Figure 3.

LpoA and LpoB are absolutely required for the in vivo function of their cognate PBP and strongly stimulate the TPase activity of their cognate PBP in vitro. A–B. Depletion of Lpo proteins in the absence of the non-cognate PBP leads to lysis. LpoA (A) and LpoB (B) were expressed from an arabinose (Ara)-inducible plasmid in mrcB⁻ and mrcA⁻ cells respectively, and depleted by dilution of stationary phase cultures into glucose-containing LB medium (repression). For LpoB-depletion, diluted cultures were first grown to OD₅₇₈=0.6 in glucose LB medium (B, blue line, inset) and then rediluted into fresh glucose LB medium to observe lysis. C–E. Morphology of Lpo-depleted cells. Cells grown with glucose to deplete LpoA (in mrcB⁻ background) and LpoB (in mrcA⁻ background), or with Ara (control), were fixed and examined by phase contrast microscopy. Lysis of LpoA- or LpoB-depleted cells began after 300 min of growth in glucose. Magnified pictures of LpoA- (D) or LpoB-depleted (E) cells at 300 min reveal the presence of lysis bulges often emerging at midcell (arrows). (F) The activity of detergent-solubilized PBP1A or PBP1B was assayed with radiolabelled lipid II in the presence or absence of their cognate Lpo protein. The PG product was digested with cellosyl and the resulting muropeptides were analysed by HPLC (for chromatograms see Fig. S3). The table shows a summary of the types of muropeptides and properties of the PG synthesized. The % peptides in cross-links was calculated as 100% − % Monomers; the degree of cross-linkage is defined as %Dimers/2 + %Trimers × 2/3 + %Tetramers × 3/4 and is equal to the percent peptides that were used as donors in TPase reactions; n.d., not detected. Both Lpo proteins increased the cross-linkage in the PG synthesized by their cognate PBP. LpoA also stimulated the PBP1A-catalysed attachment of newly made PG to sacculi (Fig. S4).