The average of two experiments is presented. (PPT 90 KB) Additional file 4: Figure S3: Densitometric analysis of MetAs in the heat-stressed cultures. The E. coli strains WE, L124 and Y229 were grown in M9 glucose medium to the exponential phase (approximately

OD600 = 0.6) at 30°C and subsequently shifted to 45°C for 30 min. Soluble (black columns) and aggregated (gray columns) fractions of MetAs were purified from 25 ml cultures as described in the Methods section. Three micrograms of total protein from the ATR inhibition insoluble and soluble fractions were subjected 17DMAG supplier to 12% SDS-PAGE, followed by Western blotting using rabbit anti-MetA antibody. The MetA in the samples was quantified through densitometry using WCIF ImageJ software and normalized to the MetA amount from

unstressed cultures, which was equal to 1. The error bars represent the standard deviations of duplicate independent cultures. Abbreviations: Ins, insoluble fraction; Sol, soluble selleckchem fraction. (PPT 110 KB) Additional file 5: Table S2: Effect of the stabilized MetA proteins on growth of the dnaK null E. coli mutants. Table S3 Effect of the stabilized MetA proteins on growth of the protease-deficient E. coli mutants. Table S4 Effect of the stabilized MetA proteins on growth of the E. coli ΔmukB mutants. (DOC 36 KB) Additional file 6: Figure S4: In vivo aggregation of the wild-type and mutated MetAs in heat-stressed cells of the ΔdnaK or protease-deficient mutant strains. Aggregates Uroporphyrinogen III synthase of the wild-type MetA (black columns), mutated I124L (gray columns) and I229Y (dark-gray columns) proteins were purified from the ΔdnaK or protease-minus mutants grown in M9 glucose medium at 32°C or 37°C, respectively, to the exponential phase

(approximately OD600 = 0.6) and transferred to 42°C for 1 h as described in the Methods section. Three micrograms of total protein from the insoluble fractions was subjected to 12% SDS-PAGE, followed by Western blotting using rabbit anti-MetA antibody. The MetAs were quantified through densitometry using WCIF ImageJ software and normalized to the wild-type MetA amount from the WE strain, which was equal to 1. The error bars represent the standard deviations of duplicate independent cultures. (PPT 88 KB) Additional file 7: Figure S5: L-methionine eliminates the growth rate difference between the wild-type and stabilized MetAs in ΔdnaK or protease-deficient mutants at non-permissive temperatures. The strains were cultured in 25 ml of M9 glucose L-methionine (50 μg/ml) medium in 125 ml Erlenmeyer flasks at 37°C (ΔdnaK mutants) or 42°C (protease-minus mutants). The average of two independent experiments is presented. Serial dilutions of cultures growing logarithmically at 30°C (ΔdnaK mutants) or 37°C (protease-minus mutants) in M9 glucose medium (OD600 of 0.5) were spotted onto M9 glucose L-methionine (50 μg/ml) agar plates. The cells were incubated for 24 h at 37°C (ΔdnaK mutants) or 42°C (protease-minus mutants).