Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria

Abstract

We have used the TEM beta-lactamase transposon Tn901, located on Escherichia coli plasmid pRI46, to introduce in vivo a genetic marker into plasmid pUH24, present in the cyanobacterial strain Anacystis nidulans R-2. Restriction enzyme analysis and heteroduplex studies of the 8.3 x 10(6)-dalton plasmids pCH1-pCH5, present in the ampicillin-resistant A. nidulans R-2 colonies obtained after transformation with pRI46, demonstrated that these plasmids consist of the complete sequence of Tn901 inserted at different places into plasmid pUH24. The pUH24::Tn901 recombinant plasmids transform A. nidulans R-2 with a frequency of 10(-4)--10(-5) per microgram of plasmid DNA and contain a single cleavage site for the restriction enzyme Xho I. From pCH1 a plasmid of 5.5 x 10(6) daltons,pUC1, was constructed with only a part of the Tn901 sequence and an additional single cleavage site for the restriction enzyme BamHI. This plasmid, as well as plasmids pCH1-pCH5, are potentially useful as vectors for cloning genes in cyanobacteria and for studying cyanobacterial plasmid biology.