Isolation, Characterisation and Complete Genome Sequence of a Tequatrovirus Phage, Escherichia phage KIT03, Which Simultaneously Infects Escherichia coli O157:H7 and Salmonella enterica.

Escherichia coli O157:H7 and Salmonella enterica subsp. enterica are the pathogens that frequently cause foodborne illness. Bacteriophage applications have been proposed as effective for preventing food contamination caused by these pathogenic bacteria. Escherichia phage KIT03 was isolated from the soil of a poultry farm in Kyoto, Japan. KIT03 can infect Escherichia coli O157:H7 and Salmonella enterica serotypes Choleraesuis and Enteritidis. One-step growth analysis revealed that KIT03 can propagate within its initial host (E. coli NBRC 3972), E. coli O157:H7 and S. Choleraesuis with an approximate burst size of 39, 51 and 37 phage particles per infected cell, respectively. The morphological type and genome annotation suggested that KIT03 belongs to the family Myoviridae, subfamily Tevenvirinae, genus Tequatrovirus. In vitro challenge tests demonstrated that KIT03 can lyse the tested bacteria and suppress their growth. Based on the susceptibility test and adsorption assay of KIT03 with E. coli K-12 BW25113 mutants, it was proposed that KIT03 may recognise and infect bacteria with a deficient outer core of lipopolysaccharides.

Sequential processing from cell lysis to protein assay on a chip enabling the optimization of an F(1)-ATPase single molecule assay condition.

We developed an integrated protein assay device, "Single Molecule MicroTAS (SMM)," which enables cell lysis, protein extraction, purification, and activity assay. The assay was achieved at the single-molecule scale for a genetically engineered protein, F(1)-ATPase, which is the smallest known rotary motor. A cell lysis condition, with a wide range of applied voltages (50-250 V) and other optimized values (pulse width: 50 micros; duty: 0.01%; electrode gap: 25 microm; total flow rate: 5 microL min(-1)) provided a high enough protein concentration for the assay. Successively, the protein was extracted and purified by specific binding in a microfluidic channel. During the assay process, the diffusion effect of lysate between a two-phase laminar flow contributes to optimizing the single-molecule assay condition, because the concentration of the original lysate from the E. coli solution is too high to assay. To achieve the most efficient assay condition, the protein diffusion effect on the assay was experimentally and numerically evaluated. The results reveal that, in our experimental conditions, concentrations of F(1) and other contaminated effluents are optimized for the F(1) rotational assay at a channel position. The adenosine triphosphate (ATP)-driven rotation speed measured in the SMM was compatible with that obtained by conventional purification and assay. Such a sequential process from cell lysis to assay proves that the SMM is an example of a sample-in-answer-out system for F(1) protein evaluation.