Coordinated peptidoglycan synthases and hydrolases stabilize the bacterial cell wall

Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), collapses rod shape despite aPBPs being non-essential for rod-like morphology in the bacterium Myxococcus xanthus. Here, we demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase. Moenomycin promotes the binding between DacB and PG and thus reduces the mobility of DacB through PBP1a2. Conversely, DacB also regulates the distribution and dynamics of aPBPs. Our findings clarify the action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.


Fig. S2 .
Fig. S2.Overall distribution and single-particle trajectories of DacB-PAmCherry and DacB S75A -PAmCherry.Overall distribution of single molecules is displayed using the composite of 100 frames.Single-particle trajectories were generated from 100 frames using the TrackMate 1 plugin in ImageJ.Individual trajectories are distinguished by colors.White arrows point to stationary particles.MOE, moenomycin, 4 µg/ml; MEC, mecillinam, 100 µg/ml.Sporulation was induced by 1M glycerol and cells were imaged after 1 h of induction.Scale bars, 5 µm.

Fig. S3 .
Fig. S3.DacB is a major PG hydrolase that dismantles rod shape during the sporulation of M. xanthus.a) The absence and overexpression of DacB delays and accelerates the sporulation of M. xanthus, respectively.Phase contrast images of cells after 2-h of glycerol induction are shown.b) The progress of sporulation is quantified by the length/width ratio of cells after 2-h of glycerol induction.Whiskers indicate the 25 th -75 th percentiles and red dots the median.The total number of cells analyzed is shown on top of each bar.p values were calculated using a one-way ANOVA test between unweighted, independent samples.c) While wild-type cells lose TADA signal near evenly during sporulation, the ∆dacB cells retain TADA at their poles.Scale bars, 5 μm.

Fig. S4 .
Fig. S4.Overall distribution and single-particle trajectories of DacB-PAmCherry in the backgrounds of aPBP mutants.Overall distribution of single molecules is displayed using the composite of 100 frames.Single-particle trajectories were generated from 100 frames using the TrackMate 1 plugin in ImageJ.Individual trajectories are distinguished by colors.White arrows point to stationary particles.MOE, moenomycin, 4 µg/ml.Scale bars, 5 µm.

Fig. S5 .
Fig. S5.The expression of PBP1a2 E97A in M. xanthus.a) Sequence alignment between M. xanths PBP1a2 (PBP1a2) and E. coli PBP1a (EcPBP1a).The catalytic active glutamate residue is marked by *. b) PBP1a2 E97A expresses as a stable protein in M.xanthus.Its stability was tested using cell lysate of a strain that expresses PBP1a2 E97A -PAmCherry as the sole source of PBP1a2 and an anti-mCherry antibody.

Fig. S6 .
Fig. S6.Overall distribution and single-particle trajectories of PAmCherry-labeled aPBPs.a) All three PAmCherry-labeled aPBPs accumulate as full-length proteins.Their stability was tested using cell lysate of the strains that each express one PAmCherry-labeled aPBP and an anti-mCherry antibody.A low molecular weight band is visible in the lane of PBP1c-PAmCherry.However, its molecular weight, ~40 kDa, does not match degraded PAmCherry (~25 kDa).b-d) PAmCherry label on aPBPs does not affect the length (b), width (c), and resistance against moenomycin (8 µg/ml, 2 h) (d).Whiskers indicate the 25 th -75 th percentiles and red dots the median.The total number of cells analyzed is shown on top of each bar.p values were calculated using the Student paired t test with a two-tailed distribution.e) Overall distribution of single molecules is displayed using the composite of 100 frames.Single-particle trajectories were generated from 100 frames using the TrackMate 1 plugin in ImageJ.Individual trajectories are distinguished by colors.Scale bars, 5 µm.