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Query: UMLS:C1832526 (PCC)
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In some strains of cyanobacteria the composition of the light-harvesting antennae is determined by the color of available light. The mechanism of this chromatic adaptation involves the regulation of gene expression by red and green light and has been most studied in Fremyella diplosiphon (Calothrix sp. PCC 7601), a filamentous cyanobacterium for which there has been no reported means of genetic manipulation. We have constructed shuttle plasmids which can be efficiently mobilized by RP4 from Escherichia coli into F. diplosiphon and which can be recovered from transconjugant F. diplosiphon and returned to E. coli by transformation. The ability of these plasmids to replicate in F. diplosiphon is conferred by an 8.0-kb DNA fragment isolated from pFDA, a plasmid native to F. diplosiphon. To create these shuttle plasmids the 8.0-kb fragment was cloned into pJCF22, a mobilizable plasmid constructed from oriV and bom from pBR322, cat from pACYC184 and aphA from pACYC177.pJCF22 lacks sites for the restriction enzymes FdiI and II. Transconjugant F. diplosiphon containing shuttle plasmid pJCF62 are resistant to chloramphenicol and highly resistant to the aminoglycosides, G418 and neomycin. When aadA from the omega interposon was incorporated into a shuttle plasmid transconjugant F. diplosiphon could also be selected with streptomycin or spectinomycin. In F. diplosiphon shuttle plasmid pJCF62 replicates with a minimum copy number of seven. The oriV for replication in F. diplosiphon was localized to a 2.8-kb region within the cyanobacterial part of pJCF62. The presence on a shuttle plasmid of a single recognition site for FdiI reduced the efficiency of mobilization into F. diplosiphon by 5- to 10-fold. Restriction at this site was prevented when the E. coli donor strain in the mating contained the enzyme Eco47II methylase.
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PMID:Construction of shuttle plasmids which can be efficiently mobilized from Escherichia coli into the chromatically adapting cyanobacterium, Fremyella diplosiphon. 823 95

Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPFT-type IV secretion systems, were able to transfer plasmid DNA from E. coli to S. elongatus by conjugation. Neither MPFF nor MPFI could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in E. coli by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between proteobacteria and cyanobacteria and point to MPFT-based interphylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of S. elongatus xenologous genes.
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PMID:Plasmid conjugation from proteobacteria as evidence for the origin of xenologous genes in cyanobacteria. 2450 15