Fig 1.
Depletion of MreC or MreD does not impair S. aureus COL growth.
(A) Growth curves for inducible mutant COLmreCDi in TSB medium in the presence (Δ) and absence (●) of 0.5 mM IPTG (a). When the culture without IPTG reached OD600nm ≈ 1, a sample was used to inoculate fresh media (b) with (□) and without (ӿ) 0.5 mM IPTG. (B and C) Growth curves for COL, COLΔmreC, COLΔmreD and COLΔmreCD in TSB rich medium (B) and SSM9PR minimal medium (C). Mutant strains have a growth rate similar to the parental strain, with a duplication time of approximately 41 min in TSB and 81 min in SSM9PR.
Fig 2.
MreC and MreD are enriched at the division septum.
S. aureus COL cells expressing sGFP fused to the N-terminal of MreC (top) or MreD (bottom) were grown in TSB supplemented with 0.2 mM IPTG and observed by SR-SIM. Left panels show differential interference contrast (DIC) images and right panels show fluorescence signal of sGFP fusion proteins. Scale bars, 0.5 μm.
Fig 3.
Profiles of peptidoglycan muropeptides remain unaltered in the absence of MreC or MreD.
HPLC profiles of peptidoglycan muropeptides from S. aureus COL, COLΔmreC, COLΔmreD and COLΔmreCD.
Fig 4.
Absence of MreC or MreD has no effect on cell morphology, cell dimensions or cell cycle progression.
(A) COL, COLΔmreC, COLΔmreD and COLΔmreCD were imaged by Superresolution Structured Illumination Microscopy (SR-SIM), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). For the SR-SIM images, cell wall and DNA were labeled with fluorescent vancomycin (Van-FL) and Hoechst 33342, respectively. Scale bars for SR-SIM and SEM images 0.5 μm and for TEM images 0.2 μm (B) Fraction of the cell cycle spent in Phase 1 (blue, before initiation of septum synthesis), Phase 2 (red, septum synthesis) and Phase 3 (green, after closure of the septum, before the two daughter cells split) [20]. Cell cycle progresses clockwise. From the inner to the outer circle the values correspond to COL, COLΔmreC, COLΔmreD and COLΔmreCD. Exponentially growing cultures were labeled with the membrane dye Nile Red and the percentage of cells in each phase of the cell cycle was determined. (C) Cellular volume distributions during each phase of the cell cycle. Cells were labeled with Nile Red and imaged by SR-SIM. The volume was calculated by approximation of the cellular shape to a prolate spheroid. n = 60 for each phase. Statistical analysis was performed using the unpaired t test and p-values were >0.05 in all cases compared to the parental strain COL, except for Phase 1 COLΔmreD (p = 0.013) and for COLΔmreCD (p = 0.014).