Fig 7. A strategy for BfmRS control of resistance to cell wall stress that is independent of its control of β-lactamase production.

(A-C) BfmRS modulates cell morphogenesis. (A) Mid-log phase bacteria were imaged with phase contrast optics. (B) Cell length from n > 300 cells per strain was quantified; Red lines indicate median values. Mutants differed significantly from WT (P <0.0001, Mann-Whitney test). (C) Chaining was quantified for bacteria counted in panel B. Histogram shows frequency of chains containing the indicated number of cells. One chain of 5 cells and one chain of 10 cells were observed with ΔbfmRS but are not included in histogram. (D) Bacteria lacking bfmRS experience cell morphology defects (arrowheads) at low concentrations of imipenem (IPM). Mid-log phase cultures were treated with imipenem at the indicated concentration for 2 hours and bacteria were imaged as in A. (E) Selective suppression of the resistance defect of ΔbfmRS bacteria with 7.5% sucrose. Bacteria were grown on solid medium containing increasing concentrations of the indicated antibiotics, as well as 0 or 7.5% sucrose, and CFE determined. Data points show geometric mean ± s.d. (n = 3). (F) pbp2 deletion causes loss of rod morphology and propagation as spheres. Bacteria were grown to log phase in antibiotic-free LB medium and imaged via phase-contrast microscopy. (G) bfmRS deletion amplifies growth defect caused by pbp2 deletion. Bacterial growth was measured as change in optical density during 37°C incubation (Materials and Methods). Data points represent mean ± s.d. (n = 3). (H) Increasing slt expression partially suppresses imipenem hypersensitivity in cells lacking BfmRS. EGA495 (ΔbfmRS) harboring vector control (pEGE305) or derivative with slt gene downstream of T5-lacp promoter (pEGE316) were grown on solid medium containing the indicated antibiotic supplemented with or without IPTG at 1mM. Data points show geometric mean of CFE ± s.d. (n = 3). 17978 WT was included as additional control (n = 2).