Small-angle neutron scattering (SANS) has been used to extend the structural characterization of the MS2 phage by examining its physical characteristics in solution. Specifically, the contrast variation technique was employed to determine the molecular weight of the individual components of the MS2 virion (protein shell and genomic RNA) and the spatial relationship of the genomic RNA to its protein shell. A consequence of this work was to evaluate a novel particle counting instrument, the integrated virus detection system (IVDS) that, in combination with SANS, has the potential to provide rapid quantitative physical characterization of unidentified viruses and phage.
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Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (≤1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified asphnE and glpT. The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E. coliK-12 phnE gene that has been shown previously to result inphnE activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation. PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with theglpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnEand multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnEgene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-p-toluidine (X-Pi). This result indicates that in E. coli, X-Pi is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems.
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ABSTRACTThis study demonstrates the characterization of the sample matrix composition of the MS2 virus using ESI-MS and IVDS detection systems. The MS2 samples were grown and purified using various techniques, which showed ESI-MS samples responding differently from IVDS samples. The LC-MS of the specific biomarker of the MS2 bacteriophage from an infected Escherichia coli (E. coli) sample was characterized in the presence of E. coli proteins. The significant impact of the sample matrix was observed upon the identification of MS2 using a database search. Escherichia coli infected with MS2 showed a different score from other uninfected scores. Only purified MS2, using CsCl and analyzed by LS-MS, showed a positive match in the database search. However, the variation in the MS2 sample matrix had no effect on the characterization of MS2.
This study was conducted to investigate the biodegradation potential of neutralized organophosphorus nerve agents: O-ethyl-S-(2-diisopropyl-aminoethyl) methylphosphonothioate (VX), Sarin (GB), Soman (GD), and O-cyclohexyl methylphosphonofluoridate (GF). Removing labile leaving groups from these compounds can be accomplished chemically (e.g. alkali) or enzymatically (by a variety of hydrolases) resulting in stable ionic methylphosphonate esters. Glyphosate utilizing Burkholderia caryophilli PG2982 was found to use for growth (as the sole phosphorus sources) low concentrations of ethyl-, isopropyl-, and pinacolyl methylphosphonates (EMPn, IMPn, and PMPn-alkali treatment products of VX, GB, and GD, respectively). Partially purified enzyme was obtained from crude extracts of the B. caryophilli PG2982 strain and tested for esterase activity against these phosphonate esters in addition to isobutyl-and cyclohexyl methylphosphonates (iBMPn and CMPn-alkali treatment products of Russian-VX and GF, respectively). Derivatized substrates and products were monitored by GC-FPD. Esterase activities were observed in the order: iBMPn>CMPn>EMPn>IMPn>PMPn. These results demonstrate the potential use of B. caryophilli derived enzyme(s) in furthering the destruction of these neurotoxic chemical compounds. This could be an important factor for the US and other nations in attempting to meet the requirements of the 1993 Chemical Weapons Convention agreement to destroy all chemical warfare agents within ten years of ratification (April 2007 for the US).
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