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DNA sequence and analysis of a cryptic 4.2-kb plasmid from the filamentous cyanobacterium, Plectonema sp. Strain PCC 6402
PLASMID
28, 170-176 (1992)
DNA Sequence and Analysis of a Cryptic 4.2-kb Plasmid from the Filamentous Cyanobacterium, Plecfonema sp. Strain PCC 6402 DOUGLAS R. PERIUNSAND~USAN R. BARNUM' Department of Botany, Miami University, Oxford, Ohio 45056 Received September 3, 199 1; revised March 1, 1992 The 4194-bp plasmid, pRFI, from Plectonema sp. Strain PCC 6402 was completely sequenced and analyzed. Seven potential open reading frames were identified. The predicted amino acid sequence of open reading frame C (ORF C) had identities of 34,29, and 25% with Rep B from the Staphylococcus aureus plasmid, PUB 110; Rep from the Bacillus amyloliquefaciens plasmid, pFTB14; and protein A from the S. aureus plasmid, pC194, respectively. A 75-amino-acid region conserved in these proteins (Rep B, Rep, and protein A) also was highly conserved in ORF C with identities of 45, 37, and 40%, respectively. Significantly, 16 of the 2 1 amino acids conserved in Rep B, Rep, and protein A were found at the same positions in ORF C. This ORF may encode a replication protein that includes a region conserved in some eubacteria. Additional structural features include a 425-bp region that contains palindromes, tandem repeats, and short direct repcats which may correspond to the origin of replication. An 1%bp inverted repeat was located between two open reading frames, A and G. o 1992 Acadrmlc PESS. 1~.
Since the first discovery of plasmids from the unicellular cyanobacterium, Anacystis nidulans (Synechococcus) (Asato and Ginoza, 1973) other strains such as Synechocystis (Lau et al., 1980; Chauvat et al., 1985; Van den Hondel et al., 1979; Schwabe el al., 1988; Bose and Carmichael, 1990), Anahaena (Lambert and Carr, 1982; Lambert et al., 1984; Simon, 1978), Oscillatoria (Friedberg and Seijffers, 1979), Nostoc (Lambert and Carr, 1982; Lambert et al., 1984; Reaston et al., 1980), Ca1othri.x (Simon, 1978; Bogorad et al., 1983), and Plectonema (Simon, 1978; Potts, 1984; Felkner and Barnum, 1988) were also found to contain plasmids. They have been characterized by restriction mapping (Kuhlemeier et al., 1981; Lambert and Can-, 1983; Van den Hondel, 1979) plasmid content and size (Felkner and Barnum, 1988; Lau et al., 1980; Potts, 1984; Simon, 1978) searching for transcripts and/or proteins encoded by plasmids (Gruber et al., 1987; Schwabe et al., 1990), and DNA sequencing (Van der Plas, 1989; Wickrema,
’ To whom correspondence 0147-619X/92
should be addressed.
$5.00
CopyrIght 0 1992 by Acadrmlc Press. Inc. 411 rights of reproduction tn any form reserved
1989). Although genes on some eubacterial plasmids have been identified, cyanobacterial plasmids remain cryptic. Some inconclusive studies have been conducted to elucidate functions for plasmids (Singh et al., 1986; Castets el al., 1986). Suggested functions include antibiotic resistance (Kushner and Breuil, 1977), transfer of restriction enzymes (Whitehead and Brown, 1985) toxin production (Hauman et al., 1982), and an increase in transformation efficiency (Chauvat et al., 1983). However, definitive experiments have not been conducted. In a first step toward studying plasmid-encoded cyanobacterial genes, the 4.2-kb plasmid, pRF1 (Felkner, 1988), from Plectonema sp. Strain PCC (Paris Culture Collection) 6402 was sequenced and analyzed. The plasmid, which was previously demonstrated to contain one EcoRI site (Felkner, 1988), was ligated into the EcoRI site of pBR322 (pRF2; Felkner, 1988) and amplified for doublestranded dideoxy sequencing (Sanger et al., 1977). Both strands of the 4194-bp plasmid (designated plus and minus) were sequenced from pRF2 with each new primer set (forward and reverse) determined by the pre170
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vious sequenceobtained. Sequenceinformation from a 50-base region, positions 2 1972247, on the minus strand could not be obtained even after dITP or 7-deaza dGTP was substituted in the reactions, the temperature of the reactions was increased, and the concentrations of template, primer, and Sequenasewere modified. However, the complementary strand was sequenced several times to check for errors. Open reading frames were identified by the presence of start (ATG) and stop codons and the potential to encode a polypeptide of at least 50 amino acids. Seven potential open reading frames (ORFs) of at least 150 bp were identified. The ORFs on the plus strand were located at positions 9661133, 1255-1473, 1382-1828, and 23483226, relative to position 1 at the EcoRI site and are referred to as ORFs A, B, C, and D, respectively (Fig. 1). The ORFs on the minus strand were located at positions 3825-3538, 1195-989, and 555-301 and are ORFs E, F, and G, respectively (Fig. 1). A 12-bp palindromic sequence,CAAAACGTTTTG, was repeated five times at positions 8, 44, 76, 168, and 236 (Fig. 1). Two 7-bp direct repeats, GATTTTT, were found at positions 292 and 33 1. An 18-bp inverted repeat, AGAAGTTTGCTTACGGAT, was located between ORFs A and G at positions 773 and 793. Four short direct repeats, CAATC, were identified at positions 4 107, 4115,4123, and 4131 and four 8-bp tandem repeats, ACTTTGTG, were located beginning at position 4 141. The complete DNA sequence of pRF1 with the ORFs and repeated sequencesunderlined is shown in Fig. 1. Interestingly, the repeats were localized mainly between positions 4 107 and 337. The fact that these repeats were found in such a small region relative to the size of the entire plasmid suggeststhat this region may be the origin of replication. The sequence CAATC, beginning at position 4 107, may be considered the beginning of the repeat region since the plasmid is a circular molecule. The nucleotide sequenceof the entire plasmid, the individual ORFs, and the deduced amino acid sequences of the ORFs were
171
aligned to bacterial sequences in the GenBank and NBRF databases. Optimal alignments were conducted both manually and with MacVector (International Biotechnologies, Inc.). The predicted amino acid sequence of ORF C was 149 residues and showed identity with two replication proteins, RepB from the staphylococcal plasmid, pUBll0 (Muller et al., 1986; McKenzie et al., 1986) and the rep product (Rep) from the Bacillus amyloliquefaciens plasmid, pFTB 14 (Murai et al., 1987). Identity was most concentrated in a 42-amino-acid region of ORF C between positions 176 and 2 17 (Fig. 2). A 75-amino-acid region of ORF C from the first methionine to the 1l-amino-acid gap corresponded to a conserved region of Rep from pFfB14 (Murai et al., 1987), protein A from the S. aureus plasmid, pC 194 (Horinouchi and Weisblum, 1982) and RepB from pUBll0 (Muller et al., 1986; McKenzie et al., 1986). Identities with ORF C were 45,37, and 40%, respectively. When conservative amino acid substitutions were included, the identities increased to 65, 52, and 57%, respectively. This was part of a larger highly conserved region of Rep, Rep B, and protein A from positions 119 to 2 17 (Murai et al., 1987; Fig. 2). We determined the identities between the highly conserved region of protein A and Rep, protein A and RepB, and Rep and Rep B to be 48,33, and 50%, respectively. Identities of the complete proteins were ORF C to Rep B, 34%; ORF C to Rep, 29%; ORF C to protein A, 25%; Rep B to Rep, 39%; protein A to Rep B, 16%;and protein A to Rep, 33%. The identities of ORF C with the replication proteins, Rep B and Rep, and protein A from pC194 were well within the range of identities seen between replication proteins. In addition, of the 21 amino acids conserved in all three proteins (Rep B, Rep, and protein A), 16 were found in ORF C at the sameposition. Basedon these values, a potential function for ORF C may be the production of a replication protein. No similarities to additional sequences were found when the entire plasmid or the other six ORFs and the translations were
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SHORT COMMUNICATIONS 70 TTCTGCTCAAAACGTTTTGAGCAACAGCGCGCGACTTCG 140 TTACCCAAAATGTTTTGGGTAGAAGCAATATCGCCGGGAA 210 CTGGCAATGGCACGATCTTGGAGTACTCAAAACGTTTTGATGC 280 GGGTTTGGTAATTCAAAGCTTGCC~CGTTTTG~C~CAGCTCGATCGAG~TTTGTGGTCG~G 350 CAACCCATGTTGATTTTTTAGAGGTTGAAGAACCTAGAAACCCACCCGA 420 AACTGTATGGAATTTTAGCTTTGATCTCTCTCCT~TACGATTCCTTGGCTGACGCAT~CATTTTCTGTTG 490 AGCTTCCTCGTAGGATTCAGCTGTGATTTCAAAACACCACCATGTCGCGCCTTTATATCGGTATTCACACAGA 3
560
AATGTTTTTTCTCGACTACATCTAGTGATTCATTTGTATT 630 ATTACGTTTATCAAGACAAGCTGGTGAGTGTGGCGCTGGTAT~~GCAGCT~T~CAGG~G~T 700 GCTGACTCTGGTGAAGGGTATAAAAACAAGCAGGATTGCAGGATTGCTAC~CGCGATCGGGATGGTTT~CGT~G 770 G09ATTCTCCTGTTTATGATCTAATTTTTGTGGGTGTTGCG~GCGGTTCGCCTTCACTT~CCT~T 840 AGAGAAGTTTGCTTACGGA~TG~TCCGT~GC~CTTCTTACAGGAC~GC~CTCACGC~TTT~GG 910 CGTAATCGAAGCGTAATTTCTGTGATGGGGGAAAGCACTAAAAAA
CCGCTGTACACTGGTAACAACCACG
A, AAACCAAGTAAATACAACGGCTTTTTTATGTCT~~C~CCCCGGAGATGCTGTTATGTCTAGTGTATCG
980 1050
TCTCGTCACGAGACAGCCTAGGACAACAGCAGCACTGTC 1120 TCACCGTAGTAGCAGCGATAAAGTTGCAGGACATTATCGC 1190 TT~ACTATTGCAGTGATTTTTTGGA~TT~~GGTTAGT~C~GAT~AGGA~AG~TC~TA~~GTTTG~GTT k-1260
-F G~CTGCACGATTTTGTCATTGCCGACAATGCATTGTATGTTCCTGGCGCAG~GTCTCGC~~G
1330 GCGAGGGCTTATAAGACTTTGCCCAAAGTAGTAGTTGCTGA~ATCCGACTGCACGATACCTTTTCCTCACGC
C
1400
TGACAGTGAAGAACTGCGCGATTACCGACCCGTCTGCGGACACTC~TGG~TCAGTCATTTTCCA 1470 GAATGACAAAGCTCAAAGATTGGCAGGCTTTGGGCTATCTATCTCCG~C~CAG~GTGACGCGG~~GAGA 1540 CGGTAAGTCAGCACACCCTCATTTCCACTGCCTATTGATGGT~G~TCCTATTTCGGTCGG~CTAC 1610 ATTTCTCAGGCTGAATGGGTAGAACTATGGAAGAAGTCGTTGCGAGTGGACTAC~CCC~~TTGGATG 1680 TTCAAGCGCTTAAAACAGAGTCTTCCTTGGTTGGTTG’PGCC 1750 AAATGATTTAGTGCTATCAGATCGTGATTGGTTTTTGGAACG 1820 ATCGCATTTGGAGGAATTTTCAAAGAGTACTTCCGTGAATTGCT 1890 TGGTCAGATAGCCCAAGCCGGAGGTTGATGAGGGACACCTCTATTTT~CTGG~CGCGT~GAG~ 1960 GAAGTACCGAATGATTGATAACAATGTATAACAGCGTTATC~TTATGTAGTTTGTGTATACC~G~ 2030 TTTGTTATCACTCTTGATAACATGGTTGAAATTCTTTCTGTACGTATATGCAGTT~CCG~GATCTGCT 2100 CATTGAATCCCCGACTGCCCGTAATGAGGGAGGGAGTCCTAG~TC~TCGTC~TC~GGGAGC~GAGAT 2170 TCTTAACAAGGTGAAAGTCTTGTTGTTTGCTGCTGTCTGGGATGG~CCCCACG~TGTGGCGGCAGAGTTGG
FIG. I, Complete nucleotide sequence of pRFl from Plectonema sp. Strain PCC 6402. Open reading frames are underlined and labeled as described in the text. Overlapping sequencesare double-underlined. Palindromes and repeats also are underlined. Direct repeats beginning at positions 292 and 33 1 are identified by a line above the sequence.
SHORT
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COMMUNICATIONS 2240
CAGATTTTTATGAAGTCTCGCTGGATGCCGTAAAAAA
CTACCAGCGGTTCAGGGATGAGTTTGAGCTTGA 2310
TGGAGTAAAAGTCGTGCGTGT~GGATTTGAAGAGGTTACCTCCTTCACTCC L TCAGGAGGTTATTTACACCCCTGCTGGTGCGTTGAGAATGTTCTCCGCGATTCACCTGTAGCG
2380 2450
AAGGCGGTTAGAACAGCAGCGATTCGAGTCATCCAAGGTGCTGCGT 2520 TTGCGAAATTGGTTGAGGGTCATTCCGGGCTTGACCAA?TTGCCGC 2590 TTTGTACGAAGAGGGGATTTTTAAGGATGCTTGCTTGCAPAGCTTTGCGGAAAAAA TATCCAGATGGMAACTT 2660 CCAGGACTGGGCGTAGAACAGTTGAAGGAATTTTTTTGC~TGTTCGCCACTTATACAGATCGTTGGGTGT 2730 TC 2800 TTCTTTTCCTGTAGAGGTTGATGGGCAAGTCAAGCAGGCAGGCTGTGATCATGT~CAGTTCTGTGATTTCCTG 2870 GTTGATGATCGGTATGTTGCTGATTGTGATAGTCGTAGATACATC~TCAGTC~G~CCTGCCTGGCG 2940 TAGATTTAGCGTGGCTGTTCTTTGTTTCCCCGTTTGGTGGTGCTACACCTGCTGCCGTG~TTTATCGAGCA 3010 GGAGATTCGCCCTGACTCTCAAGGTTACGTTACGTTATT 3080 GCTCAAGCTTCCCAGAATCGCAATAGCAATCTTCTCATTG 3150 TGGGCTATGAGATTCCGGTAGTCGAGATGGCAACTCAGTACGTTATCG 3220 ATTTGATIL4CGTTCTGGTTCTTGTAGACATCCCAGACATCCCAG~C~TTCTTGTCTTTTTCTACCTGGTGTTTCT~ 3290 GCGCTTTGAGAGAA04ATGGTGTAGAAACCTATTTGTGCTATTTGTGCTGTCTGCTTTTTTGACTGTTTAGTGTTGTTG 3360 ACTTTGTTTTTCAGTCTCTTGGGTCTTCCTGCTCTAACCTCTACTGTTCTTA 3430 GCGTTCCAAGGAATGCTCTATGAGGTAGGCTTTCTTCTTTGTTATCAGTTATGTTATC~C~CATTATC 3500 AAAGTTCTGAATGATAACTTCTTCATCCTCGTCTTTGCTACTTAGGATGTTATC~GATGGTTATCAGAT 3570 ACGATAACATCTTGAAGGTCTAATC;GAATCTGATCTATTTTTTCCTTGCTGTCCTGCTTGACGTAGATGT 3640 GTCTAGCTGACCAGCAAACACTTCTCAAATCCTGAGCGTCTCGCTCT~GATTTC~CTTTTTTTCT 3710 c 3780 TAGAGAGAGCCTTGATTAGAGTCTGTCTTCGATTCATTCGTCTGCTGTCGATACTCCTCC~GCA~ +L 3850 GTAAAGACTCTGAGGAATGCGACCGGAG AAAAAAGGTCTAGTCATGCTCGTTATCGATTTCGATAACTTA 3920 ATGATAACAGATAGTGTTCTCTTGCATCTTTCTTCGATAATC 3990 AAGGGATCGTGGAATAGCGGACTGAG~GGAGAGCATCGC~TTGACGGC~GATTGA 4060 ACCGCTACACTCTTGGGCTGTTCCGAGGGCTGATAGATGCGTTGTGTAGTTGTTATTGATTTGATAGCAG 4130 CCCTCGGAACATGCCACGCGGCGAGCGGTCTTTCTTTCTCTG~TC~GC~TCTTGC~TC~~~ 4194 CAATCTTGCAACTTTGTGACTTGTGACTTTGTGACTTTG FIG. 1-Continued
174
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ORF C rep pro A Rep B
79 ............................................................................... MYSSENDYSILEDKTATGKKRDWRGKKRRANLMAEHYEALEKRIGAPYYGKKAERLSECAEHLSFKRDPETGRLKLYQA . . . . . . . . . . . . . . . . . . . . ..MCYNMEKVnKKQR~VFQKFIKRHlGE~MDLVEDCKTFLSFVAOKnEKQKLYKA ...... .........................................................................
ORF C rep pro A Rep B
158 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I+(PSFSRMlKLKD.WQAL. HFCKVRLCPMCAWRRSLKIAYHNKLIIEEANRQYGCGWIFLTLTVRNV.ffiERLK~ISElMEG~KLFQYKKVKTSVL NSCKNRFCPVCAWRWRKDALGLSL~YIKQQEKKEFIFLnTTPNVMSDE.LENEIKRYWNSmKLIKRKKVGSVIK . . . . . . . . . . . . . . . MKHGIQSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSOICR@XRRbbQYKKINKNLV
ORF C rep pro A Rep B
GYUUXVTRGRDGKSAH WKASYF..GRNYISQAEWVELWKKSLRVDVNPlL...........IJVQAL..K ffFRALEITKNHEEDTY~HVLLPmRN~..6K~lK~SLmRAMKLDYTPIVDIRRVKGRVKIDAEQIESD GWW(LEIT(NKKRDDYWLlAYNKSYFTDKRYYl~MDLW.....RDVTGISElTQVQVQKl~NNNKELY sFMRATmlNNKDNSY~~LVCVEPTYFKNTEN~~KQWIQHWKW\MKLDYDPNV...........KVQMIRPK
ORF C rep pro A Rep B
TE..SSLVGLLAElI~CKPND..LVLSDRDW.F..L.ELTRQLHm
ORF C rep pro A Rep B
........................... EDDEVANGAFEVMAYWHPGIKNYILK. ........................... DDEKADEDGFSIIAMWNWERKNYFIKE
237
316
343
FIG. 2. Amino acid alignments of proteins (from Murai et al., 1987) Rep B (235 amino acids), Rep (339 amino acids), and protein A (229 amino acids), with the predicted amino acid sequence of ORF C. Gaps are added to improve the alignments. ORF C was aligned to Rep B (with a gap of 11 amino acids) which gave a better alignment than if a gap was not introduced. Numbering is based on the number of total amino acid positions. Identities with ORF C are shown in bold.
used in alignments. Protein A from pC 194 is coding regions, transcriptional start sites, and most likely not involved in replication of the promoter regions. plasmid (Horinouchi and Weisblum, 1982); however, protein C from pC 194, a putative ACKNOWLEDGMENTS replication protein, did not show identity with ORF C or any of the other ORFs from This work was supported by an OBOR Research ChalpRF1. The sequence of the entire plasmid lenge grant and a Miami University CFR grant to S.R.B. and the individual ORFs were compared to and a graduate student research award to D.R.P. from a the DNA sequences of pGL3 (Wickrema, Department of Botany OBOR Academic Challenge 1989) from Plectonema boryanum Strain grant. PCC 6306 and the ORFs from the 7.8-kb plasmid, pUH24 (Van der Plas, 1989) from REFERENCES Synechococcus sp. Strain PCC 7942; however, no similarities were found. ASATO, Y., AND GINOZA, H. S. (1973). Separations of small circular DNA molecules from the blue-green Seven putative gene regions have been alga Anacystis nidulans. Nature 244, 132- 133. identified, including one with a region that is BOSE, S. G., AND CARMICHAEL, W. W. (1990). Plasmid conserved in several eubacterial replication distribution among unicellular and fdamentous toxic proteins. We now are examining the ORFs in cyanobacteria. J. Appl. Phycol. 2, 13I-1 36. more detail to establish the location of actual CASTETS, A., HOUMARD, J., AND TANDEAU DE MARSAC,
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N. (1986). Is cell motility a plasmid-encoded function MCKENZIE, T., HOSHINO, T., TANAKA, T.. AND in the cyanobacterium Synechocysfis 6803? FEMS SUEOKA, N. (1986). The nucleotide sequence of Microbial. Lett. 37, 277-28 1. PUB I 10: Salient features in relation to replication and CHAUVAT,F., ASTIER,C., BEDEL,F., AND JOSET-ESPAR- its regulation. Plusmid 15, 93-103. DELLIER,F. (1983). Transformation in the cyanobac- MULLER, R. E.. ANO, T., IMANAKI, T., AND AIBA. S. terium SynechococcusR2: Improvement of efficiency; (1986). Complete nucleotide sequences of BaciNus role ofthe pUH24 plasmid. Mol. Gen. Genet. 191,39plasmids pUBl lOdB, pRBH 1 and its copy mutants. 45. Mol. Gen. Genet. 202, 169-17 I. CHAUVAT, F., DEVRIES, L. VAN DEN ENDE, A., AND MURAI, M., MIYASHITA, H., ARAKI, H., SEKI, T., AND VAN ARKEL, G. A. (1985). A host-vector system for OSHIMA,Y. (1987). Molecular structure ofthe replicagene cloning in the cyanobacterium Synechocystis tion origin of a Bacillus amyloliquefaciens plasmid PCC 6803. Mol. Gen. Genet. 204, 185-191. pFTB14. Mol. Gen. Genet. 210,92-100. FELKNER,R. H. ( 1988). “Plasmid DNA in Filamentous, PAKRASI,H., RIETHMAN, H. C., AND SHERMAN,L. A. Nonheterocystous Cyanobacteria,” M.S. thesis. (1985). Organization of pigment-proteins in the phoMiami University, Oxford, Ohio. tosystem II complex of the cyanobacterium Anacystis nidulans R2. Proc. Natl. Acad. Sci. USA 82, 6903FELKNER,R. H., AND BARNUM S. R. (1988). Plasmid 6907. content and homology of 16 strains of filamentous, nonheterocystous cyanobacteria. Curr. Microbial. 17, POTTS,M. (I 984). Distribution of plasmids in cyanobac37-41. teria of the LPP group. FEMS Microbial. Lat. 24, 35 l-354. FREIDBERG,D., AND SEIJFFERS, J. L. (1979). Plasmids in two cyanobacterial strains. FEBS Lett. 107, 165- 168. REASTON,J. R., VAN DEN HONDELC. A. M. J. J., VAN DERENDE,A., VAN ARKEL,G. A., STEWART,W. D. P., GRUBER,M. Y., GLICK, B. R., THOMPSON,J. E., AND AND HERDMAN, M. (1980). Comparison of plasmids GENDEL,S. M. (1987). In vitro expression of a cyanofrom the cyanobacterium Nostoc PCC 7524 with two bacterial plasmid. Czar. Microbial. 15, 265-268. mutant strains unable to form heterocysts. FEMS MiHAUMAN, J. H., COETZEE,W. F., AND SCOTT,W. E. crobiol. Lett. 9, 185- 188. (I 982). Isolation of plasmids from toxic Microcystis SANGER,F., NICKLEN, S., AND COULSON,A. R. ( 1977). aeruginosa. So. African J. Sci. 78, 38 1. DNA sequencing with chain-terminating inhibitors. HORINOUCHI,S., AND WEISBLUM,B. (1982). Nucleotide Proc. Natl. Acad. Sci. USA 74, 5463-5467. sequenceand functional map of pC 194,a plasmid that specifiesinducible chloramphenicol resistance.J. Bac- SCHWABE,W., WEIHE, A., BORNER,T., HENNING, M., AND KOHL, J. G. (1988). Plasmids in toxic and nonteriol. 150, 8 15-825. toxic strains of the cyanobacterium Microcystis aeruKUHLEMEIER,C. J., BORRIAS,W. E., VAN DENHONDEL, ginosa. Curr. Microbial. 17, 133-137. C. A. M. J. J. AND VAN ARKEL, G. A. (198 1). Vectors for cloning in cyanobacteria: Construction and charac- SCHWABE,W., HUBSCHMAN,T., MEIXNER, M., WEIKE, A., AND BORNER,T. ( 1990).Transcription and in vivo terization of two recombinant plasmids capable of expression of a Microcystis aeruginosa plasmid. Curr. transformation to Escherichia coli K I2 and Anacystis Microbial. 20, 365-368. nidulans R2. Mol. Gen. Genet. 184, 249-254. KUSHNER,D. J., AND BREUIL,C. (1977). Penicillase (p- SCOTT,J. R. (1984). Regulation of plasmid replication. Microbial. Rev. 48, l-23. Lactamase) formation by blue-green algae. Arch. Microbiol. 112,219-223. SIMON,R. D. ( 1978).Survey of extrachromosomal DNA found in the filamentous cyanobacteria. J. Bacteriol. LAMBERT,G. R., AND CARR,N. G. (1982). Rapid small136,414-418. scale plasmid isolation by several methods from filamentous cyanobacteria. Arch. Microbial. 133, 122- SINGH, L. J., TIWARI, D. N., AND SINGH, H. N. (1986). 125. Evidence for a genetic control of herbicide resistance in a rice field isolate of Gloeocupsa sp. capable of aeroLAMBERT,G. R., AND CARR, N. G. (1983). A restriction bic diazotrophy under photoautotrophic conditions. map of plasmid pDC1 from the filamentous cyanoJ. Gen. Microbial. 32, 81-88. bacterium Nosfoc sp. MAC PCC 8009. Plusmid 10, 196-198. TANDEAU DE MARSAC,N., AND HOUMARD, J. (1987). LAMBERT,G. R., SCOTT,J. G., AND CARR,N. G. (1984). Advances in cyanobacterial molecular genetics. In “The Cyanobacteria” (P. Fay and C. Van Baalen, Characterization and cloning of extrachromosomal Eds.), pp. 25 l-302. DNA from filamentous cyanobacteria. FEMS Microbiol. Lett. 21, 225-23 I. VAN DEN HONDEL, C. A. M. J. J., KEEGSTRA,W., BORRIAS,W. E., AND VAN ARKEL, G. A. (1979). HoLAU, R. H., SAPIENZA,C., AND DOOLITTLE, W. F. mology of plasmids in strains of unicellular cyanobac( 1980).Cyanobacterial plasmids: Their widespread octeria. Plasmid 2, 323-333. currence, and the existence of regions of homology between plasmids in the same and different species. VAN DERPLAS,J. (1989). “Gene analysis in the cyanoMol. Gen. Genet. 178, 203-2 I 1. bacterium Synechococcus sp. PCC 7942.” Disserta-
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tion, pp. 97- 128. University of Utrecht, Utrecht, The Netherlands. WHITEHEAD,P. R., AND BROWN,N. L. (1985). Three restriction endonucleases from Anabaena flos-aquae. .J. Gen. Microbial. 131,95 l-958.
WICKREMA, A. W. (1989). “Complete nucleotide sequence of a small plasmid from the cyanobacterium Plectonema boryanum.” Dissertation, pp. 46-68. Miami University, Oxford, Ohio. Communicated by D. R. Helinski
28, 170-176 (1992)
DNA Sequence and Analysis of a Cryptic 4.2-kb Plasmid from the Filamentous Cyanobacterium, Plecfonema sp. Strain PCC 6402 DOUGLAS R. PERIUNSAND~USAN R. BARNUM' Department of Botany, Miami University, Oxford, Ohio 45056 Received September 3, 199 1; revised March 1, 1992 The 4194-bp plasmid, pRFI, from Plectonema sp. Strain PCC 6402 was completely sequenced and analyzed. Seven potential open reading frames were identified. The predicted amino acid sequence of open reading frame C (ORF C) had identities of 34,29, and 25% with Rep B from the Staphylococcus aureus plasmid, PUB 110; Rep from the Bacillus amyloliquefaciens plasmid, pFTB14; and protein A from the S. aureus plasmid, pC194, respectively. A 75-amino-acid region conserved in these proteins (Rep B, Rep, and protein A) also was highly conserved in ORF C with identities of 45, 37, and 40%, respectively. Significantly, 16 of the 2 1 amino acids conserved in Rep B, Rep, and protein A were found at the same positions in ORF C. This ORF may encode a replication protein that includes a region conserved in some eubacteria. Additional structural features include a 425-bp region that contains palindromes, tandem repeats, and short direct repcats which may correspond to the origin of replication. An 1%bp inverted repeat was located between two open reading frames, A and G. o 1992 Acadrmlc PESS. 1~.
Since the first discovery of plasmids from the unicellular cyanobacterium, Anacystis nidulans (Synechococcus) (Asato and Ginoza, 1973) other strains such as Synechocystis (Lau et al., 1980; Chauvat et al., 1985; Van den Hondel et al., 1979; Schwabe el al., 1988; Bose and Carmichael, 1990), Anahaena (Lambert and Carr, 1982; Lambert et al., 1984; Simon, 1978), Oscillatoria (Friedberg and Seijffers, 1979), Nostoc (Lambert and Carr, 1982; Lambert et al., 1984; Reaston et al., 1980), Ca1othri.x (Simon, 1978; Bogorad et al., 1983), and Plectonema (Simon, 1978; Potts, 1984; Felkner and Barnum, 1988) were also found to contain plasmids. They have been characterized by restriction mapping (Kuhlemeier et al., 1981; Lambert and Can-, 1983; Van den Hondel, 1979) plasmid content and size (Felkner and Barnum, 1988; Lau et al., 1980; Potts, 1984; Simon, 1978) searching for transcripts and/or proteins encoded by plasmids (Gruber et al., 1987; Schwabe et al., 1990), and DNA sequencing (Van der Plas, 1989; Wickrema,
’ To whom correspondence 0147-619X/92
should be addressed.
$5.00
CopyrIght 0 1992 by Acadrmlc Press. Inc. 411 rights of reproduction tn any form reserved
1989). Although genes on some eubacterial plasmids have been identified, cyanobacterial plasmids remain cryptic. Some inconclusive studies have been conducted to elucidate functions for plasmids (Singh et al., 1986; Castets el al., 1986). Suggested functions include antibiotic resistance (Kushner and Breuil, 1977), transfer of restriction enzymes (Whitehead and Brown, 1985) toxin production (Hauman et al., 1982), and an increase in transformation efficiency (Chauvat et al., 1983). However, definitive experiments have not been conducted. In a first step toward studying plasmid-encoded cyanobacterial genes, the 4.2-kb plasmid, pRF1 (Felkner, 1988), from Plectonema sp. Strain PCC (Paris Culture Collection) 6402 was sequenced and analyzed. The plasmid, which was previously demonstrated to contain one EcoRI site (Felkner, 1988), was ligated into the EcoRI site of pBR322 (pRF2; Felkner, 1988) and amplified for doublestranded dideoxy sequencing (Sanger et al., 1977). Both strands of the 4194-bp plasmid (designated plus and minus) were sequenced from pRF2 with each new primer set (forward and reverse) determined by the pre170
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vious sequenceobtained. Sequenceinformation from a 50-base region, positions 2 1972247, on the minus strand could not be obtained even after dITP or 7-deaza dGTP was substituted in the reactions, the temperature of the reactions was increased, and the concentrations of template, primer, and Sequenasewere modified. However, the complementary strand was sequenced several times to check for errors. Open reading frames were identified by the presence of start (ATG) and stop codons and the potential to encode a polypeptide of at least 50 amino acids. Seven potential open reading frames (ORFs) of at least 150 bp were identified. The ORFs on the plus strand were located at positions 9661133, 1255-1473, 1382-1828, and 23483226, relative to position 1 at the EcoRI site and are referred to as ORFs A, B, C, and D, respectively (Fig. 1). The ORFs on the minus strand were located at positions 3825-3538, 1195-989, and 555-301 and are ORFs E, F, and G, respectively (Fig. 1). A 12-bp palindromic sequence,CAAAACGTTTTG, was repeated five times at positions 8, 44, 76, 168, and 236 (Fig. 1). Two 7-bp direct repeats, GATTTTT, were found at positions 292 and 33 1. An 18-bp inverted repeat, AGAAGTTTGCTTACGGAT, was located between ORFs A and G at positions 773 and 793. Four short direct repeats, CAATC, were identified at positions 4 107, 4115,4123, and 4131 and four 8-bp tandem repeats, ACTTTGTG, were located beginning at position 4 141. The complete DNA sequence of pRF1 with the ORFs and repeated sequencesunderlined is shown in Fig. 1. Interestingly, the repeats were localized mainly between positions 4 107 and 337. The fact that these repeats were found in such a small region relative to the size of the entire plasmid suggeststhat this region may be the origin of replication. The sequence CAATC, beginning at position 4 107, may be considered the beginning of the repeat region since the plasmid is a circular molecule. The nucleotide sequenceof the entire plasmid, the individual ORFs, and the deduced amino acid sequences of the ORFs were
171
aligned to bacterial sequences in the GenBank and NBRF databases. Optimal alignments were conducted both manually and with MacVector (International Biotechnologies, Inc.). The predicted amino acid sequence of ORF C was 149 residues and showed identity with two replication proteins, RepB from the staphylococcal plasmid, pUBll0 (Muller et al., 1986; McKenzie et al., 1986) and the rep product (Rep) from the Bacillus amyloliquefaciens plasmid, pFTB 14 (Murai et al., 1987). Identity was most concentrated in a 42-amino-acid region of ORF C between positions 176 and 2 17 (Fig. 2). A 75-amino-acid region of ORF C from the first methionine to the 1l-amino-acid gap corresponded to a conserved region of Rep from pFfB14 (Murai et al., 1987), protein A from the S. aureus plasmid, pC 194 (Horinouchi and Weisblum, 1982) and RepB from pUBll0 (Muller et al., 1986; McKenzie et al., 1986). Identities with ORF C were 45,37, and 40%, respectively. When conservative amino acid substitutions were included, the identities increased to 65, 52, and 57%, respectively. This was part of a larger highly conserved region of Rep, Rep B, and protein A from positions 119 to 2 17 (Murai et al., 1987; Fig. 2). We determined the identities between the highly conserved region of protein A and Rep, protein A and RepB, and Rep and Rep B to be 48,33, and 50%, respectively. Identities of the complete proteins were ORF C to Rep B, 34%; ORF C to Rep, 29%; ORF C to protein A, 25%; Rep B to Rep, 39%; protein A to Rep B, 16%;and protein A to Rep, 33%. The identities of ORF C with the replication proteins, Rep B and Rep, and protein A from pC194 were well within the range of identities seen between replication proteins. In addition, of the 21 amino acids conserved in all three proteins (Rep B, Rep, and protein A), 16 were found in ORF C at the sameposition. Basedon these values, a potential function for ORF C may be the production of a replication protein. No similarities to additional sequences were found when the entire plasmid or the other six ORFs and the translations were
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SHORT COMMUNICATIONS 70 TTCTGCTCAAAACGTTTTGAGCAACAGCGCGCGACTTCG 140 TTACCCAAAATGTTTTGGGTAGAAGCAATATCGCCGGGAA 210 CTGGCAATGGCACGATCTTGGAGTACTCAAAACGTTTTGATGC 280 GGGTTTGGTAATTCAAAGCTTGCC~CGTTTTG~C~CAGCTCGATCGAG~TTTGTGGTCG~G 350 CAACCCATGTTGATTTTTTAGAGGTTGAAGAACCTAGAAACCCACCCGA 420 AACTGTATGGAATTTTAGCTTTGATCTCTCTCCT~TACGATTCCTTGGCTGACGCAT~CATTTTCTGTTG 490 AGCTTCCTCGTAGGATTCAGCTGTGATTTCAAAACACCACCATGTCGCGCCTTTATATCGGTATTCACACAGA 3
560
AATGTTTTTTCTCGACTACATCTAGTGATTCATTTGTATT 630 ATTACGTTTATCAAGACAAGCTGGTGAGTGTGGCGCTGGTAT~~GCAGCT~T~CAGG~G~T 700 GCTGACTCTGGTGAAGGGTATAAAAACAAGCAGGATTGCAGGATTGCTAC~CGCGATCGGGATGGTTT~CGT~G 770 G09ATTCTCCTGTTTATGATCTAATTTTTGTGGGTGTTGCG~GCGGTTCGCCTTCACTT~CCT~T 840 AGAGAAGTTTGCTTACGGA~TG~TCCGT~GC~CTTCTTACAGGAC~GC~CTCACGC~TTT~GG 910 CGTAATCGAAGCGTAATTTCTGTGATGGGGGAAAGCACTAAAAAA
CCGCTGTACACTGGTAACAACCACG
A, AAACCAAGTAAATACAACGGCTTTTTTATGTCT~~C~CCCCGGAGATGCTGTTATGTCTAGTGTATCG
980 1050
TCTCGTCACGAGACAGCCTAGGACAACAGCAGCACTGTC 1120 TCACCGTAGTAGCAGCGATAAAGTTGCAGGACATTATCGC 1190 TT~ACTATTGCAGTGATTTTTTGGA~TT~~GGTTAGT~C~GAT~AGGA~AG~TC~TA~~GTTTG~GTT k-1260
-F G~CTGCACGATTTTGTCATTGCCGACAATGCATTGTATGTTCCTGGCGCAG~GTCTCGC~~G
1330 GCGAGGGCTTATAAGACTTTGCCCAAAGTAGTAGTTGCTGA~ATCCGACTGCACGATACCTTTTCCTCACGC
C
1400
TGACAGTGAAGAACTGCGCGATTACCGACCCGTCTGCGGACACTC~TGG~TCAGTCATTTTCCA 1470 GAATGACAAAGCTCAAAGATTGGCAGGCTTTGGGCTATCTATCTCCG~C~CAG~GTGACGCGG~~GAGA 1540 CGGTAAGTCAGCACACCCTCATTTCCACTGCCTATTGATGGT~G~TCCTATTTCGGTCGG~CTAC 1610 ATTTCTCAGGCTGAATGGGTAGAACTATGGAAGAAGTCGTTGCGAGTGGACTAC~CCC~~TTGGATG 1680 TTCAAGCGCTTAAAACAGAGTCTTCCTTGGTTGGTTG’PGCC 1750 AAATGATTTAGTGCTATCAGATCGTGATTGGTTTTTGGAACG 1820 ATCGCATTTGGAGGAATTTTCAAAGAGTACTTCCGTGAATTGCT 1890 TGGTCAGATAGCCCAAGCCGGAGGTTGATGAGGGACACCTCTATTTT~CTGG~CGCGT~GAG~ 1960 GAAGTACCGAATGATTGATAACAATGTATAACAGCGTTATC~TTATGTAGTTTGTGTATACC~G~ 2030 TTTGTTATCACTCTTGATAACATGGTTGAAATTCTTTCTGTACGTATATGCAGTT~CCG~GATCTGCT 2100 CATTGAATCCCCGACTGCCCGTAATGAGGGAGGGAGTCCTAG~TC~TCGTC~TC~GGGAGC~GAGAT 2170 TCTTAACAAGGTGAAAGTCTTGTTGTTTGCTGCTGTCTGGGATGG~CCCCACG~TGTGGCGGCAGAGTTGG
FIG. I, Complete nucleotide sequence of pRFl from Plectonema sp. Strain PCC 6402. Open reading frames are underlined and labeled as described in the text. Overlapping sequencesare double-underlined. Palindromes and repeats also are underlined. Direct repeats beginning at positions 292 and 33 1 are identified by a line above the sequence.
SHORT
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COMMUNICATIONS 2240
CAGATTTTTATGAAGTCTCGCTGGATGCCGTAAAAAA
CTACCAGCGGTTCAGGGATGAGTTTGAGCTTGA 2310
TGGAGTAAAAGTCGTGCGTGT~GGATTTGAAGAGGTTACCTCCTTCACTCC L TCAGGAGGTTATTTACACCCCTGCTGGTGCGTTGAGAATGTTCTCCGCGATTCACCTGTAGCG
2380 2450
AAGGCGGTTAGAACAGCAGCGATTCGAGTCATCCAAGGTGCTGCGT 2520 TTGCGAAATTGGTTGAGGGTCATTCCGGGCTTGACCAA?TTGCCGC 2590 TTTGTACGAAGAGGGGATTTTTAAGGATGCTTGCTTGCAPAGCTTTGCGGAAAAAA TATCCAGATGGMAACTT 2660 CCAGGACTGGGCGTAGAACAGTTGAAGGAATTTTTTTGC~TGTTCGCCACTTATACAGATCGTTGGGTGT 2730 TC 2800 TTCTTTTCCTGTAGAGGTTGATGGGCAAGTCAAGCAGGCAGGCTGTGATCATGT~CAGTTCTGTGATTTCCTG 2870 GTTGATGATCGGTATGTTGCTGATTGTGATAGTCGTAGATACATC~TCAGTC~G~CCTGCCTGGCG 2940 TAGATTTAGCGTGGCTGTTCTTTGTTTCCCCGTTTGGTGGTGCTACACCTGCTGCCGTG~TTTATCGAGCA 3010 GGAGATTCGCCCTGACTCTCAAGGTTACGTTACGTTATT 3080 GCTCAAGCTTCCCAGAATCGCAATAGCAATCTTCTCATTG 3150 TGGGCTATGAGATTCCGGTAGTCGAGATGGCAACTCAGTACGTTATCG 3220 ATTTGATIL4CGTTCTGGTTCTTGTAGACATCCCAGACATCCCAG~C~TTCTTGTCTTTTTCTACCTGGTGTTTCT~ 3290 GCGCTTTGAGAGAA04ATGGTGTAGAAACCTATTTGTGCTATTTGTGCTGTCTGCTTTTTTGACTGTTTAGTGTTGTTG 3360 ACTTTGTTTTTCAGTCTCTTGGGTCTTCCTGCTCTAACCTCTACTGTTCTTA 3430 GCGTTCCAAGGAATGCTCTATGAGGTAGGCTTTCTTCTTTGTTATCAGTTATGTTATC~C~CATTATC 3500 AAAGTTCTGAATGATAACTTCTTCATCCTCGTCTTTGCTACTTAGGATGTTATC~GATGGTTATCAGAT 3570 ACGATAACATCTTGAAGGTCTAATC;GAATCTGATCTATTTTTTCCTTGCTGTCCTGCTTGACGTAGATGT 3640 GTCTAGCTGACCAGCAAACACTTCTCAAATCCTGAGCGTCTCGCTCT~GATTTC~CTTTTTTTCT 3710 c 3780 TAGAGAGAGCCTTGATTAGAGTCTGTCTTCGATTCATTCGTCTGCTGTCGATACTCCTCC~GCA~ +L 3850 GTAAAGACTCTGAGGAATGCGACCGGAG AAAAAAGGTCTAGTCATGCTCGTTATCGATTTCGATAACTTA 3920 ATGATAACAGATAGTGTTCTCTTGCATCTTTCTTCGATAATC 3990 AAGGGATCGTGGAATAGCGGACTGAG~GGAGAGCATCGC~TTGACGGC~GATTGA 4060 ACCGCTACACTCTTGGGCTGTTCCGAGGGCTGATAGATGCGTTGTGTAGTTGTTATTGATTTGATAGCAG 4130 CCCTCGGAACATGCCACGCGGCGAGCGGTCTTTCTTTCTCTG~TC~GC~TCTTGC~TC~~~ 4194 CAATCTTGCAACTTTGTGACTTGTGACTTTGTGACTTTG FIG. 1-Continued
174
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ORF C rep pro A Rep B
79 ............................................................................... MYSSENDYSILEDKTATGKKRDWRGKKRRANLMAEHYEALEKRIGAPYYGKKAERLSECAEHLSFKRDPETGRLKLYQA . . . . . . . . . . . . . . . . . . . . ..MCYNMEKVnKKQR~VFQKFIKRHlGE~MDLVEDCKTFLSFVAOKnEKQKLYKA ...... .........................................................................
ORF C rep pro A Rep B
158 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I+(PSFSRMlKLKD.WQAL. HFCKVRLCPMCAWRRSLKIAYHNKLIIEEANRQYGCGWIFLTLTVRNV.ffiERLK~ISElMEG~KLFQYKKVKTSVL NSCKNRFCPVCAWRWRKDALGLSL~YIKQQEKKEFIFLnTTPNVMSDE.LENEIKRYWNSmKLIKRKKVGSVIK . . . . . . . . . . . . . . . MKHGIQSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSOICR@XRRbbQYKKINKNLV
ORF C rep pro A Rep B
GYUUXVTRGRDGKSAH WKASYF..GRNYISQAEWVELWKKSLRVDVNPlL...........IJVQAL..K ffFRALEITKNHEEDTY~HVLLPmRN~..6K~lK~SLmRAMKLDYTPIVDIRRVKGRVKIDAEQIESD GWW(LEIT(NKKRDDYWLlAYNKSYFTDKRYYl~MDLW.....RDVTGISElTQVQVQKl~NNNKELY sFMRATmlNNKDNSY~~LVCVEPTYFKNTEN~~KQWIQHWKW\MKLDYDPNV...........KVQMIRPK
ORF C rep pro A Rep B
TE..SSLVGLLAElI~CKPND..LVLSDRDW.F..L.ELTRQLHm
ORF C rep pro A Rep B
........................... EDDEVANGAFEVMAYWHPGIKNYILK. ........................... DDEKADEDGFSIIAMWNWERKNYFIKE
237
316
343
FIG. 2. Amino acid alignments of proteins (from Murai et al., 1987) Rep B (235 amino acids), Rep (339 amino acids), and protein A (229 amino acids), with the predicted amino acid sequence of ORF C. Gaps are added to improve the alignments. ORF C was aligned to Rep B (with a gap of 11 amino acids) which gave a better alignment than if a gap was not introduced. Numbering is based on the number of total amino acid positions. Identities with ORF C are shown in bold.
used in alignments. Protein A from pC 194 is coding regions, transcriptional start sites, and most likely not involved in replication of the promoter regions. plasmid (Horinouchi and Weisblum, 1982); however, protein C from pC 194, a putative ACKNOWLEDGMENTS replication protein, did not show identity with ORF C or any of the other ORFs from This work was supported by an OBOR Research ChalpRF1. The sequence of the entire plasmid lenge grant and a Miami University CFR grant to S.R.B. and the individual ORFs were compared to and a graduate student research award to D.R.P. from a the DNA sequences of pGL3 (Wickrema, Department of Botany OBOR Academic Challenge 1989) from Plectonema boryanum Strain grant. PCC 6306 and the ORFs from the 7.8-kb plasmid, pUH24 (Van der Plas, 1989) from REFERENCES Synechococcus sp. Strain PCC 7942; however, no similarities were found. ASATO, Y., AND GINOZA, H. S. (1973). Separations of small circular DNA molecules from the blue-green Seven putative gene regions have been alga Anacystis nidulans. Nature 244, 132- 133. identified, including one with a region that is BOSE, S. G., AND CARMICHAEL, W. W. (1990). Plasmid conserved in several eubacterial replication distribution among unicellular and fdamentous toxic proteins. We now are examining the ORFs in cyanobacteria. J. Appl. Phycol. 2, 13I-1 36. more detail to establish the location of actual CASTETS, A., HOUMARD, J., AND TANDEAU DE MARSAC,
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