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Nucleotide Sequence Analysis of pRS2 and pRS3, Two Small Cryptic Plasmids from Oenococcus oeni
Plasmid 46, 149–151 (2001) doi:10.1006/plas.2001.1537, available online at http://www.academicpress.com on
SHORT COMMUNICATION Nucleotide Sequence Analysis of pRS2 and pRS3, Two Small Cryptic Plasmids from Oenococcus oeni Juan M. Mesas,* M. Carmen Rodríguez,† and M. Teresa Alegre,1,‡ *Departamento de Química Analítica, Nutrición y Bromatología (Tecnología de los Alimentos), †Departamento de Biología Vegetal, and ‡Departamento de Microbiología y Parasitología, Escuela Politécnica Superior, Universidad de Santiago de Compostela, Lugo, Spain Received May 1, 2001; revised June 25, 2001 Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two subfamilies, one represented by pLo13 and pRS3, the other represented by pOg32, pRS1, and pRS2. © 2001 Academic Press Key Words: plasmids; Oenococcus oeni; nucleotide sequence; lactic acid bacteria.
Oenococcus oeni, formerly Leuconostoc oenos (Dicks et al., 1995), is a heterofermentative gram-positive coccus which grows in wine after alcoholic fermentation and is the major agent of malolactic fermentation (Davis et al., 1985; Van Vuuren and Dicks, 1993; LonvaudFunel, 1995; Versari et al., 1999). The existence of a family of small cryptic plasmids of O. oeni has been proposed (Alegre et al., 1999) on the basis of some common features; i.e., they have three major open reading frames (ORFs2) in the same strand, one coding for a replication (Rep) protein, another for a recombination (Pre) protein, and a third of unknown function; they contain the DNA sequences required for plasmid replication by the rolling-circle mechanism; and they have a CTTTTTTT(T) sequence separating the ribosome-binding sites (RBS) and the ATG start codon of their Rep proteins. To extend our knowledge of this family of plasmids, we have determined the nucleotide sequences of pRS2 and pRS3, two small cryptic 1
To whom correspondence should be addressed. Abbreviations used: ORF, open reading frame; RBS, ribosome-binding site; DSO, double-strand origin; RS, recombination-specific site; Rep, replication initiation protein. 2
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plasmids. The O. oeni strain carrying pRS2 and pRS3 was isolated from wines from Ribeira Sacra (a wine-producing region of northwestern Spain) in solid MRS broth containing 30 mL/L of tomato juice after 5–10 days of incubation at 25°C in anaerobic jars. It was identified by biochemical tests as per Holt et al. (1994) and Dicks et al. (1995). The nucleotide sequences of pRS2 and pRS3 were determined on both strands by cloning suitable overlapping restriction fragments from both plasmids, electroeluted from agarose gels, into M13mp18 and/or M13mp19. Nucleotide sequences of single-stranded DNA obtained from M13 subclones from Escherichia coli JM101 (Messing et al., 1981) were determined by the chain-termination procedure (Sanger et al., 1977) using T7 Sequenase version 2.0 DNA sequencing kits and [␣-35S]dATP from Amersham–Pharmacia Biotech. Comparison of the complete nucleotide sequence of pRS2 (2544 bp, Accession No. AJ310613) with the databases revealed 94% identity between pRS2 and pOg32, another plasmid from O. oeni (Brito et al., 1996) which has the same size as pRS2 (Fig. 1). The searches also revealed 58% identity with pRS1 0147-619X/00 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Map of pRS2 showing relevant restriction sites and location of the features derived from nucleotide sequence analysis: open reading frames (ORF), double-strand origin (DSO), recombination sequences (RS), and CTTTTTTTT sequence (CT).
(Alegre et al., 1999). pRS2 contained three major ORFs (ORF1, ORF2, and ORF3) with the same orientation, as reported for pOg32 and pRS1. Other recognizable features included RBS; (Shine and Dalgarno, 1974), a double-strand origin (DSO; Gruss and Ehrlich, 1989; Brito et al., 1996), the recombinationspecific sites RSA (Novick et al., 1984) and RSB (Gruss et al., 1987; Novick, 1989), and the CTTTTTTT(T) sequence between the RBS and the ATG start codon of ORF1 (Alegre et al., 1999). When the nucleotide sequences of pRS2 and pOg32 were compared, it was found that pRS2, like pRS1, contains eight thymines in the CTTTTTTT(T) sequence, one more than pOg32, and 137 nucleotide differences affecting the ORF2 region. The similarity between pRS2 and pOg32 argues in favor of identical or similar encoded proteins and suggests that pRS2 belongs to the family of small O. oeni plasmids which replicate by the rolling-circle mechanism (Alegre et al., 1999). The location of the divergences between pRS2 and pOg32 mainly in the ORF2 region could argue in favor of the hypothesis that these replicons consist of two relatively independent and readily exchangeable gene cassettes, one for replication and one for mobiliza-
tion (Projan and Novick, 1988; Josson et al., 1990; Ilyina and Koonin, 1992; Brito et al., 1996). Comparison of the pRS3 sequence (3948 bp, Accession No. AJ310614) with the databases revealed more than 99% identity to pLo13, also from O. oeni (Fremaux et al., 1993). When the nucleotide sequences of these plasmids were compared, only seven randomly located differences were found. It has been suggested that the small cryptic plasmids pLo13, pOg32, and pRS1 of O. oeni constitute a single family, (Alegre et al., 1999). The new plasmids described here, pRS2 and pRS3, show the three characteristics which suggest that they must be included as members of this family. However, the coexistence of both plasmids in a single strain supports the view that pRS2 and pRS3 belong to different subfamilies, one represented by pOg32, pRS1, and pRS2 and the other by pLo13 and pRS3. In support of this hypothesis, the sequences of the plasmids from the same subfamily exhibit very high identity but little identity between plasmids from different subfamilies. Finally, the function of the putative proteins encoded by the third ORF of these plasmids remains unknown; however, we have found that the putative ORF3 protein of pRS3 exhibits 48% similarity with the heat shock proteins of some higher organisms (not shown), raising the possibility that this protein could be involved in stress-resistance mechanisms. ACKNOWLEDGMENTS This work was supported by a grant from the Xunta de Galicia (PGIDT99BIO29101). We thank Dr. Jesús Cortés for his valuable critique of the manuscript.
REFERENCES Alegre, M. T., Rodríguez, M. C., and Mesas, J. M. (1999). Nucleotide sequence analysis of pRS1, a cryptic plasmid from Oenococcus oeni. Plasmid 41, 128–134. Brito, L., Vieira, G., Santos, M. A., and Paveia, H. (1996). Nucleotide sequence analysis of pOg32, a cryptic plasmid from Leuconostoc oenos. Plasmid 36, 49–54. Davis, C. R., Wibowo, D., Eschenbruch, R., Lee, T. H., and Fleet, G. H. (1985). Practical implications of malolactic fermentation: A review. Am. J. Enol. Vitic. 36, 290–301. Dicks, L. M. T., Dellaglio, F., and Collins, M. D. (1995). Proposal to reclassify Leuconostoc oenos as Oenococcus
SHORT COMMUNICATION oeni [corrig.] gen. nov., comb. nov. Int. J. Syst. Bacteriol. 45, 395–397. Fremaux, C., Aigle, M., and Lonvaud-Funel, A. (1993). Sequence analysis of Leuconostoc oenos DNA: Organization of pLo13, a cryptic plasmid. Plasmid 30, 212–223. Gruss, A. D., and Ehrlich, S. D. (1989). The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol. Rev. 53, 231–241. Gruss, A. D., Ross, H. F., and Novick, R. P. (1987). Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc. Natl. Acad. Sci. USA 84, 2165–2169. Holt, J., Krieg, N., Sneath, P., Staley, J., and Williams, S. (Eds.) (1994). “Bergey’s Manual of Determinative Bacteriology,” 9th ed. Williams and Wilkins, Baltimore. Ilyina, T. V., and Koonin, E. V. (1992). Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res. 20(13), 3279–3285. Josson, K., Soetaert, P., Michiels, F., Joos, H., and Mahillon, J. (1990). Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other Gram-positive organisms. J. Bacteriol. 172, 3089–3099. Lonvaud-Funel, A. (1995). Microbiology of the malolactic fermentation: Molecular aspects. FEMS Microbiol. Lett. 126, 209–214.
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Messing, J., Crea, R., and Seeburg, P. H. (1981). A system for shotgun DNA sequencing. Nucleic Acids Res. 9, 309–321. Novick, R. P. (1989). Staphylococcal plasmids and their replication. Annu. Rev. Microbiol. 43, 537–565. Novick, R. P., Projan, S. J., Rosenblum, W., and Edelman, I. (1984). Staphylococcal plasmid cointegrates are formed by host- and phage-mediated general rec systems that act on short region of homology. Mol. Gen. Genet. 195, 374–377. Projan, S. J., and Novick, R. (1988). Comparative analysis of five related staphylococcal plasmids. Plasmid 19, 203–221. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Shine, J., and Dalgarno, L. (1974). The 3⬘-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to non-sens triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342–1346. Van Vuuren, H. J. J. and Dicks, L. M. T. (1993). Leuconostoc oenos: A review. Am. J. Enol. Vitic. 44, 99–112. Versari, A., Parpinello, G. P., and Cattaneo, M. (1999). Leuconostoc oenos, and malolactic fermentation in wine: A review. J. Ind. Microbiol. Biotechnol. 23, 447– 455.
Communicated by J. I. Rood
SHORT COMMUNICATION Nucleotide Sequence Analysis of pRS2 and pRS3, Two Small Cryptic Plasmids from Oenococcus oeni Juan M. Mesas,* M. Carmen Rodríguez,† and M. Teresa Alegre,1,‡ *Departamento de Química Analítica, Nutrición y Bromatología (Tecnología de los Alimentos), †Departamento de Biología Vegetal, and ‡Departamento de Microbiología y Parasitología, Escuela Politécnica Superior, Universidad de Santiago de Compostela, Lugo, Spain Received May 1, 2001; revised June 25, 2001 Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two subfamilies, one represented by pLo13 and pRS3, the other represented by pOg32, pRS1, and pRS2. © 2001 Academic Press Key Words: plasmids; Oenococcus oeni; nucleotide sequence; lactic acid bacteria.
Oenococcus oeni, formerly Leuconostoc oenos (Dicks et al., 1995), is a heterofermentative gram-positive coccus which grows in wine after alcoholic fermentation and is the major agent of malolactic fermentation (Davis et al., 1985; Van Vuuren and Dicks, 1993; LonvaudFunel, 1995; Versari et al., 1999). The existence of a family of small cryptic plasmids of O. oeni has been proposed (Alegre et al., 1999) on the basis of some common features; i.e., they have three major open reading frames (ORFs2) in the same strand, one coding for a replication (Rep) protein, another for a recombination (Pre) protein, and a third of unknown function; they contain the DNA sequences required for plasmid replication by the rolling-circle mechanism; and they have a CTTTTTTT(T) sequence separating the ribosome-binding sites (RBS) and the ATG start codon of their Rep proteins. To extend our knowledge of this family of plasmids, we have determined the nucleotide sequences of pRS2 and pRS3, two small cryptic 1
To whom correspondence should be addressed. Abbreviations used: ORF, open reading frame; RBS, ribosome-binding site; DSO, double-strand origin; RS, recombination-specific site; Rep, replication initiation protein. 2
149
plasmids. The O. oeni strain carrying pRS2 and pRS3 was isolated from wines from Ribeira Sacra (a wine-producing region of northwestern Spain) in solid MRS broth containing 30 mL/L of tomato juice after 5–10 days of incubation at 25°C in anaerobic jars. It was identified by biochemical tests as per Holt et al. (1994) and Dicks et al. (1995). The nucleotide sequences of pRS2 and pRS3 were determined on both strands by cloning suitable overlapping restriction fragments from both plasmids, electroeluted from agarose gels, into M13mp18 and/or M13mp19. Nucleotide sequences of single-stranded DNA obtained from M13 subclones from Escherichia coli JM101 (Messing et al., 1981) were determined by the chain-termination procedure (Sanger et al., 1977) using T7 Sequenase version 2.0 DNA sequencing kits and [␣-35S]dATP from Amersham–Pharmacia Biotech. Comparison of the complete nucleotide sequence of pRS2 (2544 bp, Accession No. AJ310613) with the databases revealed 94% identity between pRS2 and pOg32, another plasmid from O. oeni (Brito et al., 1996) which has the same size as pRS2 (Fig. 1). The searches also revealed 58% identity with pRS1 0147-619X/00 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Map of pRS2 showing relevant restriction sites and location of the features derived from nucleotide sequence analysis: open reading frames (ORF), double-strand origin (DSO), recombination sequences (RS), and CTTTTTTTT sequence (CT).
(Alegre et al., 1999). pRS2 contained three major ORFs (ORF1, ORF2, and ORF3) with the same orientation, as reported for pOg32 and pRS1. Other recognizable features included RBS; (Shine and Dalgarno, 1974), a double-strand origin (DSO; Gruss and Ehrlich, 1989; Brito et al., 1996), the recombinationspecific sites RSA (Novick et al., 1984) and RSB (Gruss et al., 1987; Novick, 1989), and the CTTTTTTT(T) sequence between the RBS and the ATG start codon of ORF1 (Alegre et al., 1999). When the nucleotide sequences of pRS2 and pOg32 were compared, it was found that pRS2, like pRS1, contains eight thymines in the CTTTTTTT(T) sequence, one more than pOg32, and 137 nucleotide differences affecting the ORF2 region. The similarity between pRS2 and pOg32 argues in favor of identical or similar encoded proteins and suggests that pRS2 belongs to the family of small O. oeni plasmids which replicate by the rolling-circle mechanism (Alegre et al., 1999). The location of the divergences between pRS2 and pOg32 mainly in the ORF2 region could argue in favor of the hypothesis that these replicons consist of two relatively independent and readily exchangeable gene cassettes, one for replication and one for mobiliza-
tion (Projan and Novick, 1988; Josson et al., 1990; Ilyina and Koonin, 1992; Brito et al., 1996). Comparison of the pRS3 sequence (3948 bp, Accession No. AJ310614) with the databases revealed more than 99% identity to pLo13, also from O. oeni (Fremaux et al., 1993). When the nucleotide sequences of these plasmids were compared, only seven randomly located differences were found. It has been suggested that the small cryptic plasmids pLo13, pOg32, and pRS1 of O. oeni constitute a single family, (Alegre et al., 1999). The new plasmids described here, pRS2 and pRS3, show the three characteristics which suggest that they must be included as members of this family. However, the coexistence of both plasmids in a single strain supports the view that pRS2 and pRS3 belong to different subfamilies, one represented by pOg32, pRS1, and pRS2 and the other by pLo13 and pRS3. In support of this hypothesis, the sequences of the plasmids from the same subfamily exhibit very high identity but little identity between plasmids from different subfamilies. Finally, the function of the putative proteins encoded by the third ORF of these plasmids remains unknown; however, we have found that the putative ORF3 protein of pRS3 exhibits 48% similarity with the heat shock proteins of some higher organisms (not shown), raising the possibility that this protein could be involved in stress-resistance mechanisms. ACKNOWLEDGMENTS This work was supported by a grant from the Xunta de Galicia (PGIDT99BIO29101). We thank Dr. Jesús Cortés for his valuable critique of the manuscript.
REFERENCES Alegre, M. T., Rodríguez, M. C., and Mesas, J. M. (1999). Nucleotide sequence analysis of pRS1, a cryptic plasmid from Oenococcus oeni. Plasmid 41, 128–134. Brito, L., Vieira, G., Santos, M. A., and Paveia, H. (1996). Nucleotide sequence analysis of pOg32, a cryptic plasmid from Leuconostoc oenos. Plasmid 36, 49–54. Davis, C. R., Wibowo, D., Eschenbruch, R., Lee, T. H., and Fleet, G. H. (1985). Practical implications of malolactic fermentation: A review. Am. J. Enol. Vitic. 36, 290–301. Dicks, L. M. T., Dellaglio, F., and Collins, M. D. (1995). Proposal to reclassify Leuconostoc oenos as Oenococcus
SHORT COMMUNICATION oeni [corrig.] gen. nov., comb. nov. Int. J. Syst. Bacteriol. 45, 395–397. Fremaux, C., Aigle, M., and Lonvaud-Funel, A. (1993). Sequence analysis of Leuconostoc oenos DNA: Organization of pLo13, a cryptic plasmid. Plasmid 30, 212–223. Gruss, A. D., and Ehrlich, S. D. (1989). The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol. Rev. 53, 231–241. Gruss, A. D., Ross, H. F., and Novick, R. P. (1987). Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc. Natl. Acad. Sci. USA 84, 2165–2169. Holt, J., Krieg, N., Sneath, P., Staley, J., and Williams, S. (Eds.) (1994). “Bergey’s Manual of Determinative Bacteriology,” 9th ed. Williams and Wilkins, Baltimore. Ilyina, T. V., and Koonin, E. V. (1992). Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res. 20(13), 3279–3285. Josson, K., Soetaert, P., Michiels, F., Joos, H., and Mahillon, J. (1990). Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other Gram-positive organisms. J. Bacteriol. 172, 3089–3099. Lonvaud-Funel, A. (1995). Microbiology of the malolactic fermentation: Molecular aspects. FEMS Microbiol. Lett. 126, 209–214.
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Messing, J., Crea, R., and Seeburg, P. H. (1981). A system for shotgun DNA sequencing. Nucleic Acids Res. 9, 309–321. Novick, R. P. (1989). Staphylococcal plasmids and their replication. Annu. Rev. Microbiol. 43, 537–565. Novick, R. P., Projan, S. J., Rosenblum, W., and Edelman, I. (1984). Staphylococcal plasmid cointegrates are formed by host- and phage-mediated general rec systems that act on short region of homology. Mol. Gen. Genet. 195, 374–377. Projan, S. J., and Novick, R. (1988). Comparative analysis of five related staphylococcal plasmids. Plasmid 19, 203–221. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Shine, J., and Dalgarno, L. (1974). The 3⬘-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to non-sens triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342–1346. Van Vuuren, H. J. J. and Dicks, L. M. T. (1993). Leuconostoc oenos: A review. Am. J. Enol. Vitic. 44, 99–112. Versari, A., Parpinello, G. P., and Cattaneo, M. (1999). Leuconostoc oenos, and malolactic fermentation in wine: A review. J. Ind. Microbiol. Biotechnol. 23, 447– 455.
Communicated by J. I. Rood