As shown in Figure 3, the I124L and I229L MetA mutants were approximately 2-3-fold more stable than native MetA, with half-lives (t1/2) of 87 min (I124L) and 107 min (I229L) at 37°C and 52 min (I124L) and 57 min (I229L) at 44°C, respectively; the half-life of the native MetA was 36 min at 37°C and 25 min at 44°C. Figure 3 In vivo stability of MetA mutants. Cells of the strains WE, L124 and Y229 exponentially

growing (OD600 = 0.3) at 37°C in M9 medium were treated with 200 μg/ml of chloramphenicol. The cultures were divided; one half of each culture was maintained at 37°C (solid symbols), and VRT752271 chemical structure the other half of the culture was shifted to 44°C (open symbols). The samples were collected at the indicated time points and analyzed through Western blotting as described in the see more Methods section. Densitometry results were normalized after setting the MetA amount before chloramphenicol addition equal to 100%. Stabilized MetAs partially compensate the growth defects of the ΔdnaK mutants MetA has been suggested to be classified as a Class III substrate for chaperones because this enzyme is extremely prone to aggregation [10]. Under physiological heat stress conditions, the DnaK system PKC inhibitor is the most effective chaperone for preventing the aggregation of thermolabile proteins [14]. Thus, the ΔdnaK52 mutant strain

displays a slower growth rate at 37°C and no growth at 42°C [15]. Because MetA is one of the most thermolabile proteins, we determined the growth profiles of dnaK null mutants expressing stabilized MetAs. We constructed

the WE∆dnaK, L124∆dnaK and Y229∆dnaK mutant strains and cultured these cells in M9 (-)-p-Bromotetramisole Oxalate glucose medium at 37°C. As shown in Figure 4, the mutant strain Y229∆dnaK grew 26% faster than the control strain WE∆dnaK, with a growth rate of 0.48 h-1 for Y229∆dnaK and 0.38 h-1 for WE∆dnaK (see Additional file 5: Table S2 for the specific growth rates). The mutant strain L124∆dnaK grew at the same rate as Y229∆dnaK. We observed an increased accumulation of insoluble wild-type MetA in heat-stressed ∆dnaK cells compared with the mutated I124L and I229Y enzymes, which had relative amounts of 57% and 33% of the wild-type enzyme, respectively (Additional file 6: Figure S4). This finding might partially explain the slower growth of the WE∆dnaK strain due to an increased aggregation of the wild-type MetA compared with the I124L and I229Y mutants. Figure 4 Effect of stable MetA mutants on the growth of dnaK null and protease-deficient mutants of the E. coli strains WE and Y229. The strains were cultured in 25 ml of M9 glucose medium in 125 ml Erlenmeyer flasks at 37°C (∆dnaK mutants) or 42°C (protease-minus mutants). To measure the growth, the optical density was monitored at 600 nm every 1 h. The average of two independent experiments is presented.