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A Continuation of the Analysis of the Host Range Determinants of the P2 Related Phage Wphi James Kokorelis, Dr. Louis Temple, Dr. Gail Christie 08 June 2006 Microbial evolution is largely perpetuated by bacteriophages (phages) for they facilitate in the transferring of genetic material from one bacterium to another. Some phages, temperate phages, enter the bacteria causing relatively negligible harm. These phages integrate their genetic material into the chromosomal DNA of the host bacterium1 Although these phages can integrate their DNA, they are however restricted to their host range- the selective array of cellular organisms that a virus is capable of infecting. Viruses may encompass host ranges that include multiple species, though usually the more closely related the host speciesare , the more likely that they will share a given virus2. I am specifically working with the temperate bacteriophage Wphi, which was isolated from an E. coli strain W. Wphi has prominent genetic and morphological similarities to the well characterized temperate phage P2. However, despite these similarities, there is a notable difference in whether or not the phage will grow when plated on various E. coli. Testing was then done by growing Wphi on various E. coli strains. It appeared that the phage would plate on some restriction-minus E. coli K-12 strains, but not on others. Genotypic analysis of these strains revealed that one common attribute of those that failed to plate on Wphi was the presence of a mutation in a gene named rfbD (a specific rfb gene in the O antigen cluster). This mutation in the rfbD gene is purported to alter the structure of the host LPS which is recognized by many phages like P1, P2 and Mu as their specialized receptor. Thus, confirmation of this hypothesis will require testing Wphi growth on a pair of otherwise isogenic E. coli K-12 strains, one with the rfbD mutation and one without. Andrew Archer showed that phage absorption specificity is generally controlled by the phage tail fibers. In previous studies, Wphi growth on E. coli B strains is due to a defect in adsorption and not to immunity or to exclusion as a result of the cryptic P2related prophage present in E. coli B. In conjunction, spontaneous mutants of Wphi that will plate on E. coli B were isolated. Sequence analysis of the tail fiber gene of one of these mutants revealed four amino acid changes near the C-terminal end. It is likely that these changes confirm the adsorption specificity of the mutant phage. Over the 2005 academic year, progress was made in quantifying the plating efficiencies, titers, of phages P2, 186, PSP3, Wphi and the Wphi host range mutant on E. coli C, B, W and several K12 strains. It was seen that P2 and Wphi host range mutant were able to infect all of the aforementioned E. coli strains; whereas, Wphi was limited to C-1A, RR1, and W3850. This indicates that further analysis into the genetic differences between the H gene of P2 and W phi is warranted. My research is now narrowed into two related aims: 1.confirming the role of E. coli gene rfbD in phage Wphi host range, 2. confirming the role of C-terminal amino acids in the phage tail fiber gene in determining host range specificity. The first aim involves testing phage plating on a pair of otherwise isogenic strains, one with the rfbD mutation and one without. This pair of strains was successfully constructed for the study. PCR primers were designed to amplify and sequence the rfbD gene from the mutant strain. Primers were then designed to detect the rfbD1 mutation. The mutation lacks any sort of easily measurable phenotype. This will be used to confirm the identity of the allele in the isogenic pair which will be constructed by P1 transduction using a closely linked TCR marker. For the second aim, a complementation will be then used to confirm the role of C-terminal amino acids in the tail fiber gene. The H gene which codes for the tail fibers was amplified using PCR after primers were designed. The product was purified then cut with EcoR1 and Bgl11. The cut H gene is to be inserted into the vector puHE242, a plasmid chosen for it contains a site that codes for Ampacillin resistance and has a highly active transcription initiator signal. The plasmid has been isolated and cut; progress is underway to ligate the H gene into the plasmid. When the H protein is provided on a plasmid, phage particles resulting should be able to infect E. coli B, through direct use of H protein or recombination of the H gene into the phage genome.