Characterization of the spontaneous elimination of streptomycin sensitivity (Sm^s) on high-copy-number plasmids: Sm^s-enforcement cloning vectors with a synthetic rpsL gene

The strA^s or rpsL^+ gene, encoding a ribosomal protein, S12, expresses its streptomycin-sensitivity (Sm^s) phenotype dominantly over strA^R or rpsL^- gene. Therefore, strA^R cells that harbor plasmids with strA^s alleles are phenotypically Sm^s. It was found that the Sm^s phenotype is unstable, and...
Ausführliche Beschreibung

The strA^s or rpsL^+ gene, encoding a ribosomal protein, S12, expresses its streptomycin-sensitivity (Sm^s) phenotype dominantly over strA^R or rpsL^- gene. Therefore, strA^R cells that harbor plasmids with strA^s alleles are phenotypically Sm^s. It was found that the Sm^s phenotype is unstable, and such cells eventually switch to the Sm-resistance (Sm^R) phenotype, especially when the strA^s gene was cloned on high-copy-number (HCN) plasmids. It seemed that the strA gene cloned on HCN plasmids was toxic to Escherichia coli host cells and, during prolonged cultivation, plasmids with an inactivated strA^5 gene, mostly carrying insertion sequence elements, such as IS1, IS5 and γδ, were selected. The instability of the strA gene was particularly enhanced when the Val^5^1 residue in the middle of S12 protein was replaced by Leu, suggesting enhanced toxicity of the altered S12. Since the strA^S gene was stably maintained throughout approx. 100 cell doublings when its expression was abolished, most probably it is the gene product rather than the nucleotide sequence itself that is responsible for the instability of strA gene on HCN plasmids. To improve the stability of the Sm^s phenotype, the previously reported ampicillin-resistance-conferring and Sm^s-enforcing plasmid vector, pHSG670, was reconstructed. The resulting vector, pHSG683, confers chloramphenicol resistance, enforces Sm^s on strA^R and supE^- host bacteria, and has multiple cloning sites within the coding region of synthetic rpsL gene. When pHSG683 DNA was prepared from strA^R and sup^+ cells grown in tryptophan-rich medium with Cm and Sm, less than 10^-^6 plasmids failed to enforce Sm^s on strA^R and supE^- cells in tryptophan-less medium with Cm. This vector is extremely powerful and useful for efficient detection of the rare incidence of DNA recombination or genetic alterations in vitro and in vivo. Ausführliche Beschreibung