Abstract
Bacterial viruses (bacteriophages) recognize their host cells with the help of specialized Receptor-Binding
Proteins (RBPs) that emanate from the ‘tail’, a host attachment organelle of the phage. RBPs are either long and
slender fibers devoid of enzymatic activity or shorter and stockier tailspikes that can digest or modify
polysaccharide molecules that extend from or cover the surface of a bacterial cell. Upon host attachment, the
tail creates a conduit between the phage capsid and the host cell cytoplasm allowing phage DNA and proteins
to be delivered into the cell. Several aspects of this process, especially those that concern the transition from
the initial recognition event to irreversible attachment, remain poorly understood. As components of the phage
particle, tailspike RBPs are required for both the initial recognition and irreversible attachment. However, isolated
tailspike RBPs destroy the cell surface receptor and make the cell resistant to the phage carrying those RBPs.
Furthermore, tail fiber RBPs bind to the host cell weakly, but this binding triggers a conformational change in the
particle committing it to irreversible attachment. Our goal is to describe this transition and the associated
structural transformation of the virus particle for bacteriophage G7C, a virus that infects Escherichia coli and
Shigella dysenteriae. G7C has a short tail, 24 tailspike RBPs of two different types, and contains several large
proteins inside the capsid. In Aim 1, we will examine the role of different domains of the two tailspike RBPs in
host cell recognition and attachment. We will measure the energy of binding of G7C RBPs to their O-antigen
substrates. We will also establish the number of RBPs per particle required for infection. We will develop a
protocol for fluorescence/phase contrast imaging and computer processing of attachment of the phage to the
host cell in a single cell and ensemble modes. In Aim 2, we will examine the structural transformation of the
phage particle upon irreversible attachment with the help of cryo-electron microscopy, cryo-electron tomography,
and X-ray crystallography. In Aim 3, we will identify the outer membrane receptor for G7C that causes opening
of the tail channel and DNA release and examine the structure of G7C bound to that receptor. In summary, the
overarching goal of this proposal is to quantitively describe how bacteriophages commit themselves to
irreversible attachment, what kind of factors are involved, what is the source of energy that activates the particle
for irreversible attachment. The results of the proposed work will lay a foundation for quantitative description of
attachment in other phages and, possibly, in other viruses.