Figure 2
The ugtP mutant requires LytE to maintain rod-shape. (A) Spot plate assay showing the lethality of the ugtP lytE double knockouts when cells were grown on PAB media. The ectopic expression of UgtP or supplementing the plates with magnesium sulphate allowed BSB1 ΔugtP ΔlytE to grow on PAB. (B) Fluorescence microscopy using brightfield and membrane stain to study the morphology of the mutants. BSB1 ΔugtP ΔlytE showed severe shape defects during exponential phase when grown in LB media. The complementation of ugtP recovered this phenotype. I− without IPTG, I+ with IPTG. Scale bar 3 µm. (C) TEM analysis of the mutants revealed the severity of the ugtP lytE morphology defects (arrows) where cells showed loss of rod-shape and membrane integrity. Arrows indicate ruptures in the cell wall (D) HPLC analysis of muropeptides isolated from BSB1 ΔugtP ΔlytE and BSB1 ΔugtP ΔcwlO showed alterations in the amidation patterns compared to BSB1. These alterations were mainly detected in the muropeptides Tri-(NH2) (muropeptide 3), TetraTri-(NH2) (muropeptide 15) and TetraTri-(NH2)2 (muropeptide 21). (E) Diagram representing the difference in the level of selected muropeptides in mutants compared to BSB1. 100% indicates the level of a muropeptide in BSB1. *p ≤ 0.05.
