MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for chromosome partitioning in Escherichia coli. We report that overproduction of MukBEF results in marked chromosome condensation. This condensation is rapid and precedes the effects of overproduction on macromolecular synthesis. Condensed nucleoids are often mispositioned; however, cell viability is only mildly affected. The overproduction of MukB leads to a similar chromosome condensation, even in the absence of MukE and MukF. Thus, the non-SMC subunits of MukBEF play only an auxiliary role in chromosome condensation. MukBEF, however, was often a better condensin than MukB. Furthermore, the chromosome condensation by MukB did not rescue the temperature sensitivity of MukEF-deficient cells, nor did it suppress the high frequency of anucleate cell formation. We infer that the role of MukBEF in stabilizing chromatin architecture is more versatile than its role in controlling chromosome size. We further propose that MukBEF could be directly involved in chromosome segregation.
MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of the Escherichia coli chromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold.
In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44°C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetA E. coli protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44°C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetA E. coli to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetA E. coli enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.
BackgroundThe growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway. MetA is also unstable under other stressful conditions, such as weak organic acids and oxidative stress. The MetA protein unfolds, even at 25°C, forms considerable aggregates at 37°C and completely aggregates at 44°C.ResultsWe extended the MetA mutation studies using a consensus concept based on statistics and sequence database analysis to predict the point mutations resulting in increased MetA stability. In this study, four single amino acid substitutions (Q96K, I124L, I229Y and F247Y) in MetA designed according to the consensus concept and using the I-mutant2.0 modeling tool conferred accelerated growth on the E. coli strain WE at 44°C. MetA mutants that enabled E. coli growth at higher temperatures did not display increased melting temperatures (Tm) or enhanced catalytic activity but did show improved in vivo stability at mild (37°C) and elevated (44°C) temperatures. Notably, we observed that the stabilized MetA mutants partially recovered the growth defects of E. coli mutants in which ATP-dependent proteases or the DnaK chaperone was deleted. These results suggest that the impaired growth of these E. coli mutants primarily reflect the inherent instability of MetA and, thus, the methionine supply. As further evidence, the addition of methionine recovered most of the growth defects in mutants lacking either ATP-dependent proteases or the DnaK chaperone.ConclusionsA collection of stable single-residue mutated MetA enzymes were constructed and investigated as background for engineering the stabilized mutants. In summary, the mutations in a single gene, metA, reframe the window of growth temperature in both normal and mutant E. coli strains.
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