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Results for “temperature-sensitive mutations”

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PubMed2023

Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

Escherichia coli↗

PubMed2025

Mutation of the gidB gene causes intrinsic streptomycin resistance in Bacillus velezensis.

Bacillus velezensis strain DMB07, isolated from the traditional fermented Korean soybean meju, exhibits resistance to streptomycin [minimum inhibitory concentration (MIC) 128 mg/L]. To shed light on the genetic background behind this phenotype, this study determined the complete genome sequence of strain DMB07 and compared it with the genomes of two B. velezensis strains that are sensitive to streptomycin. Compared with the streptomycin-sensitive strains, in strain DMB07 there was a mutation of a nucleotide (C58T) of the 16 S rRNA (guanine527-N7)-methyltransferase gene (gidB) that leads to a change in the amino acid sequence of the protein (Arg20Cys). This sequence of gidB gene was previously linked with streptomycin resistance. To test the hypothesis that this change in the gidB gene sequence of strain DMB07 confers streptomycin resistance, a temperature-sensitive plasmid, pIMAY-tgidBT58C, was constructed for site-directed mutation (from thymine to cytosine) of nucleotide 58 of gidB in strain DMB07. The resulting strain, DMB07gidBT58C, showed the decreased MIC value (32 mg/L) against streptomycin. Furthermore, introduction of the wild-type gidB gene into strain DMB07gidBT58C resulted in recovery of the MIC for streptomycin to 128 mg/L. Thus, a single mutation of the nucleotide sequence of the gidB gene can confer resistance to streptomycin.

Streptomycin↗

PubMed2025

Approaches to Study Proteins Encoded by Essential Genes.

Although the phenotypes and functions of nonessential proteins can be studied by deletion of their coding sequences (both gene copies in diploid organisms), essential genes cannot be deleted unless loss of the encoded protein can be bypassed. Bypass is often achieved by supplementation with the product of the enzyme. However, supplementation cannot bypass loss of essential genes such as those encoding enzymes of DNA or RNA synthesis. To study proteins encoded by essential genes that cannot be bypassed, the mutations must be conditional in nature. The mutant cells must be able to grow under a permissive condition, but fail to grow under a different condition, the nonpermissive condition. Several methods have been developed to obtain conditional mutations in essential genes. Mutations that result in proteins abnormally sensitive to high temperatures are called temperature-sensitive (Ts) mutants and are a widely used type of conditional mutation. An alternative to Ts mutants is the "degron" system to target proteins for destruction by cellular proteases. Approaches to conditionally control the functions of proteins encoded by essential genes, plus the advantages and disadvantages of these and other approaches, will be considered.

Genes, Essential↗