Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae.
Attempts to clone wild-type DNA containing the malM gene of Streptococcus pneumoniae in plasmid pBR322 of Escherichia coli were unsuccessful. However, it was possible to clone a PstI fragment of DNA containing this gene in a plasmid of S. pneumoniae. Cells carrying the recombinant plasmid produced large amounts of the malM product, amylomaltase, and a fragment of the protein coded by the adjacent malX gene, apparently as a result of transcription in opposite directions from strong promoters located between the two genes in the plasmid insert. Under derepressed conditions these products represented 10% of the total protein. No transcription terminators appeared to be included within the cloned segment. The effect of various mutations in the segment on its ability to be cloned in pBR322 was examined. Of those tested, only a down promoter mutation that affected production of both the amylomaltase and the X-protein rendered the segment clonable in E. coli. Fragments of the S. pneumoniae vector, pMV158, which appear to lack strong promoters, were readily cloned in the pBR322-E. coli system. Although it is possible that large amounts of the X-fragment are toxic for E. coli, a more general explanation would be that excessive transcription of the pBR322 vector portion interferes with maintenance of the recombinant plasmid.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D009876:Operon; D010957:Plasmids; D013296:Streptococcus pneumoniae / Q000235:genetics*; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes