A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism

Abstract

Cyanobacteria are gram-negative prokaryotes that carry out oxygenic photosynthesis. In their function and ultrastructure, they have numerous similarities to chloroplasts in higher plants (19). These similarities and their prokaryotic nature make cyanobacteria good model systems for molecular genetic studies of chloroplasts. Many cyanobacterial strains contain endogenous plasmids of various sizes (2,6,8,28,29,31,39,40,43,(46)(47)(48)52), but thus far all of these plasmids have proven to be cryptic.Central to the genetic study of cyanobacteria has been the development of efficient cloning vectors that can transform cyanobacteria. However, the construction of these cloning and expression vectors is greatly dependent upon an understanding of DNA replication of cyanobacterial plasmids. At present, no cyanobacterial plasmids are found to replicate in Escherichia coli (27, 53), and no E. coli plasmids except a promiscuous IncQ plasmid (24) are known to replicate within cyanobacteria (26,45,58). Therefore, many shuttle vectors comprising E. coli plasmid and cyanobacterial DNA have been constructed (4, 9-13, 16, 22, 27, 30). However, these chimeric plasmids usually do not replicate efficiently in cyanobacteria (6, 45, 55), leading to difficulties in identifying these plasmids directly and employing them as efficient expression vectors in cyanobacteria. It appears that the copy numbers of the plasmids in cyanobacteria are stringently controlled. Furthermore, in many cases, shuttle vectors tend to integrate into chromosomal DNA and recombine with resident plasmids (26,49,55,57 been established that most plasmids from gram-negative bacteria (for example, F, pSC101, R6K, R1, and ColEl-type plasmids) replicate by a theta-form mechanism. In contrast, plasmids of less than 10 kbp from gram-positive bacteria, such as the pT181 and pUB110 families, replicate by a rolling circle mechanism via a single-stranded (ss) intermediate (17,25) and therefore are referred to as ssDNA plasmids (17). One exception is the recent discovery that plasmid pKYM from the gram-negative bacterium Shigella sonnei replicates by a rolling circle instead of a theta-form mechanism (59).With the exception of ColEl-type plasmids (23), most of the characterized plasmids encode a specific replication (Rep) protein which, in addition to the host proteins, is necessary for their replication (17,44). In contrast to the situation with E. coli, studies of the replication of cyanobacterial plasmids at the molecular level have never been performed. To enhance the understanding of the replication, genomic organization, and function of cyanobacterial plasmids, we report here the complete nucleotide sequence of a small plasmid, pCA2.4, isolated from the unicellular cyanobacterium Synechocystis strain PCC 6803. This plasmid contains an open reading frame (ORF) which, on the basis of the homology studies, probably encodes a replication protein (RepA). This putative RepA protein has been expressed in E. coli, and the gene product has been characterized. We also report the ...

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…Since no RNA-seq data were available for these plasmids (pCA2.4, pCB2.4, and pCC5.2), we retrieved their annotated sequences from GenBank and identified two regions on each plasmid without annotated transcripts or repeated and/or inverted regions of DNA, which have previously been suggested to have significant functions (8)(9)(10)(11) (Table 1).…”

“…strain PCC 6803 is a naturally transformable cyanobacterial chassis for synthetic biology. This strain carries several endogenous plasmids in addition to its single chromosome (8)(9)(10)(11). Since there are a limited number of self-replicating exogenous plasmids for the expression of heterologous genes in Synechocystis PCC 6803 (12) and other cyanobacteria, these endogenous plasmids may prove to be attractive parts for synthetic biology (13).…”

Transfer of a genetic marker from a megaplasmid of Anabaena sp. strain PCC 7120 to a megaplasmid of a different Anabaena strain

“…1, arrow b (10,17,26). All cyanobacterial plasmids heretofore studied remain cryptic; i.e., beyond replication functions (20,(24)(25)(26)30), there are no known functions encoded by them except that a normally silent copy of psbG has been found to be plasmid localized in Synechocystis sp. strain PCC 6803 (22).…”

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

“…There are 7 plasmids in Synechocystis sp. PCC 6803 (Kaneko et al, 2003;Xu and McFadden, 1997;Yang and McFadden, 1993;Yang and McFadden, 1994) and 6 in Nostoc (also known as Anabaena) sp. PCC 7120 (Kaneko et al, 2001).…”