Fig. 2.

PBP1a* variants bypass the need for LpoA for function in vivo. (A). Overnight cultures of MM20 [∆lpoA P_ara::ponB] containing the plasmids pJLB16 [P_lac::ponA], pJLB20 [P_lac::ponA(G62A)], pJLB21 [P_lac::ponA(G62S)], pJLB22 [P_lac::ponA(L146I)], pJLB25 [P_lac::ponA(W494G)], or pJLB29 [P_lac::ponA(T38P)] were grown in M9-arabinose-Cam medium at 30 °C. Cells were then pelleted, washed once in M9 salts, and resuspended in M9 salts to an OD₆₀₀ of 1.0. The cell suspensions were then serially diluted, and 5 μL of each dilution was spotted onto M9-arabinose-Cam agar or LB-Cam agar containing IPTG, as indicated. Plates were incubated at 30 °C overnight prior to imaging. Note that we use M9 arabinose as our permissive condition because we find that the P_ara::ponB construct is expressed better in this medium than in LB. (B) Overnight cultures of MM20 [∆lpoA P_ara::ponB], JLB128 [∆lpoA ponA(G62S) P_ara::ponB], and JLB127 [∆lpoA ponA(T38P) P_ara::ponB] were grown and serially diluted as in A, except the medium lacked Cam. The dilutions were spotted onto M9-arabinose or LB-glucose agar as indicated. Plates were incubated at 30 °C overnight prior to imaging. (C). Overnight cultures of PA686 cells [∆ponB ∆lpoA] harboring the plasmids pPSV38 [P_lacUV5::empty], pNG91 [P_lacUV5::ponA], pNG94 [P_lacUV5::ponA(E158K)], pNG95 [P_lacUV5::ponA(G62S)], or pNG96 [P_lacUV5::ponA(G284D)] were grown in LB no salt with Gent at 30 °C. Cells were then pelleted, washed twice in an equal volume of VBMM media, and resuspended in VBMM at an OD₆₀₀ of 1.0. The cell suspensions were then diluted and plated as above on VBMM agar plates containing the indicated concentration of IPTG. Plates were incubated at 37 °C overnight prior to imaging.