FIG 2.

Counterselection of E. coli hemA dapA donor on BHIB agar. (A) Schematics depicting the reactions catalyzed by HemA (left side) and DapA (right side), enzymes required for the biosynthesis of heme and peptidoglycan, respectively. Complementation of growth medium with the metabolic intermediates depicted in red is used to support the growth of hemA and dapA mutants. (B) Growth of wild-type S. glossinidius, E. coli hemA (MP1182), and E. coli dapA (BW29427) on BHIB agar lacking or containing DAP. Plates were photographed after 8 days of incubation at 27°C under microaerophilic conditions. Red arrows indicate residual growth. (C) Growth of wild-type E. coli (MG1655) and S. glossinidius following 120 min of incubation, at room temperature, in 10 mM MgCl₂ or 10 mM MgCl₂ containing phage T7. Cell suspensions were diluted, and 5 μl was spotted on plates. Escherichia coli was incubated at 37°C on LB for 16 h. Sodalis glossinidius was incubated under microaerophilic conditions at 27°C on BHIB for 8 days. (D) Growth of E. coli dapA hemA (MP1554) on BHIB agar with various combinations of ALA and DAP. Cells were incubated at room temperature for 120 min in 10 mM MgCl₂ or 10 mM MgCl₂ containing phage T7. Cultures were diluted, and 5 μl was plated on BHIB agar. Plates were incubated at 37°C for 16 h. Red arrows indicate residual growth. (E) Growth of S. glossinidius and E. coli dapA hemA (MP1554) on BHIB agar lacking ALA and DAP. Bacteria were grown separately on plates in a mock conjugation experiment, subsequently exposed to phage T7, washed, diluted, and spotted on BHIB as described for panel C. Plates were incubated for 8 days at 27°C under microaerophilic conditions. Images depict representative plates of at least 3 independent experiments.