| Summary: | 博士 === 國立陽明大學 === 微生物暨免疫學研究所 === 87 === AR1, a coliphage whose morphology and some essential genes are similar to those of T4 phage, infects Escherichia coli O157:H7 with high specificity. We characterized its two tail fiber proteins gp37 and gp38, and investigated host factors that confer this infection specificity. The host range determinants of T4-like phages and T2-like phages are gp37 and gp38, respectively. It is known that g37 of AR1 does not share similarity with that of any T-even phages except for the 5'' region coding for the first 56 amino acid residues. In the phage particles, gp38 could be detected in the T2-like phages, but not in the T4-like phages. Using specific antibodies, we demonstrated that gp38 was also detected in AR1 particle, a result supporting the previous genetic data showing that g38 is distantly related to that of T2-like phages, particularly in the glycine-rich coding regions. Therefore, we concluded that molecularly AR1 is closer to T2 than T4.
We also demonstrated that the outer membrane porin OmpC of E. coli O157:H7 is an important factor that determines the AR1 specificity. OmpC of E. coli O157:H7 was shown to differ from the counterpart of E. coli K-12 that is impermissive to the AR1 infection. A 7% variation of the two OmpC molecules may partly explain the adverse host differences in the AR1 susceptibilities. We deduced this phage-host interaction by (1) transposon mutagenesis to select hosts with potentially critical gene(s) mutated; (2) complementation in the Tn-mutants with plasmids that expresses the putative candidate; (3) generation of knockout mutants by specific gene replacement and further confirming the gene-phenotype relationship using the complementation assay.
In addition to OmpC, a gene of waaJ homolog was demonstrated to be important for supporting the infection of AR1. The gene product of waaJ is suggested to be a glucosyltransferase involved in the biosynthesis of R3 type LPS. So far, the LPS type of E. coli O157:H7 has not been characterized. The presence of a waaJ homolog suggests that the LPS organization of E. coli O157:H7 may belong to the R3 type. Furthermore, disturbing the expression of waaJ interfered with the synthesis of OmpC and consequently resulted in the loss of susceptibility to AR1. LPS per se may also contribute to a minor supporting factor of the AR1 infection as mutation affecting the integrity of LPS decreased slightly the plaque forming efficiency of AR1.
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