of this organism, which isclassified as a restrictive methylotroph . Here we describe theDNA sequence and characterization of a 19,167-bp circular plasmid,designated pBM19, isolated from B . methanolicus MGA3 . Sequenceanalysis of pBM19 demonstrated the presence of the methanoldehydrogenase gene, mdh, which is crucial for methanol consumptionin this bacterium . In addition, five genes [pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase] with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19 . A shuttle vector, pTB1.9, harboringthe pBM19 minimal replicon [repB and ori] was constructed and used to transform MGA3 . Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not ableto grow on methanol . A pTB1.9 derivative harboring the completemdh gene could not restore growth on methanol when it was introducedinto the pBM19-cured strain, suggesting that additional pBM19genes are required for consumption of this carbon source . Screeningof 13 thermotolerant B . methanolicus wild-type strains showedthat they all harbor plasmids similar to pBM19, and this isthe first report describing plasmid-linked methylotrophy inany microorganism . Our findings should have an effect on futuregenetic manipulations of this organism, and they contributeto a new understanding of the biology of methylotrophs. Assessment of Methods for Detection of Infectious Cryptosporidium Oocysts and Giardia Cysts in Reclaimed Effluents. W. Quintero-Betancourt, 2003.This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used . The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions . (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested . Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method . Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability . The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential . Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively . The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation .
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