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Genetic Analysis and Complete Nucleotide Sequence of a 2-kb Cryptic Plasmid from the Marine Methylotroph Methylophaga thalassica S1
Plasmid 44, 105–109 (2000) doi:10.1006/plas.2000.1479, available online at http://www.idealibrary.com on
SHORT COMMUNICATION Genetic Analysis and Complete Nucleotide Sequence of a 2-kb Cryptic Plasmid from the Marine Methylotroph Methylophaga thalassica S1 Andrei Y. Chistoserdov 1 Marine Sciences Research Center, State University of New York at Stony Brook, Stony Brook, New York 11794-5000 Received February 22, 2000; revised April 12, 2000 A small cryptic plasmid (pMTS1) was isolated from the marine methylotroph Methylophaga thalassica S1. Sequence analysis showed that pMTS1 has a 1995-bp genome encoding three putative polypeptides and containing two intergenic regions. The longest open reading frame encodes a polypeptide of 325 amino acid (37,079 Da). This polypeptide shares similarity and signature amino acids with the rep proteins of the pC194 group replicons, suggesting that pMTS1 replicates by the rolling-cycle mechanism. Insertional mutagenesis confirmed that the repA gene product is obligatory for replication of pMTS1 in M. thalassica S1. A pC194-type dso was identified in the second intergenic area. It appears that this intergenic region contains both the nic and bind sites. It is hypothesized that sso of pMTS1 is located in the first intergenic region. Two smaller open reading frames (orf-1 and orf-2) encode transcriptionally coupled polypeptides of 123 and 101 amino acids (14,486 and 11,241 Da, respectively) with unknown biological function. © 2000 Academic Press
The Methylophaga spp. play an important role in marine coastal environments, participating in remineralization of organic carbon and, particularly, biogenic amines (Sieburth, 1988). The genetics of the Methylophaga genus is essentially unknown and no cloning vectors have been described for this genus. One strain, M. thalassica S1, harbors a small 2-kb plasmid, called pMTS1. M. thalassica S1 Rif R was grown in a mineral medium (Fulton et al., 1984) with 1% of methanol as a source of carbon and 200 g/ml of rifamycin or 150 g/ml of kanamycin when needed. General cloning procedures such as plasmid isolation (boiling method), restriction endonuclease digest and mapping, agarose gel electrophoresis, ligation, and transformation were carried out according to Maniatis et al. (1982). pUC19 derivatives were isolated for sequencing using Qiagen columns according to the protocol of the manufacturer (Qiagen, Chatsworth, CA). This is Contribution No. 1183 from Marine Sciences Research Center. 1 To whom correspondence should be addressed. Fax: (516) 632-8820. E-mail: [email protected].
The complete nucleotide sequence of pMTS1 was determined and contains 1995 bp (Fig. 1). Three open reading frames encoding polypeptides of 325 (37,079 Da), 123 (14,486 Da), and 101 (11,241 Da) amino acids were identified. The three genes are transcribed in the same direction and the two shorter open reading frames, designated as orf-1 and orf-2, are transcriptionally coupled and thus comprise an operon. A search in the GenBank databases revealed that the 325-amino-acid polypeptide, encoded by the largest open reading frame, shares similarity with replication proteins of several small rolling-cycle replication plasmids and Orf-2 from the cryptic plasmid pAYL found in Nitrosomonas sp. Strain ENI-11 (Yamagata et al., 1999). Therefore, this open reading frame was named repA. The RepA polypeptide shares the highest level of identity (49.4%) with Orf-2 from pAYL, followed by the replication protein of the Bacillus sp. plasmid pBAA1 (31.6%). An alignment of several RepA-like proteins from pMTS1, pAYL, pBBAA1, and several rollingcycle replication plasmids belonging to the pC194 family is shown in Fig. 2A. These proteins share conserved motifs (del Solar et al.,
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0147-619X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Complete nucleotide sequences of pMTS1 and the polypeptides it encodes (GenBank Accession No. AF233873). Inverted repeats are indicated with arrows (IR stands for inverted repeat). ⫺35 and ⫺10 regions of putative promoters are double underlined, and ribosome-binding sites are underlined. Recognition sites of selected restriction endonucleases are shown in bold. The DNA sequence highly similar in pMTS1 and pAYL is italicized, and the nick site is indicated with a vertical arrow. A putative membrane-spanning stretch of Orf-1 is italicized as well.
1998), suggesting that both pMTS1 and pAYL are members of the pC194 family. A potential promoter has been identified upstream of the repA gene, designated P3 in Fig. 1. Unlike repA
genes from the majority of pC194 family plasmids (del Solar et al., 1998), repA from pMTS1 has an efficient ribosome binding site. The two hairpin structures immediately downstream of
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FIG. 1—Continued
the repA gene (IR1 and IR2) may function as -independent terminators. No sequences homologous to either Orf-1 or Orf-2 were found in the GenBank database. The genes for these two polypeptides are preceded by two putative promoters (shown as P1 and P2 in Fig. 1). In addition to the three open reading frames, the pMTS1 genome contains two intergenic regions: the region between the 3⬘ terminus of the repA and the orf-1/orf-2 operon was called the
intergenic region I and the DNA stretch between the 3⬘ terminus of orf-2 and the beginning of repA comprises the intergenic region II. pAYL and pMTS1 shared a short near identical sequence (see Fig. 2B), which is highly similar to the nic area of pC194. This further confirms the notion that pMTS1 belongs to the pC194 family of rolling-cycle replication plasmids. dso of rolling-cycle replicating plasmids contains two distinct loci, the bind and nic regions. The pC194 family plasmids have the bind region as
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FIG. 2. (A) Alignment of deduced amino acid sequences of replicative proteins from plasmids: (A) pLB1 (GenBank Accession No. P16953), (B) pLAB1000 (P35857), (C) pFTB14 (P13963), (D) pBAA1 (P36229), (E) pAYL (AF018481), and (F) pMTS1 (AF233873). Identical amino acids are indicated with asterisks and similar amino acids are indicated with dots. The conserved motifs, Motif 2 and Motif 3, in initiator proteins of the pC194 family of rolling-cycle replication plasmids are deduced according to del Solar et al. (1998) and are shown in bold. (B) Alignment of conserved DNA sequences from (A) pMTS1, (B) pAYL, and (C) the consensus nic sequence for pC194 family plasmids according to del Solar et al. (1998). The putative cleavage sites in pAYL and pMTS1 are indicated by vertical arrows.
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an inverted repeat contiguous to the nick site (del Solar et al., 1998). Therefore, we hypothesize that IR3 (see Fig. 1) is in fact the bind region of pMTS1. The nic region, which covalently binds RepA, overlaps with the putative repA promoter, thus suggesting a possible feedback mechanism of pMTS1 replication regulation. The lagging strand replication in rollingcycle plasmids is initiated in a specific noncoding region called sso (del Solar et al., 1998). The only possible location remaining for sso within the pMTS1 genome is the intergenic region I. Indeed, a ssDNA/RNA secondary structure analysis shows that this area forms an extended imperfect hairpin structure commonly associated with sso loci and the portion of the intergenic region I (273–353 bp) is highly ATrich (⬎80%). The promoter P1 sequence is included in this structure and therefore may be responsible for synthesis of a primer RNA during lagging strand replication. To elucidate possible functions of the genes found in pMTS1, it was linearized with different restriction endonucleases (KpnI, SphI, and NarI) and inserted into the multiple cloning site of pUC19 (New England BioLab, MA). The resulting two-replicon plasmids had the repA gene, orf-2, and the intergenic region I interrupted, respectively. Since the pUC19::pMTS1 fusions could not be mobilized directly into the methylotroph, the 1.2-kb HindIII fragment from the plasmid pSUP5011 (Simon, 1984), which carries an origin of transfer (mob) and a Km R marker, was blunted and inserted into the Ecl136I restriction site of the three two-replicon plasmids. The resulting plasmids, called pAYC1119a, pAYC1118, and pAYC1121a, respectively, all have the mob ⫹bla(Ap R)aphC(Km R) genotype and therefore can be mobilized into M. thalassica S1 Rif R. pAYC1118 and pAYC1121a were mobilized from Escherichia coli S17-1 (F ⫺ ⌬proAB thi-1 recA56 RP4-2(Tc R::Mu Km R::Tn7 (Tp RSm R))
integrated into chromosome; Simon, 1994) into M. thalassica S1 Rif R with similar frequencies of about 2 ⫻ 10 ⫺4 per donor cell. However, no transconjugates were obtained during pAYC1119a mobilization experiments. Since at least 10 9 donor cells were present in a conjugation mix, it was concluded that this plasmid is not able to replicate in the methylotrophic host, thus further proving that RepA is indeed a replication protein. Both pAYC1118 and pAYC1121a were able to replicate in M. thalassica S1. Apparently, Orf-2 and IR1 are not essential for pMTS1 replication in its host. ACKNOWLEDGMENT This work was supported by NIH Grant GM52316.
REFERENCES del Solar, G., Giralo, R., Ruiz-Echevarria, M. J., Espinosa, M., and Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microb. Mol. Biol. Rev. 62, 434 – 464. Devine, K. M., Hogan, S. T., Higgins, D. J., and McConnell, D. J. (1989). Replication and segregational stability of Bacillus plasmid pBAA1. J. Bacteriol. 171, 1166 –1172. Fulton, G. L., Nunn, D. N., and Lidstrom, M. E. (1984). Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph. J. Bacteriol. 160, 718 –723. Maniatis, T., Fritsch, E. F., and SamBrook, J. (1982). ‘‘Molecular Cloning: A Laboratory Manual.’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sieburth, J. M. (1988). The tropic role of bacteria in marine ecosystems is complicated by synergistic consorsia and mixotrophic cometabolism. Limnol. Oceanog. 21, 117– 128. Simon, R. (1984). High-frequency mobilization of Gramnegative bacterial replicons by the in vitro constructed Tn5-mob transposon. Mol. Gen. Genet. 196, 413– 420. Yamagata, A., Kato, J., Hirota, R., Kuroda, A., Ikeda, T., Takiguchi, H., and Ohtake, H. (1999). Isolation and characterization of two cryptic plasmids in the ammoniaoxidizing bacterium Nitrosomonas sp. strain ENI-11. J. Bacteriol. 181, 3375–3381. Communicated by A. Chakrabarty
SHORT COMMUNICATION Genetic Analysis and Complete Nucleotide Sequence of a 2-kb Cryptic Plasmid from the Marine Methylotroph Methylophaga thalassica S1 Andrei Y. Chistoserdov 1 Marine Sciences Research Center, State University of New York at Stony Brook, Stony Brook, New York 11794-5000 Received February 22, 2000; revised April 12, 2000 A small cryptic plasmid (pMTS1) was isolated from the marine methylotroph Methylophaga thalassica S1. Sequence analysis showed that pMTS1 has a 1995-bp genome encoding three putative polypeptides and containing two intergenic regions. The longest open reading frame encodes a polypeptide of 325 amino acid (37,079 Da). This polypeptide shares similarity and signature amino acids with the rep proteins of the pC194 group replicons, suggesting that pMTS1 replicates by the rolling-cycle mechanism. Insertional mutagenesis confirmed that the repA gene product is obligatory for replication of pMTS1 in M. thalassica S1. A pC194-type dso was identified in the second intergenic area. It appears that this intergenic region contains both the nic and bind sites. It is hypothesized that sso of pMTS1 is located in the first intergenic region. Two smaller open reading frames (orf-1 and orf-2) encode transcriptionally coupled polypeptides of 123 and 101 amino acids (14,486 and 11,241 Da, respectively) with unknown biological function. © 2000 Academic Press
The Methylophaga spp. play an important role in marine coastal environments, participating in remineralization of organic carbon and, particularly, biogenic amines (Sieburth, 1988). The genetics of the Methylophaga genus is essentially unknown and no cloning vectors have been described for this genus. One strain, M. thalassica S1, harbors a small 2-kb plasmid, called pMTS1. M. thalassica S1 Rif R was grown in a mineral medium (Fulton et al., 1984) with 1% of methanol as a source of carbon and 200 g/ml of rifamycin or 150 g/ml of kanamycin when needed. General cloning procedures such as plasmid isolation (boiling method), restriction endonuclease digest and mapping, agarose gel electrophoresis, ligation, and transformation were carried out according to Maniatis et al. (1982). pUC19 derivatives were isolated for sequencing using Qiagen columns according to the protocol of the manufacturer (Qiagen, Chatsworth, CA). This is Contribution No. 1183 from Marine Sciences Research Center. 1 To whom correspondence should be addressed. Fax: (516) 632-8820. E-mail: [email protected].
The complete nucleotide sequence of pMTS1 was determined and contains 1995 bp (Fig. 1). Three open reading frames encoding polypeptides of 325 (37,079 Da), 123 (14,486 Da), and 101 (11,241 Da) amino acids were identified. The three genes are transcribed in the same direction and the two shorter open reading frames, designated as orf-1 and orf-2, are transcriptionally coupled and thus comprise an operon. A search in the GenBank databases revealed that the 325-amino-acid polypeptide, encoded by the largest open reading frame, shares similarity with replication proteins of several small rolling-cycle replication plasmids and Orf-2 from the cryptic plasmid pAYL found in Nitrosomonas sp. Strain ENI-11 (Yamagata et al., 1999). Therefore, this open reading frame was named repA. The RepA polypeptide shares the highest level of identity (49.4%) with Orf-2 from pAYL, followed by the replication protein of the Bacillus sp. plasmid pBAA1 (31.6%). An alignment of several RepA-like proteins from pMTS1, pAYL, pBBAA1, and several rollingcycle replication plasmids belonging to the pC194 family is shown in Fig. 2A. These proteins share conserved motifs (del Solar et al.,
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0147-619X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Complete nucleotide sequences of pMTS1 and the polypeptides it encodes (GenBank Accession No. AF233873). Inverted repeats are indicated with arrows (IR stands for inverted repeat). ⫺35 and ⫺10 regions of putative promoters are double underlined, and ribosome-binding sites are underlined. Recognition sites of selected restriction endonucleases are shown in bold. The DNA sequence highly similar in pMTS1 and pAYL is italicized, and the nick site is indicated with a vertical arrow. A putative membrane-spanning stretch of Orf-1 is italicized as well.
1998), suggesting that both pMTS1 and pAYL are members of the pC194 family. A potential promoter has been identified upstream of the repA gene, designated P3 in Fig. 1. Unlike repA
genes from the majority of pC194 family plasmids (del Solar et al., 1998), repA from pMTS1 has an efficient ribosome binding site. The two hairpin structures immediately downstream of
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FIG. 1—Continued
the repA gene (IR1 and IR2) may function as -independent terminators. No sequences homologous to either Orf-1 or Orf-2 were found in the GenBank database. The genes for these two polypeptides are preceded by two putative promoters (shown as P1 and P2 in Fig. 1). In addition to the three open reading frames, the pMTS1 genome contains two intergenic regions: the region between the 3⬘ terminus of the repA and the orf-1/orf-2 operon was called the
intergenic region I and the DNA stretch between the 3⬘ terminus of orf-2 and the beginning of repA comprises the intergenic region II. pAYL and pMTS1 shared a short near identical sequence (see Fig. 2B), which is highly similar to the nic area of pC194. This further confirms the notion that pMTS1 belongs to the pC194 family of rolling-cycle replication plasmids. dso of rolling-cycle replicating plasmids contains two distinct loci, the bind and nic regions. The pC194 family plasmids have the bind region as
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FIG. 2. (A) Alignment of deduced amino acid sequences of replicative proteins from plasmids: (A) pLB1 (GenBank Accession No. P16953), (B) pLAB1000 (P35857), (C) pFTB14 (P13963), (D) pBAA1 (P36229), (E) pAYL (AF018481), and (F) pMTS1 (AF233873). Identical amino acids are indicated with asterisks and similar amino acids are indicated with dots. The conserved motifs, Motif 2 and Motif 3, in initiator proteins of the pC194 family of rolling-cycle replication plasmids are deduced according to del Solar et al. (1998) and are shown in bold. (B) Alignment of conserved DNA sequences from (A) pMTS1, (B) pAYL, and (C) the consensus nic sequence for pC194 family plasmids according to del Solar et al. (1998). The putative cleavage sites in pAYL and pMTS1 are indicated by vertical arrows.
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an inverted repeat contiguous to the nick site (del Solar et al., 1998). Therefore, we hypothesize that IR3 (see Fig. 1) is in fact the bind region of pMTS1. The nic region, which covalently binds RepA, overlaps with the putative repA promoter, thus suggesting a possible feedback mechanism of pMTS1 replication regulation. The lagging strand replication in rollingcycle plasmids is initiated in a specific noncoding region called sso (del Solar et al., 1998). The only possible location remaining for sso within the pMTS1 genome is the intergenic region I. Indeed, a ssDNA/RNA secondary structure analysis shows that this area forms an extended imperfect hairpin structure commonly associated with sso loci and the portion of the intergenic region I (273–353 bp) is highly ATrich (⬎80%). The promoter P1 sequence is included in this structure and therefore may be responsible for synthesis of a primer RNA during lagging strand replication. To elucidate possible functions of the genes found in pMTS1, it was linearized with different restriction endonucleases (KpnI, SphI, and NarI) and inserted into the multiple cloning site of pUC19 (New England BioLab, MA). The resulting two-replicon plasmids had the repA gene, orf-2, and the intergenic region I interrupted, respectively. Since the pUC19::pMTS1 fusions could not be mobilized directly into the methylotroph, the 1.2-kb HindIII fragment from the plasmid pSUP5011 (Simon, 1984), which carries an origin of transfer (mob) and a Km R marker, was blunted and inserted into the Ecl136I restriction site of the three two-replicon plasmids. The resulting plasmids, called pAYC1119a, pAYC1118, and pAYC1121a, respectively, all have the mob ⫹bla(Ap R)aphC(Km R) genotype and therefore can be mobilized into M. thalassica S1 Rif R. pAYC1118 and pAYC1121a were mobilized from Escherichia coli S17-1 (F ⫺ ⌬proAB thi-1 recA56 RP4-2(Tc R::Mu Km R::Tn7 (Tp RSm R))
integrated into chromosome; Simon, 1994) into M. thalassica S1 Rif R with similar frequencies of about 2 ⫻ 10 ⫺4 per donor cell. However, no transconjugates were obtained during pAYC1119a mobilization experiments. Since at least 10 9 donor cells were present in a conjugation mix, it was concluded that this plasmid is not able to replicate in the methylotrophic host, thus further proving that RepA is indeed a replication protein. Both pAYC1118 and pAYC1121a were able to replicate in M. thalassica S1. Apparently, Orf-2 and IR1 are not essential for pMTS1 replication in its host. ACKNOWLEDGMENT This work was supported by NIH Grant GM52316.
REFERENCES del Solar, G., Giralo, R., Ruiz-Echevarria, M. J., Espinosa, M., and Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microb. Mol. Biol. Rev. 62, 434 – 464. Devine, K. M., Hogan, S. T., Higgins, D. J., and McConnell, D. J. (1989). Replication and segregational stability of Bacillus plasmid pBAA1. J. Bacteriol. 171, 1166 –1172. Fulton, G. L., Nunn, D. N., and Lidstrom, M. E. (1984). Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph. J. Bacteriol. 160, 718 –723. Maniatis, T., Fritsch, E. F., and SamBrook, J. (1982). ‘‘Molecular Cloning: A Laboratory Manual.’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sieburth, J. M. (1988). The tropic role of bacteria in marine ecosystems is complicated by synergistic consorsia and mixotrophic cometabolism. Limnol. Oceanog. 21, 117– 128. Simon, R. (1984). High-frequency mobilization of Gramnegative bacterial replicons by the in vitro constructed Tn5-mob transposon. Mol. Gen. Genet. 196, 413– 420. Yamagata, A., Kato, J., Hirota, R., Kuroda, A., Ikeda, T., Takiguchi, H., and Ohtake, H. (1999). Isolation and characterization of two cryptic plasmids in the ammoniaoxidizing bacterium Nitrosomonas sp. strain ENI-11. J. Bacteriol. 181, 3375–3381. Communicated by A. Chakrabarty