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PenG Prevents the L-Form Switch from the Walled State

(A) Schematic representation of peptidoglycan (PG) synthesis in B. subtilis and its inhibition by antibiotics. The PG wall is built from long glycan strands composed of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) crosslinked to each other by short peptide bridges. The precursor for PG is initially synthesized in the cytoplasm by the action of MurAA, MurAB, MurC, MurD, MurE, and MurF enzymes. MurNAc-pentapeptide is coupled to a membrane carrier, undecaprenyl pyrophosphate, by MraY, and GlcNAc is added by MurG to form lipid II, which is then transferred to the outside of the cytoplasmic membrane. Newly synthesized PG is incorporated into the existing PG meshwork by a combination of transglycosylation and transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) and RodA. The antibiotics fosfomycin and D-cycloserine inhibits MurA and Ddl, respectively. The β-lactam antibiotics including penicillins (e.g., penicillin G) and cephalosporins (e.g., cephalexin) target the PBPs.

(B–D) Effects of antibiotics on L-form switch. (B) B. subtilis strains wild-type (168CA) and ispA mutant (RM81) were grown on NA/MSM plates with or without 200 μg/mL D-cycloserine (DCS) (with 1 μg/mL of FtsZ inhibitor 8j to prevent the rare reversion to walled cells) or 200 μg/mL penicillin G (PenG) at 30°C for 2–3 days. (C) PC micrographs of ispA mutant cells with or without DCS taken from the cultures shown in (B). (D) B. subtilis L-form strain (LR2; Pxyl-murE ispA−) was grown on NA/MSM plates with or without PenG in the absence of xylose.

(E–G) Effects of PenG on L-form emergence from the parental walled cells in LR2 (Pxyl-murE ispA−; E and F) and RM81 (ispA−murE+; G). Individual frames are extracted from example movies of time-lapse experiments (see also Movies S1 and S2). Numbers in the bottom right corner of each frame represent time (min) elapsed in the movies. Examples of L-form emergence are shown by blue arrows.

See also Figures S1 and ​S2.