1022-7954/04/4007- © 2004
Russian Journal of Genetics, Vol. 40, No. 7, 2004, pp. 813–816. Translated from Genetika, Vol. 40, No. 7, 2004, pp. 997–1001.
Original Russian Text Copyright © 2004 by Samoilenko, Syrchin, Tsimbal, Mendzhul.
Cyanobacteria are a unique group of microorgan-
isms that are morphologically similar to Gram-negative
bacteria but are capable of oxygenic photosynthesis
characteristic of higher plants. Like the vast majority of
prokaryotes, cyanobacteria contain a broad set of
endogenous plasmids varying in copy number and size.
Notwithstanding the unique taxonomic position of these
microorganisms and prospects of using their plasmids in
gene engineering, cyanobacterial plasmids have not been
studied in sufﬁcient detail. Only a few plasmids are now
known, and only some of these have been examined for
structural and functional organization [1–8].
The major objective of studies with cyanobacterial
plasmids is to construct effective vectors of cloning
DNA fragments in cyanobacteria. To ensure its stable
replication in cyanobacterial cells, a vector must be
provided with an autonomous replicon isolated from an
endogenous plasmid of the host. At present, a well-
known cyanobacterial replicon is a 1.75-kb fragment of
plasmid pDU1 of
PCC 7524 . The fragment
contains a gene whose expression product is the repli-
cation-associated Rep protein. Genes for similar pro-
teins are contained in other cyanobacterial plasmids
such as pCA2.4, pCB2.4, and pCC5.2 of
PCC 6803 [3–5]; pSY10 of
042902 ; pMA1 of
Kutsing ; and some others.
By speciﬁcs of their replication, these plasmids have
been assigned to the rolling circle replication (RCR)
type [3–10]. On the other hand, substantial homology
to small plasmids of Gram-positive bacteria has made it
possible to class some of the plasmids to a broad
pUB110/pC194 family [9, 10]. This family is now rap-
idly accumulating atypical, including cyanobacterial,
plasmids. Their assignment to the RCR type was earlier
based on speciﬁc features of the origin of replication
) [9, 10], yet analysis of the amino acid sequence of
Rep proteins proved to be more reliable .
Several properties of cyanobacterial plasmid pSM1,
which has been isolated from
CALU 465, are indicative of its rolling circle replica-
tion . We have constructed vectors on the basis of
pSM1 and preliminarily localized its
and a gene for
the Rep protein [13–15]. Plasmid pSM1 is one of the
smallest known natural replicons, which allowed us to
obtain a relatively small shuttle vector.
The objectives of this work were to establish the nucle-
otide sequence of the
gene of pSM1 and to compare
the amino acid sequence of the Rep protein with
sequences encoded by related cyanobacterial plasmids.
To sequence the
gene, we used recombinant
plasmid pBSM1, which represents multicopied vector
pBluescriptII SK(+) (Stratagene) with cyanobacterial
pSM1 cloned into its unique
I site . Sequencing
was carried out in a CEQ 2000 XL automated sequencer
To identify the open reading frame in the sequence
established, we used the BCM GeneFinder program avail-
able from the Baylor College of Medicine Search
Launcher/). A search for homologous nucleotide and
amino acid sequences in databases was performed with
the BLASTN and BLASTP programs (National Center
for Biotechnology Information, http://www.ncbi.nih.
gov/BLAST/). Nucleotide and amino acid sequence align-
ments were obtained with the ClustalW program .
To identify the open reading frame corresponding to a
gene for the Rep protein, we sequenced a pSM1 fragment
sized more than 1100 bp. Sequence analysis took account
of the well known fact that the region upstream of the
gene often lacks a ribosome-binding site . This struc-
tural feature was also observed for plasmid pSM1.
Note that the Rep proteins of RCR plasmids each con-
sist of approximately 300 amino acid residues [9, 10].
This was also taken into account in identifying the open
reading frame in the sequenced pSM1 fragment. In ﬁve
out of six possible variants, translation of the nucleotide
sequence yielded a polypeptide chain consisting of
somewhat more than 100 residues. Only a single variant
The Nucleotide Sequence of the
of Cyanobacterial Plasmid pSM1
V. A. Samoilenko, S. A. Syrchin, N. V. Tsimbal, and M. I. Mendzhul
Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kiev, 03143 Ukraine;
Received June19, 2003; in ﬁnal form December 5, 2003
—The nucleotide sequence was established for the
gene of plasmid pSM1 isolated from cyano-
CALU 465. Both nucleotide sequence and the encoded amino acid sequences
showed 98% homology to the corresponding sequences of small plasmids pPF1, pGL3, pPBS1, pBLX, and
pPB1. An active center was identiﬁed in the replicative protein sequences.