Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics

Abstract

The peptidoglycan (PG) layers surrounding bacterial cells play an important role in determining cell shape. The machinery controlling when and where new PG is made is not understood, but is proposed to involve interactions between bacterial actin homologs such as Mbl, which forms helical cables within cells, and extracellular multiprotein complexes that include penicillin-binding proteins. It has been suggested that labeled antibiotics that bind to PG precursors may be useful for imaging PG to help determine the genes that control the biosynthesis of this polymer. Here, we compare the staining patterns observed in Bacillus subtilis using fluorescent derivatives of two PG-binding antibiotics, vancomycin and ramoplanin. The staining patterns for both probes exhibit a strong dependence on probe concentration, suggesting antibiotic-induced perturbations in PG synthesis. Ramoplanin probes may be better imaging agents than vancomycin probes because they yield clear staining patterns at concentrations well below their minimum inhibitory concentrations. Under some conditions, both ramoplanin and vancomycin probes produce helicoid staining patterns along the cylindrical walls of B. subtilis cells. This sidewall staining is observed in the absence of the cytoskeletal protein Mbl. Although Mbl plays an important role in cell shape determination, our data indicate that other proteins control the spatial localization of the biosynthetic complexes responsible for new PG synthesis along the walls of B. subtilis cells.

Fig. 1.

Structures and cellular targets of vancomycin and ramoplanin. (A) Structures of compounds discussed in the text. (B) The extracellular stage of PG biosynthesis. Vancomycin recognizes d-Ala-d-Ala (red); ramoplanin recognizes diphospho-MurNAc (blue). In B. subtilis, L-Lys is replaced with meso-diaminopimelic acid.

Fig. 2.

Staining of B. subtilis PY79 with ramoplanin analogs. (A) Examples of sidewall staining observed with 2a:2b. (B–H Left) Probe-treated cells. (B–H Center) TMA-DPH-treated cells (mb, membrane stain). (B–H Right) Overlays of probe-treated (green) and TMA-DPH-treated (red) cells. Arrowheads and arrows point to old division sites (poles) and new division sites (septa), respectively. (A) Fluorescent images of 1:1 mixture of 2a:2b at 0.5 μg/ml each, along with a schematic representation of the helix. (B) 2b at 0.1× MIC (2 μg/ml). (C) 2b at 1.0× MIC (20 μg/ml). (D) A 1:1 mixture of 2a:2c at 0.5 μg/ml each (higher concentrations look similar). (E) A 1:1 mixture of 2a:2b at 0.5 μg/ml each. (F) A 1:1 mixture of 2a:2b at 1.0 μg/ml each. (G) A 1:1 mixture of 2a:2b at 2.5 μg/ml each. (H) 2d at 0.1× MIC (1.0 μg/ml). (Scale bars, 2 μm.) See Fig. 5, which is published as supporting information on the PNAS web site, for larger fields.

Fig. 3.

Staining of B. subtilis PY79 with vancomycin analogs. (Left) Probe-treated cells. (Center) TMA-DPH-treated cells. mb, membrane stain. (Right) Overlays of probe-treated (green) and TMA-DPH-stained cells (red). Arrowheads and arrows point to old division sites (poles) and new division sites (septa), respectively. (A) A 1:1 mixture of 1a:1b at 0.13 μg/ml each. (B) A 1:1 mixture of 1a:1b at 0.4 μg/ml. (C) 1d at 0.4× MIC (1.0 μg/ml). (D) A 1:1 mixture of 1a:1d at 0.4 μg/ml. (Scale bars, 2 μm.) See Fig. 7, which is published as supporting information on the PNAS web site, for larger fields.

Fig. 4.

Comparison of mblΔ and WT strains stained with 2d (Upper) or a 1a:1d mixture (Lower). (A Left) mblΔ stained with 2d (0.2 μg/ml). (A Right) WT stained with 2d (1.0 μg/ml). (B Left) mblΔ stained with 1a:1d (1:1 ratio at 0.08 μg/ml). (B Right) WT stained with 1:1 mixture of 1a:1d (1:1 ratio at 0.5 μg/ml). (Scale bars, 2 μm.)