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

摘要: The strAS or rpsL+ gene, encoding a ribosomal protein, S12, expresses its streptomycin-sensitivity (SmS) phenotype dominantly over strAR rpsL- gene. Therefore, cells that harbor plasmids with alleles are phenotypically SmS. It was found the SmS is unstable, and such eventually switch to Sm-resistance (SmR) phenotype, especially when gene cloned on high-copy-number (HCN) plasmids. seemed strA HCN toxic Escherichia coli host and, during prolonged cultivation, an inactivated mostly carrying insertion sequence elements, as IS1, IS5 gamma delta, were selected. instability of particularly enhanced Val51 residue in middle S12 protein replaced by Leu, suggesting toxicity altered S12. Since stably maintained throughout approx. 100 cell doublings expression abolished, most probably it product rather than nucleotide itself responsible for To improve stability previously reported ampicillin-resistance-conferring SmS-enforcing plasmid vector, pHSG670, reconstructed. resulting pHSG683, confers chloramphenicol resistance, enforces supE- bacteria, has multiple cloning sites within coding region synthetic rpsL When pHSG683 DNA prepared from sup+ grown tryptophan-rich medium Cm Sm, less 10(-6) failed enforce tryptophan-less Cm.(ABSTRACT TRUNCATED AT 250 WORDS)