Download A Continuation of the Analysis of the Host Range

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.