Restoration of Growth Phenotypes of Escherichia coli DH5α in Minimal Media through Reversal of a Point Mutation in purB

Abstract

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5␣ and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.The Escherichia coli DH5␣ strain possesses many beneficial genotypes (recA, deoR, gyrA, and endA1) and has been widely used for many purposes, such as gene cloning and protein production (5). However, E. coli DH5␣ also exhibits inferior growth phenotypes, especially in minimal media, compared to other E. coli strains. As such, the utilization of this bacterium has been limited to the laboratory despite its numerous advantages. We can assume that these inferior growth phenotypes have resulted from unknown accumulated mutations during the strain development process (5). Some of those mutations, which might impact growth in minimal media, have been characterized, including the phenotypes for thiamine requirement and relaxed amino acid synthesis (5). Still, there may be other uncharacterized mutations whose interactions hamper the growth of E. coli DH5␣ in minimal media.Based on successful identifications (6, 7) of gene targets for metabolic engineering (3), we performed serial subcultures of E. coli DH5␣ transformants with an E. coli K-12 genomic library based on a multicopy plasmid (9) to isolate genes that improve growth phenotypes in minimal media. The M9 minimal medium and R medium (11) were chosen for enrichment experiments because of their popular use in metabolic engineering (1, 2, 7) and in high-cell-density fermentation (8, 10, 11). After 11 serial transfers of the transformants in the M9 medium, and 27 transfers in the R medium, cultured cells were diluted and plated onto LB agar for single-colony isolation. Although more than 10 colonies were picked, only three distinctive plasmids, containing different inserts, were isolated from the transformants enriched in M9 medium. In the case of R medium enrichment, all isolated plasmids were identical. Sequencing of the isolated plasmids revealed the exact genome coordinates of each insert. A diagram of the inserts in the context of the E. coli genome sequence is shown in Fig. 1. Interestingly, all of the isolated plasmids contained similar regions of genomic DNA. mnmA (tRNA 5-methylaminomethyl-2-thiouridylate-methyltransferase), purB (adenylosuccinate lyase), and hflD (lysogenization regulator) were the annotated genes in the overlapping region among distinctive isolated fragments. However, since the N-terminal portions of mnmA and hflD were truncated in some of the inserts, we selected only the M3 and R1 plasmids for further experimentation. These two plasmids were retransformed into E. coli DH5␣ for confirmation of their beneficial effects on growth of E. coli in minimal media. The newly transformed strains showed growth phenotypes almost identical to those of the previously isolated transfor...

Complete Genome Sequence of Escherichia coli ER1821R, a Laboratory K-12 Derivative Engineered To Be Deficient in All Methylcytosine and Methyladenine Restriction Systems

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Efficient Production of Single-Stranded Phage DNA as Scaffolds for DNA Origami

“…The low maximum growth rate is due to a point mutation (E115 K) in purB gene, coding for adenylosuccinate lyase. 38 The maximum growth rate of infected host cells was reduced to 0.24 h –1 in batch fermentation in stirred-tank reactors under comparable conditions regarding temperature, pH and dissolved oxygen concentration. Although phage infection reduces maximal growth rate, a predefined exponential feeding with a specific growth rate of 0.15 h –1 led to no difference of growth comparing infected and uninfected host-cell in the first 6 h after infection.…”

Engineered acetoacetate-inducible whole-cell biosensors based on the AtoSC two-component system

“…The acetoacetate sensor worked in both E. coli DH5α and E. coli Nissle 1917 but performed differently on each host and plasmid vector. Growth rates of E. coli DH5α were slow due to mutations in its genome that cause inferior growth phenotypes [33]. Median specific growth rates (µ) ranged between 0.0014-0.0022 for E. coli DH5α while 0.0035-0.0053 for E. coli Nissle 1917.…”