Extended Data Fig. 8: MurA(G58D) and MurA(E406K) impact binding and activation of PaLpxC.
From: Coordination of bacterial cell wall and outer membrane biosynthesis
(a) Anti-FLAG immunoblot detecting F-PaMurA variants after 1 h of induction with 1 mM IPTG. A corresponding blot for RpoA was used as a loading control. Data are representative of 3 biological replicates. (b) MurA activity assay in which purified PaMurA variants (100 nM) were mixed with UDP-GlcNAc (1 mM) and PEP (0.5 mM) and the release of Pi was measured by Lanzetta assay. Dots indicate the values obtained for three individual replicates, bars indicate the mean, and error bars represent their standard deviation. The dashed line indicates the average catalytic activity of PaMurA(C117S) observed in Fig S7C. (c) in vitro pulldowns in which purified F-PaLpxC and H-MurA variants were mixed in a 1:1 ratio in the presence or absence of CHIR-090 and processed as in Extended Data Fig. 4b. Data are representative of at least two replicates. (d) Spot titer assay in which serial dilutions of a PAO1 strain harboring a PamurA deletion or PamurA(C117S) allele at the native locus complemented by a chromosomally-integrated, IPTG-inducible copy of PamurA(WT) were plated on LB agar with the indicated supplements. As indicated, the strains also contained an empty plasmid or one encoding PamurA(G58D) under arabinose-inducible control. Plates were incubated at 37oC for 20 h before being photographed. Data are representative of 3 biological replicates. For gel source data, see Supplementary Fig. 1.
