Fig 2.

Deletion of alp7R destabilizes mini-pLS20 and is lethal to cells carrying the plasmid. (A) LB agar plates containing 10 μg ml⁻¹ tetracycline with or without 0.5% d-xylose were incubated at 30°C for 24 h. Lower left, JP3133 (PY79 carrying mini-pLS20 [pLS20 origin of replication and alp7AR operon]); lower right, JP3171 (PY79 carrying mini-pLS20 Δalp7AR [pLS20 origin of replication only]); top, JP3302 (PY79 with an integrated xylose-inducible copy of alp7R, carrying mini-pLS20 Δalp7R [pLS20 origin of replication and alp7AR operon lacking alp7R]). (B) Exponential cultures grown in the absence of selection were sampled at regular intervals for plasmid retention as described in Materials and Methods. Black, JP3133; blue, JP3171; red, JP3302. Open squares, hourly supplementation with xylose to 0.5%; filled circles, no xylose supplementation. (C to E) Fluorescence microscopy images of strains supplemented or not supplemented with xylose. Exponential cultures grown in the absence of selection as in the plasmid stability assay were sampled for microscopy. Membranes were stained with FM4-64 (red), and DNA was stained with DAPI (blue). Scale bar equals 1 μm; all panels are at the same scale. (C) JP3302, with no xylose supplementation, at 4 h. Arrows indicate anucleate cells. (D) JP3302, supplemented hourly with 0.5% xylose, at 4 h. (E) JP3133, supplemented hourly with xylose to 0.5%, at 3 h.