Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.

Clean food contact surfaces are important in reducing the likelihood of foodborne disease transmission. The goal of this study was to assess and compare baseline cleanliness of food contact and environmental surfaces in retail food establishments by using ATP bioluminescence (ATP-B), visual assessment, and surface contact plates. Four hundred eighty-nine surface samples were collected from three food service establishments at the University of Minnesota-Twin Cities (Minneapolis) and analyzed for either ATP (252) or total aerobic plate count bacteria (237). ATP levels ranged from a minimum of 4 relative light units (RLU; 0.60 log RLU) on a clean slicer to a maximum of 506,618 RLU (5.77 log RLU) on a dirty cutting board. The overall mean was 1,950 RLU (3.29 log RLU). Cutting boards had the highest ATP levels (mean, 5,495 RLU or 3.74 log RLU; median, 6,761 RLU or 3.83 log RLU). Of the 128 samples judged visually clean at the time of sampling, 70.3 % failed ATP-B testing. Sixty-one (26 % ) of the 237 total aerobic plate count samples yielded counts of over 125 CFU/50 cm(2) (failed), and of those that failed, 40 % were assessed as visually clean before sampling. The highest average counts in CFU/50 cm(2) were found on slicers (104) and cutting boards (87). The results of this study suggest that the current practice of evaluating food contact surface cleanliness by sight and touch to meet regulatory requirements might be inadequate. ATP-B testing may be an efficient tool to facilitate creation, implementation, and validation of more effective food contact surface cleaning in food establishments.

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The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-kappaB (nuclear factor kappaB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).

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