PhyA from Selenomonas ruminantium (PhyAsr), is a bacterial protein tyrosine phosphatase (PTP)-like inositol polyphosphate phosphatase (IPPase) that is distantly related to known PTPs. PhyAsr has a second substrate binding site referred to as a standby site and the P-loop (HCX 5 R) has been observed in both open (inactive) and closed (active) conformations. Site-directed mutagenesis and kinetic and structural studies indicate PhyAsr follows a classical PTP mechanism of hydrolysis and has a broad specificity toward polyphosphorylated myo-inositol substrates, including phosphoinositides. Kinetic and molecular docking experiments demonstrate PhyAsr preferentially cleaves the 3-phosphate position of Ins P 6 and will produce Ins(2)P via a highly ordered series of sequential dephosphorylations: D-Ins(1,2,4,5,6)P 5 , Ins(2,4,5,6)P 4 , D-Ins(2,4,5)P 3 , and D-Ins(2,4)P 2 . The data support a distributive enzyme mechanism and suggest the PhyAsr standby site is involved in the recruitment of substrate. Structural studies at physiological pH and high salt concentrations demonstrate the ''closed'' or active P-loop conformation can be induced in the absence of substrate. These results suggest PhyAsr should be reclassified as a D-3 myo-inositol hexakisphosphate phosphohydrolase and suggest the PhyAsr reaction mechanism is more similar to that of PTPs than previously suspected.Keywords: inositol polyphosphate phosphatase; protein tyrosine phosphatase; phosphoinositide phosphatase; phytase; myo-inositol; P-loop; hydrolysis pathway Supplemental material: see www.proteinscience.org Protein tyrosine phosphatase (PTP) superfamily enzymes have been discovered in a range of prokaryotes, and most appear to serve roles that mimic their better-known eukaryotic counterparts as regulators of cellular function (Shi et al. 1998;Kennelly and Potts 1999). Some bacteria have adapted PTPs as ''molecular missiles,'' secreted into the infected host where they assist in the progression of infection (Bliska et al. 1991;Fu and Galan 1998;Bretz et al. 2003). The recently described PTP-like inositol polyphosphate phosphatase (IPPase) from Selenomonas ruminantium, PhyAsr, contains a PTP-like active site signature sequence (HCEAGVGR) but lacks significant primary sequence identity with known IPPases and PTPs (<20%). While its biological function is unclear, it is the first example of a PTP-like enzyme with activity toward myo-inositol hexakisphosphate (Ins P 6 , Fig. 1), one of the most abundant inositol polyphosphates (IPPs) in a majority of prokaryotic and eukaryotic cells (Sasakawa et al. 1995).The X-ray crystallographic structure of PhyAsr (Chu et al. 2004) reveals a PTP-like fold and several novel 4 These authors contributed equally to this work. Reprint requests to: Steven C. Mosimann, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada; e-mail: steven.mosimann@uleth.ca; fax: (403) 329-2057.Article published online ahead of print. Article and publication date are at http://www.proteinscien...

Food is a vulnerable target for potential bioterrorist attacks; therefore, a critical mitigation strategy is needed for the rapid concentration and detection of biothreat agents from food matrices. Magnetic beads offer a unique advantage in that they have a large surface area for efficient capture of bacteria. We have demonstrated the efficient capture and concentration of Bacillus anthracis (Sterne) spores using immunomagnetic beads for a potential food application. Magnetic beads from three different sources, with varying sizes and surface chemistries, were functionalized with monoclonal antibodies and polyclonal antibodies from commercial sources and used to capture and concentrate anthrax spores from spiked food matrices, including milk, apple juice, bagged salad, processed meat, and bottled water. The results indicated that the Pathatrix beads were more effective in the binding and capture of anthrax spores than the other two bead types investigated. Furthermore, it was observed that the use of polyclonal antibodies resulted in a more efficient recovery of anthrax spores than the use of monoclonal antibodies. Three different magnetic capture methods, inversion, the Pathatrix Auto system, and the new iCropTheBug system, were investigated. The iCropTheBug system yielded a much higher recovery of spores than the Pathatrix Auto system. Spore recoveries ranged from 80 to 100% for the iCropTheBug system when using pure spore preparations, whereas the Pathatrix Auto system had recoveries from 20 to 30%. Spore capture from food samples inoculated at a level of 1 CFU/ml resulted in 80 to 100% capture for milk, bottled water, and juice samples and 60 to 80% for processed meat and bagged salad when using the iCropTheBug system. This efficient capture of anthrax spores at very low concentrations without enrichment has the potential to enhance the sensitivity of downstream detection technologies and will be a useful method in a foodborne bioterrorism response.

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Aims: Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10-fold serial dilutions of Bacillus anthracis spores using quantitative real-time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6Á5 9 10 1 and 1Á3 9 10 2 CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). Methods and Results: The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 AE 14, 68 AE 8, 66 AE 4 and 52 AE 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. Conclusions: Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. Significance and Impact of the Study: The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.