OmpA and OmpC are critical host factors for bacteriophage Sf6 entry in Shigella.

Single step growth curves S. flexneri (WT, ompA - , ompC - , and ompA - C - ) were grown in LB at 30 °C to a concentration of 2×10 8 cells per mL. Phage infection was initiated at an MOI of 15 for the WT, ompA -, and ompC - strains, and an MOI of 30 was used for the ompA - C - strain. At times ranging from zero to three hours after infection was initiated, aliquots of infected cells were diluted with phage buffer containing saturating amounts of chloroform to lyse the cells. Lysates were plated and the number of phage per cell at each time point was calculated as described previously ( Parent et al., 2004 ). Aliquots of the cells were assayed before infection and ten minutes post infection to ensure that >95% of the cells were infected, and therefore burst size determination was not affected by a second round of infected cells. These experiments were repeated in triplicate and representative plots are shown in Figure 2A . Adsorption rates of Sf6 to Shigella Initial Sf6 adsorption to Shigella was monitored by adding phage to cells at high MOI, taking aliquots at various times (ranging from zero to 20 minutes at 30 °C), spinning out the cells and attached phage for 30 seconds in a microfuge, and titering the supernatant. The concentration of phage was held constant in each experiment (∼1 × 10 9 phage per mL). We also accurately determined the cell concentration for each sample by plating and counting CFUs before the addition of phage. The fit of these data was done using Kaleidagraph software and only data sets that demonstrated linear kinetics were included in further analysis.

Fluorescence microscopy Fluorescent

Sf6 were made by incubating a high-titer lysate (>10 9 PFU/ml) with 1 μM of Sytox green nucleic acid stain at 4°C for 24 hours. Overnight cultures of Shigella flexneri (WT, ompA - , ompC - , and ompA - C - ) were diluted in LB and grown to mid-log at 30°C with aeration. Cells (1 mL) were pelleted with 1 minute of centrifugation and resuspended in approximately 50μL of fresh LB. Five μL of a DNase I mixture (two units DNase I (New England Biolabs), in 1× supplied DNase I buffer, in ddH 2 O) were added to the cell re-suspension. A five μL aliquot of the cell suspension was mixed with one μL of Sytox-stained phage (at an MOI