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

Abstract

The strAS or rpsL+ gene, encoding a ribosomal protein, S12, expresses its streptomycin-sensitivity (SmS) phenotype dominantly over strAR or rpsL- gene. Therefore, strAR cells that harbor plasmids with strAS alleles are phenotypically SmS. It was found that the SmS phenotype is unstable, and such cells eventually switch to the Sm-resistance (SmR) phenotype, especially when the strAS 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 strAS gene, mostly carrying insertion sequence elements, such as IS1, IS5 and gamma delta, were selected. The instability of the strA gene was particularly enhanced when the Val51 residue in the middle of S12 protein was replaced by Leu, suggesting enhanced toxicity of the altered S12. Since the strAS 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 SmS phenotype, the previously reported ampicillin-resistance-conferring and SmS-enforcing plasmid vector, pHSG670, was reconstructed. The resulting vector, pHSG683, confers chloramphenicol resistance, enforces SmS on strAR and supE- host bacteria, and has multiple cloning sites within the coding region of synthetic rpsL gene. When pHSG683 DNA was prepared from strAR and sup+ cells grown in tryptophan-rich medium with Cm and Sm, less than 10(-6) plasmids failed to enforce SmS on strAR and supE- cells in tryptophan-less medium with Cm.(ABSTRACT TRUNCATED AT 250 WORDS)