Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin

Abstract

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.