In the methionine- supplemented medium, the ∆dnaK mutants grew at equal rates, and only slightly slower growth than the dnaK + strains was observed (Additional file 5: Table S2; Additional file 7: Figure S5). These findings suggest that a malfunction of the methionine biosynthetic PRN1371 solubility dmso enzymes, including MetA, is primarily responsible for the impaired growth of the ∆dnaK mutant strains at 37°C. At temperatures higher than 37°C, defects in other factors, such as chromosomal partitioning, extensive filamentation and increased levels of heat-shock protein (HSP) biosynthesis, might significantly hamper the growth of the ΔdnaK mutants, as previously shown for the ΔdnaK52
mutant strain [15]. L-methionine also eliminated the difference in the growth rates between the protease- deficient control WE(P-) and mutant Y229(P-) strains (0.58 and 0.59 h-1, respectively) at 42°C (Additional file 5: Table S3; Additional file 7: Figure S5). However, the protease-negative mutants grew 25% slower than the parent strains in the presence of L-methionine (Additional file 5: Table S3; Additional file 7: Figure S5), potentially reflecting the accumulation
of other protein aggregates [17]. A partial complementation of the impaired growth of the ∆dnaK and protease-negative strains GSK126 manufacturer through stabilized MetAs indicates that the inherent instability Seliciclib mouse of MetA plays a significant role in the growth defects observed in these mutant strains. Discussion The growth of E. coli strains at elevated temperatures in a defined medium is impaired by the extreme instability of the first enzyme in the methionine biosynthetic pathway, homoserine o-succinyltransferase (MetA) [18]. Although
the key role of Fluorometholone Acetate the MetA protein in E. coli growth under thermal stress has been known for 40 years [8], it is unclear which residues are involved in the inherent instability of MetA. Previously, we identified two amino acid substitutions, I229T and N267D, responsible for MetA tolerance to both thermal and acid stress [11]. In this study, we employed several approaches to design more stable MetA proteins. Using the consensus concept approach [12], stabilization was achieved through three single amino acid substitutions, Q96K, I124L and F247Y. We hypothesized that a combination of these amino acid substitutions might significantly increase MetA stability compared with the single mutants we identified in the randomly mutated thermotolerant MetA-333 [11]. The new MetA mutant enzymes were more resistant to heat-induced aggregation in vitro (Figure 2). The enhanced in vivo stabilities of the MetA mutants were also demonstrated through the immunodetection of residual MetA protein after blocking protein synthesis (Figure 3). However, the melting temperature, a good indicator of thermal stability [19], was only slightly increased.