The stabilized MetA mutant enzymes at least partially recovered the growth defects of mutant E. coli strains with deletions of either ATP-dependent proteases or the DnaK chaperone. These results suggest that the growth defects of ΔdnaK or protease-deficient mutants primarily reflect malfunctioning MetA at 37°C,

a standard Pexidartinib manufacturer physiological temperature. Consistently, the addition of methionine recovered the temperature-dependent growth defects of these mutants. Results Mutant MetAs enable E. coli growth at elevated temperatures Previously, we identified two amino acid substitutions, I229T and N267D, which conferred stability to the MetA protein [11]. To obtain additional stable MetA mutants, we employed a multiple alignment approach and identified eight amino acid residues present in all thermophilic MetAs but absent in E. CHIR-99021 datasheet coli MetA (Additional file 1: Figure S1). The metA mutations that resulted in the corresponding amino acid substitutions Q96K, L110V, I124L, R160L, A195T, A200E, D218G and F247Y were integrated into the E. coli JW3973 (∆metA) chromosome to yield the strains K96,

V110, L124, L160, T195, E200, G218 and Y247, respectively. Among the constructed strains, three mutants, K96, L124 and Y247, demonstrated accelerated growth at 44°C in M9 glucose medium (Figure 1; Additional file 2: Table S1) compared with the control strain WE, which harbored the wild-type metA gene from the E. coli K-12 strain W3110 [11]. Figure 1 Stabilized MetA mutants stimulate growth of the E. coli WE strain at 44°C. The strains were cultured

in M9 glucose medium in a TVS126MB automatic growth-measuring incubator at 44°C. The optical densities of the growing cultures were measured at 600 nm every 10 min. The average of two independent experiments is presented. Serial dilutions of cultures growing logarithmically at 30°C in M9 glucose medium (OD600 of 0.5) were spotted on M9 glucose see more and M9 glucose L-methionine (50 μg/ml) agar plates. The cells were incubated for 24 h at 44°C. Using the I-Mutant2.0 modeling tool [13] for protein stability prediction, the I229Y mutation was predicted to improve MetA stability and accelerate growth at 44°C (Figure 1; Additional file 2: Table S1). To confirm the enhanced thermo-tolerant growth of the L124, Y229 and Y247 mutants, the serially diluted cultures were incubated on solid M9 glucose plates at 44°C (Figure 1). The viability of the mutant strains was increased by at least one to two orders of magnitude compared with the wild-type strain (Figure 1). Supplementation of the culture medium with L-methionine stimulated the growth of the wild-type and the mutant strains at 44°C to the same extent, thus abolishing the differences between the wild-type and mutant strains (Figure 1). The mutant strains L124 and Y229, which displayed the higher growth rates at 44°C (Additional file 2: Table S1), were selected for further analysis.