Journal of Experimental Microbiology and Immunology (JEMI) Vol. 7:62-67 Copyright © April 2005, M&I UBC Roles of Lipopolysaccharide and the Outer Membrane Protein OmpC on Bacteriophage T4 Infectivity in Escherichia coli WILEY CHUNG, JOHN SIU, KATE TANNER Department of Microbiology and Immunology, UBC Although previous studies have demonstrated that lipopolysaccharide (LPS) and the outer membrane protein OmpC are required for bacteriophage T4 infectivity in Escherichia coli, it is unclear whether they played a role in binding or in the downstream viral processes that followed. Such processes may vary from phage DNA injection to viral gene expression, assembly, or release. To investigate this further, phage binding and overlay plaque assays were performed on E. coli strains C149 (wildtype), C845 (LPS mutant), C842 (LPS mutant), and C157 (OmpC mutant). In this study, we report that LPS and OmpC play an essential role in one or more viral processes that follow binding. In addition, it was discovered that LPS and OmpC mutants C842 and C157, respectively, did not alter phage binding affinity but decreased phage binding stability. Moreover, the LPS mutant, C845, increased binding affinity to T4 phage but decreased binding stability. All these results suggest that modifications to LPS can influence both the affinities and stabilities of phage binding. _________________________________________________________________ The T4 bacteriophage, a member of the T-even phage group, is an efficient infectious agent of Escherichia coli (E. coli) (4). Its structure consists of a DNA-containing head, a tail, as well as a baseplate which is attached to both short and long tail fibres (12). T4 phage employ a very complex entry mechanism, involving an ATP-dependent DNA injection process (7). Bacteriophages remain attached to the outer cell surface during infection and have evolved a mechanism to use their tail fibers for host cell recognition, attachment, and genome delivery (7). T4 phage contain six tail fibers which are symmetrically organized around a hexagonal baseplate (1,3). Initial attachment to a host cell receptor is reversible, weak, and dependent upon the interaction between at least three long tail fibers and a glucose residue on of the outer core of the lipopolysaccharide (LPS) of the host cell surface (1). Previous studies have shown that these binding interactions are weak and may vary from hydrogen bonding to hydrophobic and electrostatic interactions (4). After the tail fiber binding has been consolidated, the baseplate then comes in contact with the host cell surface, causing the short tail fibers to eject and bind irreversibly to the heptose section of the inner core of the LPS (1). This transition from reversible to irreversible binding involves a number of host and phage components (3,7). After this conformational change, the tail sheath contracts, causing the tail tube to be forced through the outer cell membrane of the host (1). The tail tube then reaches the inner membrane, aided by the tail lysozyme, glycoprotein 5. The phage genome is then injected into the host cytoplasm (1). It is important to note that the irreversibly bound state can exist in two phases, one where the phage DNA has not been internalized and one where it has. Following DNA injection, the host cell transcription and translation machinery is used to produce phage specific early mRNA and proteins (1). Eventually, late mRNA and proteins are produced (1). These proteins include phage structural proteins as well as proteins involved in cellular lysis (1). Ultimately, infectious phage particles are assembled and the host cell undergoes lysis and releases phage progeny (11). Previous studies have demonstrated that LPS and the outer membrane protein OmpC are required for bacteriophage T4 infectivity in E. coli (9,11,12). LPS is located exclusively in the outer leaflet of the outer membrane of gram negative bacteria (11). LPS exhibits features of both lipids and polysaccharides and its structure is divided into three regions: Lipid A, R- core, and O-polysaccharide (11). OmpC is an outer membrane protein that is involved in regulating the response of E. coli to the osmolarity of its environment (2). It is highly expressed during conditions of high osmolarity and repressed during conditions of low osmolarity (2). Particular studies found that E. coli K- 12 strains that contained a mutation in their OmpC or LPS structures were resistant to T4 phage infection (9,12). Although these studies have helped identify that both LPS and OmpC are required for infectivity, it was unclear whether these structures played a role in binding or in downstream viral processes that followed. Such processes may vary from phage DNA injection to viral gene expression, assembly, or even release. The purpose of this experiment was to determine whether LPS and OmpC are involved in the initial binding of phage or a viral process that followed 62