.
../ 1990 Oxford University Press
Nucleic Acids Research, Vol. 18, No. 23 7177
Nucleotide sequence and expression of the copy number control gene (cop) of the incFVII plasmid pSU233 J.L6pez, I.Andres, J.M.Ortiz and J.C.Rodriguez Departamento de Biologia Molecular, Universidad de Cantabria, Poligono de Cazona s/n, 39011 Santander, Spain Submitted October 18, 1990
The replication of plasmid replicons belonging to the RepFIIA family is regulated by the availability of the protein encoded by the repA gene. The control of repA is exerted by at least two other genes present in the replicon, namely copB (cop) and copA
(inc) (1).
By hybridising with a probe containing the cop gene of the IncFIII plasmid pSU316 (2) we have isolated the cop gene from the IncFVllI plasmid pSU233 in a Sau3A fragment 398 bp in size. In a minicell system this Sau3A fragment expressed a protein identical in size to that expressed by the cop gene of the plasmid pSU212 (not shown), which belongs to the same family of replicons as pSU233 (RepFHA family). The nucleotide sequence of the cop gene of pSU233 was determined and compared to the prototypes of the plasmids belonging to the RepFIIA family. The comparison is shown in figure 1. The cop gene of pSU233 has very low similarity with the cop genes of other plasmids belonging to the RepFIIA family and thus defines a new allele. The fragment starts with a region of almost identity, which includes the amino-terminal part of the protein. Then the homology decreases to a minimum in the region coding for the carboxyl terminus of the protein and the -35 region of the RepA promoter. In the plasmid RI these regions are respectively the active part and the target of the Cop protein (3). Thus, our results support the hypothesis of Saadi et al. (4) that the modules that form the RepFIIA replicons are constituted by both a gene and the target of the product of that gene, which strongly suggests that RepFIIA replicons have evolved by homologous recombination of these modules (see also reference
EMBL accession no. X55893
REFERENCES 1. Nordstr6m,K., Wagner,E.G.H., Persson,C., Blomberg,P. and Ohman,M. (1988) Gene 72, 237-240. 2. Andres,I., Rodrfguez,J.C., Barbadillo,J. and Ortiz,J.M. (1987) J. Bacteriol.
169, 2405-2409. 3. Riise,E. and Molin,S. (1986) Plasmid 15, 163-171. 4. Saadi,S., Maas,W.K., Hill,D.F. and Bergquist,P.L. (1987) J. Bacteriol. 169, 1836-1846. 5. L6pez,J., Crespo,P., Rodriguez,J.C., Andres,I. and Ortiz,J.M. (1989) Gene 78, 183-187. 6. Takeshita,S., Sato,M., Toba,M., Masahashi,W. and Hashimoto-Gotoh,T. (1987) Gene 61, 63-74. 7. Ryder,T.B., Davison,D.B., Rosen,J.I., Ohtsubo,E. and Ohtsubo,H. (1982) Gene 17, 299-316. CopB Pr.
-35
-10
.... A.T .. ........... i.................. ................ P307 ... S U31 6 ... RIOO GATCTTCGTCACAATTCTCAA CGCTG TTTCAAAAAACTGTA ATCCTGCGAAAC
.
C
-
.....................................
R1
p
pSU233
A
........................................................ ... GATTCCTGTTTGAGTATTGAGGAGGCGAG TGTCGCAGACAGAAAATGCAGTGACTTCCT .
.
etSerGlnThrGluAsnAlaValThrSerS
C.. G.TC ..GCCAG...GA.A ..AA AC . T . C.G . ......... . GTTTGTGCGC.G TGACT.T ... TGA G. .TT.TGA .... GAAA.AAA .......... .... .... A.. G
......A...GA
..CA .TG.CG
..GAAA ..AA
CATCAGaTACAAAGCGAGCATACAGAAAAGGTAATCCTCTGACGCTTGCAGAACGTCAGC
erSerGlyThrLysArgAlaTyrArgLysGlyAsnProLeuThrLeuAlaGluArgGlnG
G .. T. .T..A. .TC.C.G.A.CAAC..T ..... G.CTT.TCAT.C.G.T. C.AG ..CG T CG.TG.T ..... TCT . C ..... GA.A.A... T ..TGTCAAAAA.C GATT AG.. ...CCT .A. T.CTTC ..G . G.A.A ...A.TC. .GAAC.AA
AGGCATCCCTGGCAAGAAAACGAGCTACACATAAAGAACTCAGAGTGTTCATTCCTGCTG
lnAlaSerLeuAlaArgLysArgAlaThrHisLysGluLeuArgValPhel leProAlaA
5).
G .. ACAGGC ..AGT.AAC. .GCA ..T.AG..A..TA .. GC. C .TC.G ... AT.TCA ..GTT.A.TAC. C .G.GA. GA.A. ... C.. TCG A. AT .G. .CATGC.CAT.CAA ...... C.T.AA .T..TC.G. .. AG.TC
ACKNOWLEDGEMENTS This work was supported by grant 0007/85 from the Comision Asesora de Investigacion Cientifica y Tecnica (CAICYT). J.L. is a predoctoral recipient of a grant from the Spanish Ministry of Education (Plan de Formacion del Personal Investigador).
CGTTAAAAGCGCAATTGCAGGTGATGTGTGAAGCTGAGGGCGTTACCCAGGCTGAGATGA laLeuLysAlaGlnLeuGlnValMetCysGluAlaGluGlyValThrGlnAlaGluMetI
TT. CATTC. ...AAA ....... TG.ATCAG. .CTGAAACG ... AGCGACTTTG.A ...AGA. AC. C. .AG.TG. .CTGCAGAGACTGG.TA. .CT.AAG GAC.TTAC. AA.TG.AGCGCAAAAACGA.GC TGTG. .GA ... AC. CTTAC.TTC TTGCCGAACTGATAAAGCAGAAGAGTGCTTTTAGCTA TGTAGCGACATCACAT leAlaGluLeuI leLysGlnLysSerAlaPheSerEn [
C.T~TTCTCAGGC.TGA
... .....
.TG.GI.GTAGCA
LGA.TD TCAGAA.A.TA
..
.......
.A
A.....C
'Ft1rTGC~3TTTCTGGTGTGCGTG ¶AGAT GCTGATC -35
RepA Pr.
-10
Figure 1. Comparison of the cop gene of pSU233 and other replicons belonging to the RepFILA family. The sequences are from P307 (IncFI, (4)), RI and R100 (IncFII, (7)).
../ 1990 Oxford University Press
Nucleic Acids Research, Vol. 18, No. 23 7177
Nucleotide sequence and expression of the copy number control gene (cop) of the incFVII plasmid pSU233 J.L6pez, I.Andres, J.M.Ortiz and J.C.Rodriguez Departamento de Biologia Molecular, Universidad de Cantabria, Poligono de Cazona s/n, 39011 Santander, Spain Submitted October 18, 1990
The replication of plasmid replicons belonging to the RepFIIA family is regulated by the availability of the protein encoded by the repA gene. The control of repA is exerted by at least two other genes present in the replicon, namely copB (cop) and copA
(inc) (1).
By hybridising with a probe containing the cop gene of the IncFIII plasmid pSU316 (2) we have isolated the cop gene from the IncFVllI plasmid pSU233 in a Sau3A fragment 398 bp in size. In a minicell system this Sau3A fragment expressed a protein identical in size to that expressed by the cop gene of the plasmid pSU212 (not shown), which belongs to the same family of replicons as pSU233 (RepFHA family). The nucleotide sequence of the cop gene of pSU233 was determined and compared to the prototypes of the plasmids belonging to the RepFIIA family. The comparison is shown in figure 1. The cop gene of pSU233 has very low similarity with the cop genes of other plasmids belonging to the RepFIIA family and thus defines a new allele. The fragment starts with a region of almost identity, which includes the amino-terminal part of the protein. Then the homology decreases to a minimum in the region coding for the carboxyl terminus of the protein and the -35 region of the RepA promoter. In the plasmid RI these regions are respectively the active part and the target of the Cop protein (3). Thus, our results support the hypothesis of Saadi et al. (4) that the modules that form the RepFIIA replicons are constituted by both a gene and the target of the product of that gene, which strongly suggests that RepFIIA replicons have evolved by homologous recombination of these modules (see also reference
EMBL accession no. X55893
REFERENCES 1. Nordstr6m,K., Wagner,E.G.H., Persson,C., Blomberg,P. and Ohman,M. (1988) Gene 72, 237-240. 2. Andres,I., Rodrfguez,J.C., Barbadillo,J. and Ortiz,J.M. (1987) J. Bacteriol.
169, 2405-2409. 3. Riise,E. and Molin,S. (1986) Plasmid 15, 163-171. 4. Saadi,S., Maas,W.K., Hill,D.F. and Bergquist,P.L. (1987) J. Bacteriol. 169, 1836-1846. 5. L6pez,J., Crespo,P., Rodriguez,J.C., Andres,I. and Ortiz,J.M. (1989) Gene 78, 183-187. 6. Takeshita,S., Sato,M., Toba,M., Masahashi,W. and Hashimoto-Gotoh,T. (1987) Gene 61, 63-74. 7. Ryder,T.B., Davison,D.B., Rosen,J.I., Ohtsubo,E. and Ohtsubo,H. (1982) Gene 17, 299-316. CopB Pr.
-35
-10
.... A.T .. ........... i.................. ................ P307 ... S U31 6 ... RIOO GATCTTCGTCACAATTCTCAA CGCTG TTTCAAAAAACTGTA ATCCTGCGAAAC
.
C
-
.....................................
R1
p
pSU233
A
........................................................ ... GATTCCTGTTTGAGTATTGAGGAGGCGAG TGTCGCAGACAGAAAATGCAGTGACTTCCT .
.
etSerGlnThrGluAsnAlaValThrSerS
C.. G.TC ..GCCAG...GA.A ..AA AC . T . C.G . ......... . GTTTGTGCGC.G TGACT.T ... TGA G. .TT.TGA .... GAAA.AAA .......... .... .... A.. G
......A...GA
..CA .TG.CG
..GAAA ..AA
CATCAGaTACAAAGCGAGCATACAGAAAAGGTAATCCTCTGACGCTTGCAGAACGTCAGC
erSerGlyThrLysArgAlaTyrArgLysGlyAsnProLeuThrLeuAlaGluArgGlnG
G .. T. .T..A. .TC.C.G.A.CAAC..T ..... G.CTT.TCAT.C.G.T. C.AG ..CG T CG.TG.T ..... TCT . C ..... GA.A.A... T ..TGTCAAAAA.C GATT AG.. ...CCT .A. T.CTTC ..G . G.A.A ...A.TC. .GAAC.AA
AGGCATCCCTGGCAAGAAAACGAGCTACACATAAAGAACTCAGAGTGTTCATTCCTGCTG
lnAlaSerLeuAlaArgLysArgAlaThrHisLysGluLeuArgValPhel leProAlaA
5).
G .. ACAGGC ..AGT.AAC. .GCA ..T.AG..A..TA .. GC. C .TC.G ... AT.TCA ..GTT.A.TAC. C .G.GA. GA.A. ... C.. TCG A. AT .G. .CATGC.CAT.CAA ...... C.T.AA .T..TC.G. .. AG.TC
ACKNOWLEDGEMENTS This work was supported by grant 0007/85 from the Comision Asesora de Investigacion Cientifica y Tecnica (CAICYT). J.L. is a predoctoral recipient of a grant from the Spanish Ministry of Education (Plan de Formacion del Personal Investigador).
CGTTAAAAGCGCAATTGCAGGTGATGTGTGAAGCTGAGGGCGTTACCCAGGCTGAGATGA laLeuLysAlaGlnLeuGlnValMetCysGluAlaGluGlyValThrGlnAlaGluMetI
TT. CATTC. ...AAA ....... TG.ATCAG. .CTGAAACG ... AGCGACTTTG.A ...AGA. AC. C. .AG.TG. .CTGCAGAGACTGG.TA. .CT.AAG GAC.TTAC. AA.TG.AGCGCAAAAACGA.GC TGTG. .GA ... AC. CTTAC.TTC TTGCCGAACTGATAAAGCAGAAGAGTGCTTTTAGCTA TGTAGCGACATCACAT leAlaGluLeuI leLysGlnLysSerAlaPheSerEn [
C.T~TTCTCAGGC.TGA
... .....
.TG.GI.GTAGCA
LGA.TD TCAGAA.A.TA
..
.......
.A
A.....C
'Ft1rTGC~3TTTCTGGTGTGCGTG ¶AGAT GCTGATC -35
RepA Pr.
-10
Figure 1. Comparison of the cop gene of pSU233 and other replicons belonging to the RepFILA family. The sequences are from P307 (IncFI, (4)), RI and R100 (IncFII, (7)).
