Figure Legend Snippet: Inactivation of the two known acetylation mechanisms in E. coli eliminates the majority of acetylation. Wild-type (WT) E. coli (strain BW25113) and an isogenic Δ pta yfiQ acs cobB mutant (Gutted) were aerated in TB7 supplemented with 0.4% glucose for 10 h. Whole-cell lysates were analyzed (A) by Coomassie blue-stained SDS-polyacrylamide gel to ensure equivalent loading and (B) by anti-acetyllysine Western blotting.

Techniques Used: Mutagenesis, Staining, Western Blot

Figure Legend Snippet: Overexpression of five GNAT family members results in altered lysine acetylation patterns by anti-acetyllysine Western blotting. The gutted strain (BW25113 Δ pta yfiQ acs cobB ) was transformed with the pCA24n vector control (negative [−] control) or pCA24n containing the indicated genes under an IPTG-inducible promoter . As a positive (+) control, an isogenic strain that retained the WT allele of acs (Δ pta yfiQ cobB ) was transformed with pCA24n containing YfiQ. The resulting strains were aerated in TB7 supplemented with 0.4% glucose, 50 μM IPTG, and 25 μg/ml chloramphenicol for 10 h. Whole-cell lysates were analyzed (right panels) by Coomassie blue-stained SDS-polyacrylamide gel electrophoresis to ensure equivalent loading and (left panels) by anti-acetyllysine Western blotting. Note that the band in RimJ was not reproducible. The positive control contains one additional YfiQ-dependent band around 72 kDa, which corresponds to Acs . YncA and AstA each produce an acetylated band that can be observed in the Coomassie blue-stained gel at the expected molecular weight of these proteins.

Techniques Used: Over Expression, Western Blot, Transformation Assay, Plasmid Preparation, Negative Control, Positive Control, Staining, Polyacrylamide Gel Electrophoresis, Molecular Weight

Figure Legend Snippet: Mutation of conserved catalytic amino acids prevents RimI-, PhnO-, YjaB-, and YiaC-dependent acetylation. The gutted strain (BW25113 Δ pta yfiQ acs cobB ) was transformed with the pCA24n vector control, pCA24n carrying the wild-type allele for each putative KAT, or mutant alleles for each putative KAT with alanine substitutions of the indicated residues. The resulting strains were grown in TB7 supplemented with 0.4% glucose, 100 μM IPTG, and 25 μg/ml chloramphenicol for 8 h. Crude lysates harvested after 4 h were analyzed for expression of the KAT proteins. Whole-cell lysates harvested after 8 h were analyzed for acetylation. Coomassie blue-stained SDS-PAGE gels (A and C) served as loading controls for anti-His (B) and anti-acetyllysine (D) Western blot analysis.

Techniques Used: Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Staining, SDS Page, Western Blot