Cloning and expression in streptomyces lividans of antibiotic resistance genes derived from Escherichia coli.
Hybrid plasmids that replicate in both Escherichia coli and Streptomyces lividans were constructed in vitro by joining the E. coli-derived plasmid pACYC184 or pACYC177, at their BamHI or PstI restriction site, respectively, to S. lividans plasmid pSLP111. After introduction of the composite replicons into S. lividans by transformation, chloramphenicol (Cm) resistance encoded by pACYC184 and kanamycin resistance encoded by pACYC177 were phenotypically expressed in the S. lividans host. A Sau3A restriction endonuclease-generated deoxyribonucleic acid fragment from pACYC184 containing the entire structural gene for the Cm acetyltransferase enzyme, but lacking the nucleotide sequence ordinarily serving as the Cm resistance gene promoter, also specified resistance to Cm when introduced in either orientation into the BamHI or BclI endonuclease cleavage site of pSLP111 or into corresponding sites of the analogous plasmid pSLP101. These findings make it unlikely that the biologically active CM acetyltransferase was being made in S. lividans as part of a fused protein, but instead indicate that the ATG start codon used for initiation of translation of the Cm resistance gene in E. coli was also utilized in S. lividans. In contrast, the synthesis of messenger ribonucleic acid that encodes the Cm acetyltransferase in S. lividans was, in at least one instance, apparently initiated at nucleotide sequences within the S. lividans plasmid vector, with resulting transcriptional read-through into the E. coli-derived deoxyribonucleic acid segment.
D000123:Acetyltransferases / Q000235:genetics; D002701:Chloramphenicol / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase; D004274:DNA, Recombinant; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D007612:Kanamycin / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D010957:Plasmids; D013302:Streptomyces / Q000235:genetics*; D014169:Transformation, Bacterial
D004274:DNA, Recombinant; D007612:Kanamycin; D002701:Chloramphenicol; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase