A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism.
Different cryptic plasmids are widely distributed in many strains of cyanobacteria. A small cryptic plasmid, pCA2.4, from Synechocystis strain PCC 6803 was completely sequenced, and its replication mode was determined. pCA2.4 contained 2,378 bp and encoded a replication (Rep) protein, designated RepA. An analysis of the deduced amino acid sequence revealed that RepA of pCA2.4 has significant homology with Rep proteins of pKYM from Shigella sonnei, a pUB110 plasmid family from gram-positive bacteria, and with a protein corresponding to an open reading frame in a Nostoc plasmid and open reading frame C of Plectonema plasmid pRF1. pKYM and pUB110 family plasmids replicate by a rolling circle mechanism in which a Rep protein nicks the origin of replication to allow the generation of a single-stranded plasmid as a replication intermediate. RepA encoded by pC2.4 was expressed in Escherichia coli cells harboring a vector, pCRP336, containing the entire repA gene. The observed molecular weight of RepA was consistent with the value of 39,200 calculated from its deduced amino acid sequence, as was the N-terminal sequence analysis done through the 12th residue. Single-stranded plasmid DNA of pCA2.4 that was specifically degraded by S1 nuclease was detected in Synechocystis cells by Southern hybridization. These observations suggest that pCA2.4 replicates by a rolling circle mechanism in Synechocystis cells.
Yang
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Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
McFadden
B A
BA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics*; D004265:DNA Helicases; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011506:Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators
D000596:Amino Acids; D001426:Bacterial Proteins; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D011506:Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases