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Bacteroides Shuttle Vectors
Plasmid 45, 227–232 (2001) doi:10.1006/plas.2000.1515, available online at http://www.academicpress.com on
SHORT COMMUNICATION Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors Derry K. Mercer,1 Saleem Patel, and Harry J. Flint Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Great Britain Received October 20, 2000; revised December 20, 2000 pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4. © 2001 Academic Press Key Words: Prevotella ruminicola; shuttle vector; cryptic plasmid; rumen; Rep; Pre.
Prevotella spp. occur in many regions of the gastrointestinal tract of mammals, including the oral cavity, rumen, and hind gut. In the rumen, Prevotella spp. represent a high proportion of bacterial diversity based both on cultivation and on direct analysis of 16S rRNA sequences (Bryant et al., 1958; Van Gylswyk et al., 1990; Wood et al., 1998; Whitford et al., 1998; Tajima et al., 1999; Ramsak et al., 2000). Understanding of gene transfer in these organisms is still very limited, but plasmids have been reported from a few strains (Flint et al., 1987, 1988; Ogata et al., 1996). P. ruminicola 223/M2/7 carries three plasmids, including a self-transmissible 19.5-kb plasmid that encodes tetracycline resistance via a tetQ determinant (Flint et al., 1988; Nikolich et al., 1994) and two smaller cryptic plasmids of 7.0 and 3.4 kb (Flint et al., 1988). The smallest of these, pRRI2, has been used to construct the shuttle vectors pRRI207 1 To whom correspondence should be addressed. Fax: 01224 716687. E-mail: [email protected].
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and pRH3 by fusion with existing Escherichia coli vectors pHG165 and pBluescript SK⫹ and incorporation of selectable erm or tetQ marker genes from Bacteroides spp., respectively (Thomson et al., 1992; Daniel et al., 1995). These vectors were able to replicate in several Bacteroides species, demonstrating that pRRI2 has a fairly broad host range (Flint et al., 2000). pRH3 has also been used to express cloned glycoside hydrolase genes derived from P. ruminicola 23 in Bacteroides spp. (Daniel et al., 1995). Until now, however, there has been no detailed molecular information on plasmids of rumen Prevotella spp., apart from one report on the plasmid pRAM4 of P. ruminicola T31 (Ogata et al., 1996). Here we present sequence analysis of pRRI2 and use this information to interpret the behavior of pRRI2 vector constructs. The cryptic plasmid pRRI2 (Accession No. AJ278872) was isolated from P. ruminicola 223/M2/7. This is only the second plasmid so far sequenced from Prevotella spp. (Ogata et al., 0147-619X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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1996). pRRI2 has a GC content of 41.0%, which is lower than the average GC content of coding regions of P. ruminicola (47.2%) or the genomic GC content of P. ruminicola 223/M2/7 (49.1%) (Avgustin et al., 1994). Two unambiguous open reading frames are visible, ORF1 (1068–1913 bp) and ORF2 (2278–2856 bp). ORF1 and ORF2 encode polypeptides with molecular sizes of 33.2 and 22.4 kDa, respectively. Upstream of ORF1 is a region containing a number of inverted and tandem repeats (722–857), with an AT-rich region immediately upstream (665–721, 78.4%) and one immediately downstream (858–904, 70.2%) of this region. These features are characteristic of replication origins (Komberg and Baker, 1992). Within this region are three tandem DNA repeats (iterons) (AGTATTAAATTTGTAAT) which correspond to possible binding sites for Rep proteins. Such sequences are common to plasmids replicating by any of the three mechanisms of replication for circular plasmids and are essential for replication and its control (del Solar et al., 1998). Within this region is a 21-bp region (840–861) that shows 55% identity with a nic site of the Staphylococcus aureus plasmid pC194, a region essential for DNA strand cleavage during replication of rolling-circle plasmids (del Solar et al., 1998). Also within this region (722–857) are three sets of inverted repeats. ORF1, named rep, shows 48% amino acid sequence identity with the repA gene of the B. vulgatus 5-nitroimidazole resistance plasmid pIP417, which encodes a putative replication protein, over a 247-amino-acid region, and 47% amino acid sequence identity to the repA gene of the B. fragilis 5-nitroimidazole resistance plasmid pIP421 over a 246-amino-acid region (Haggoud et al., 1995). ORF1 also shows lower sequence homology to the RepA protein of a cryptic plasmid of Campylobacter hyointestinalis (25% over 267 amino acids) (Waterman et al., 1993). A ProDom NCBI–BLASTP2 search (Corpet et al., 1999) with the ORF1 amino acid sequence revealed 48% amino acid identity over a 154-amino-acid overlap with a member of the RepB protein family, which is an initiator of plasmid replication and possesses nicking–closing (topoisomerase I)-like activity (del Solar et
al., 1998). The Rep protein of pRRI2 shows no significant homology with the Rep protein of the only other sequenced Prevotella spp. plasmid, pRAM4. Similarly, there is no significant homology between ORF2 of pRRI2 and ORF2 of pRAM4 (Ogata et al., 1996). ORF2, a putative plasmid recombination/mobilization protein (192 amino acids), shows greatest amino acid identity (34% over a 144-amino-acid overlap) with a putative plasmid recombination protein from the plasmid pFL1 of Flavobacterium spp. KP1 (Asiuchi et al., 1999). ORF2 also shows 30% amino acid identity with the mobilization protein BmpH of the B. fragilis transposon Tn5520 (Vedantam et al., 1999). Lower amino acid identities (23–25%) over 172- to 177-amino-acid overlaps are also observed with a number of mobilization/recombination proteins, including PreT of Bacillus spp. plasmid pTB53 (Accession No. D14852), Mob of Bacillus subtilis plasmid pTB19 (Oskam et al., 1991), and Pre of the S. aureus plasmids pUB110, pSK41, and pSV41 (Bashkirov et al., 1986; Berg et al., 1998). Immediately upstream of the putative plasmid recombination/mobilization protein are two stem–loop structures (Fig. 1) that might play a role in the regulation of gene expression. Plasmid recombination proteins are normally associated with the recombination site (RSA) and examination of the sequence upstream of ORF2 (2130–2148) revealed a recombination site RSA-like sequence, which contains a short (7 bp) inverted repeat and shows 45% homology with RSA sequences from pFL1 of Flavobacterium spp. KP1 (Asiuchi et al., 1999), pOM1 of Butyrivibrio fibrisolvens (Hefford et al., 1997), and pT181 of S. aureus (Kahn and Novick, 1983). RSA sequences are characteristic of plasmids that demonstrate a rolling-circle mechanism of replication (Hefford et al., 1997). pRRI2 contains a unique HindIII restriction site (1632–1637) and when pRRI2 was cut with HindIII and ligated with the E. coli plasmid vector pHG165, the resulting plasmid was capable of replication in E. coli but could not replicate when transferred by electroporation into B. uniformis (Thomson et al., 1992). The authors con-
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FIG. 1. Putative regulatory region upstream of the putative plasmid recombination/mobilization protein gene (ORF2) of pRRI2. The two stem loops have free energies (⌬G) of ⫺15.4 and ⫺16.6 kcal/mol, respectively. The start codon of the putative plasmid recombination/mobilization protein gene (ORF2) is underlined.
cluded that the HindIII site must fall within a site necessary for plasmid replication or maintenance in Bacteroides spp. and the sequence data confirm that the HindIII site falls within a gene involved in plasmid replication, rep. When pRRI2 was cut with EcoRI* (Gardner et al., 1982) and ligated with pHG165 plus an erythromycin/clindamycin resistance marker to form pRRI207, this construct was capable of replication in both E. coli and several Bacteroides species. This indicates that EcoRI* cleavage did not destroy functions necessary for replication in Bacteroides spp. (Thomson et al., 1992). Consistent with this, EcoRI* does not cut within rep, but within ORF2, the putative plasmid recombination/mobilization protein. pRRI207 was mobilizable from E. coli to E. coli or Bacteroides spp. recipients using the helper plasmid pRK2013 (which acts on the pHG165 section of the plasmid), but was not mobilizable by the IncP helper plasmid, which has been shown previously to mobilize a number of other Bacteroides spp. plasmids (Pheulpin et al., 1988; Shoemaker et al., 1986). It is possible that an intact ORF2 might extend the mobilization potential of constructs based on pRRI2. Restriction analysis of pRRI2 reveals unique ClaI (0), NcoI (368), and StuI (623) sites that fall outside both ORFs and
OriR. These sites may be used to construct other shuttle vectors able to replicate and perhaps be mobilized by native plasmids in the Bacteroides/Prevotella group. Another consequence of the disruption of ORF2 in pRRI2 is that the putative ORF2 promoter is placed immediately upstream of a cloning site. This promoter may therefore have contributed to expression of xylanase activity from P. ruminicola inserts cloned into this site in the Bacteroides/Prevotella shuttle vector pRH3, which contains the complete sequence of pRRI2 (Daniel et al., 1995). One of the major stumbling blocks in further molecular analysis of Prevotella spp. is the lack of systems for the genetic manipulation of these bacteria in the laboratory. An important step toward solving this problem is the further development of Prevotella/E. coli shuttle vectors and the concomitant development of systems for the introduction of such constructs into the cells. The use of pRRI2 in the construction of the Bacteroides/Prevotella shuttle vector pRH3 (Fig. 2), despite its origin in P. ruminicola 223/M2/7, has shown that it has potential for use in the genera Prevotella and Bacteroides. The complete nucleotide sequence of this plasmid offers the potential to develop more widely applicable shuttle vector systems for Prevotella spp., for
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FIG. 2. Plasmid map of pRRI2 and pRH3, a shuttle vector derived from pRRI2. pRRI2 was cut with EcoRI* and inserted into pBluescript SK⫹ (Stratagene) at the EcoRI site. The tet(Q) gene from a Bacteroides spp. isolate (Stevens et al., 1990) was inserted at the SstI site to give the plasmid pRH2. pRH2 was cleaved with SalI and religated to form the vector pRH3. Xylanase genes of P. ruminicola were inserted using the unique KpnI and SalI sites and were expressed, presumably making use of the putative plasmid recombination/mobilization protein gene promoter (Daniel et al., 1995).
example, by restoring plasmid mobilization. Conjugal transfer has proved, so far, to be the only means for delivering plasmids into certain Prevotella species [e.g., P. bryantii B14 (Gardner et al., 1996], and the ability to mobilize constructs with a wider range of helper elements is likely to be crucial. ACKNOWLEDGMENTS We acknowledge the support of the Scottish Executive Rural Affairs Department and the UK Food Standards Agency. Saleem Patel was
funded by a UK Biotechnology and Biological Science Research Council Ph.D. studentship. REFERENCES Asiuchi, M., Zakaria, M. M., Sakaguchi, Y., and Yagi, T. (1999). Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium. FEMS Microbiol. Lett. 170, 243–249. Avgustin, G., Wright, F., and Flint, H. J. (1994). Genetic diversity and phylogenetic relationships among strains of Prevotella (Bacteroides) ruminicola from the rumen. Int. J. Syst. Bacteriol. 44, 246–255. Bashkirov, V. I., Mil’Shina, N. V., and Prozorov, A. A. (1986). Nucleotide sequence and physical map of
SHORT COMMUNICATION kanamycin-resistant plasmid RT pUB110 from Staphylococcus aureus. Genetika 22, 1081–1092. Berg, T., Firth, N., Apisiridej, S., Hettiaratchi, A., Leelaporn, A., and Skurray, R. A. (1998). Complete nucleotide sequence of pSK41: Evolution of staphylococcal conjugative multiresistance plasmids. J. Bacteriol. 180, 4350–4359. Bryant, M. P., Small, N., Bouma, C., and Chu, H. (1958). Bacteroides ruminicola n. sp. and Succinogenes amylolytica: The new genus and species. J. Bacteriol. 76, 15–23. Corpet, F., Gouzy, J., and Kahn, D. (1999). Recent improvements of the ProDom database of protein families. Nucleic Acids Res. 27, 263–267. Daniel, A. S., Martin, J., Vanat, I., Whitehead, T. R., and Flint, H. J. (1995). Expression of a cloned cellulase/xylanase gene from Prevotella ruminicola in Bacteroides vulgatus, Bacteroides uniformis and Prevotella ruminicola. J. Appl. Bacteriol. 79, 417–424. del Solar, G., Giraldo, R., Ruiz-Echevarría, M. J., Espinosa, M., and Díaz-Orajas, R. (1998). Replication and control of circular bacterial plasmids. Microbiol. Mol. Biol. Rev. 62, 434–464. Flint, H. J., Martin, J. C., and Thomson, A. M. (2000). Prevotella bryantii, P. ruminicola and Bacteroides strains. In “Electrotransformation of Bacteria” (V. Teissie and N. Rynard, Eds.), pp. 138–149. Springer-Verlag, Berlin/New York. Flint, H. J., Thomson, A. M., and Bisset, J. (1988). Plasmidassociated transfer of tetracycline resistance in Bacteroides ruminicola. Appl. Environ. Microbiol. 54, 855–860. Flint, H. J., and Stewart, C. S. (1987). Antibiotic resistance patterns and plasmids of ruminal strains of Bacteroides ruminicola and Bacteroides multiacidus. Appl. Microbiol. Biotechnol. 26, 450–455. Gardner, R. C., Howarth, A. J., Messing, J., and Shepherd, R. J. (1982). Cloning and sequencing of restriction fragments generated by Eco RI*. DNA 1, 109–115. Gardner, R. G., Russell, J. B., Wilson, D. B., Wang, G.-R., and Shoumaker, N. B. (1996). Use of a modified Bacteroides–Prevotella shuttle vector to transfer a reconstructed -1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B14. Appl. Environ. Microbiol. 62, 196–202. Haggoud, A., Trinh, S., Moumni, M., and Reysset, G. (1995). Genetic analysis of the minimal replicon of plasmid pIP417 and comparison with the other encoding 5-nitroimidazole resistance plasmids from Bacteroides spp. Plasmid 4, 132–143. Hefford, M. A., Kobayashi, Y., Allard, S. E., Forster, R. J., and Teather, R. M. (1997). Sequence analysis and characterisation of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for butyrivibrios. Appl. Environ. Microbiol. 63, 1701–1711. Kahn, S. A., and Novick, R. P. (1983). Complete nucleotide sequence of pT181, a tetracycline resistance plasmid from Staphylococcus aureus. Plasmid 10, 251–259.
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Kornberg, A., and Baker, T. A. (1992). “DNA Replication,” 2nd ed. Freeman, New York. Nikolich, M. P., Hong, G., Shoemaker, N. B., and Salyers, A. A. (1994). Evidence for natural horizontal transfer of tetQ between bacteria that normally colonise humans and bacteria that normally colonise livestock. Appl. Environ. Microbiol. 60, 3255–3260. Ogata, K., Aminov, R. I., Nagamine, T., Benno, Y., Sekizaki, T., Mitsumori, M., Minato, H., and Itabashi, H. (1996). Structural organisation of pRAM4, a cryptic plasmid from Prevotella ruminicola. Plasmid 35, 91–97. Oskam, L., Hillenga, D. J., Venema, G., and Bron, S. (1991). The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions. Plasmid 26, 30–39. Pheulpin, P., Tierny, Y., Béchet, M., and Guillaume, J.-B. (1988). Construction of new shuttle plasmid vectors for Escherichia coli–Bacteroides transgeneric cloning. FEMS Microbiol. Lett. 55, 15–22. Priebe, S. D., and Lacks, S. A. (1989). Region of the streptococcal plasmid pMV158 required for conjugative mobilisation. J. Bacteriol. 171, 4778–4784. Ramsˇ ak, A., Peterka, M., Tajima, K., Martin, J. C., Wood, J., Johnston, M. E. A., Aminov, R. I., Flint, H. J., and Avgusˇ tin, G. (2000). Unravelling the genetic diversity of ruminal bacteria belonging to the CFB phylum. FEMS Microbiol. Ecol. 33, 69–79. Shoemaker, N. B., Getty, C., Guthrie, E. P., and Salyers, A. A. (1986). Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli–Bacteroides shuttle vectors to be mobilised by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element. J. Bacteriol. 166, 959–965. Tajima, K., Aminov, R. I., Nagamine, T., Ogata, K., Nakamura, M., Matsui, H., and Benno, Y. (1999). Rumen bacterial diversity as determined by sequence analysis of 16S rDNA libraries. FEMS Microbiol. Ecol. 29, 159–169. Thomson, A. M., Flint, H. J., Béchet, M., Martin, J., and Dubourguier, H.-C. (1992). A new E. coli:Bacteroides shuttle vector pRRI207 based on the Bacteroides ruminicola plasmid replicon pRRI2. Curr. Microbiol. 24, 49–54. van Dijl, J. M., de Jong, A., Vehmaanperä, J., Venema, G., and Bron, S. (1992). Signal peptidase I of Bacillus subtilis: Patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases. EMBO J. 11, 2819–2828. van Gylswyk, N. O. (1990). Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass-silage based diets. FEMS Microbiol. Ecol. 73, 243–254. Vedantam, G., Novicki, T. J., and Hecht, D. W. (1999). Bacteroides fragilis transfer factor Tn5520: The smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli. J. Bacteriol. 181, 2564–2571. Waterman S. R., Hackett J. A., and Manning P. A. (1993). Characterization of the replication region of the small
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cryptic RT plasmid of Campylobacter hyointestinalis. Gene 125, 11–17. Whitford, M. F., Forster, R. J., Beard, C. E., Gong, J., and Teather, R. M. (1998). Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes. Anaerobe 4, 153–163. Wood, J., Scott, K. P., Avgusˇ tin, G., Newbold, C. J., and
Flint, H. J. (1998). Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences. Appl. Environ. Microbiol. 64, 3683–3689. Communicated by C. J. Smith
SHORT COMMUNICATION Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors Derry K. Mercer,1 Saleem Patel, and Harry J. Flint Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Great Britain Received October 20, 2000; revised December 20, 2000 pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4. © 2001 Academic Press Key Words: Prevotella ruminicola; shuttle vector; cryptic plasmid; rumen; Rep; Pre.
Prevotella spp. occur in many regions of the gastrointestinal tract of mammals, including the oral cavity, rumen, and hind gut. In the rumen, Prevotella spp. represent a high proportion of bacterial diversity based both on cultivation and on direct analysis of 16S rRNA sequences (Bryant et al., 1958; Van Gylswyk et al., 1990; Wood et al., 1998; Whitford et al., 1998; Tajima et al., 1999; Ramsak et al., 2000). Understanding of gene transfer in these organisms is still very limited, but plasmids have been reported from a few strains (Flint et al., 1987, 1988; Ogata et al., 1996). P. ruminicola 223/M2/7 carries three plasmids, including a self-transmissible 19.5-kb plasmid that encodes tetracycline resistance via a tetQ determinant (Flint et al., 1988; Nikolich et al., 1994) and two smaller cryptic plasmids of 7.0 and 3.4 kb (Flint et al., 1988). The smallest of these, pRRI2, has been used to construct the shuttle vectors pRRI207 1 To whom correspondence should be addressed. Fax: 01224 716687. E-mail: [email protected].
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and pRH3 by fusion with existing Escherichia coli vectors pHG165 and pBluescript SK⫹ and incorporation of selectable erm or tetQ marker genes from Bacteroides spp., respectively (Thomson et al., 1992; Daniel et al., 1995). These vectors were able to replicate in several Bacteroides species, demonstrating that pRRI2 has a fairly broad host range (Flint et al., 2000). pRH3 has also been used to express cloned glycoside hydrolase genes derived from P. ruminicola 23 in Bacteroides spp. (Daniel et al., 1995). Until now, however, there has been no detailed molecular information on plasmids of rumen Prevotella spp., apart from one report on the plasmid pRAM4 of P. ruminicola T31 (Ogata et al., 1996). Here we present sequence analysis of pRRI2 and use this information to interpret the behavior of pRRI2 vector constructs. The cryptic plasmid pRRI2 (Accession No. AJ278872) was isolated from P. ruminicola 223/M2/7. This is only the second plasmid so far sequenced from Prevotella spp. (Ogata et al., 0147-619X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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1996). pRRI2 has a GC content of 41.0%, which is lower than the average GC content of coding regions of P. ruminicola (47.2%) or the genomic GC content of P. ruminicola 223/M2/7 (49.1%) (Avgustin et al., 1994). Two unambiguous open reading frames are visible, ORF1 (1068–1913 bp) and ORF2 (2278–2856 bp). ORF1 and ORF2 encode polypeptides with molecular sizes of 33.2 and 22.4 kDa, respectively. Upstream of ORF1 is a region containing a number of inverted and tandem repeats (722–857), with an AT-rich region immediately upstream (665–721, 78.4%) and one immediately downstream (858–904, 70.2%) of this region. These features are characteristic of replication origins (Komberg and Baker, 1992). Within this region are three tandem DNA repeats (iterons) (AGTATTAAATTTGTAAT) which correspond to possible binding sites for Rep proteins. Such sequences are common to plasmids replicating by any of the three mechanisms of replication for circular plasmids and are essential for replication and its control (del Solar et al., 1998). Within this region is a 21-bp region (840–861) that shows 55% identity with a nic site of the Staphylococcus aureus plasmid pC194, a region essential for DNA strand cleavage during replication of rolling-circle plasmids (del Solar et al., 1998). Also within this region (722–857) are three sets of inverted repeats. ORF1, named rep, shows 48% amino acid sequence identity with the repA gene of the B. vulgatus 5-nitroimidazole resistance plasmid pIP417, which encodes a putative replication protein, over a 247-amino-acid region, and 47% amino acid sequence identity to the repA gene of the B. fragilis 5-nitroimidazole resistance plasmid pIP421 over a 246-amino-acid region (Haggoud et al., 1995). ORF1 also shows lower sequence homology to the RepA protein of a cryptic plasmid of Campylobacter hyointestinalis (25% over 267 amino acids) (Waterman et al., 1993). A ProDom NCBI–BLASTP2 search (Corpet et al., 1999) with the ORF1 amino acid sequence revealed 48% amino acid identity over a 154-amino-acid overlap with a member of the RepB protein family, which is an initiator of plasmid replication and possesses nicking–closing (topoisomerase I)-like activity (del Solar et
al., 1998). The Rep protein of pRRI2 shows no significant homology with the Rep protein of the only other sequenced Prevotella spp. plasmid, pRAM4. Similarly, there is no significant homology between ORF2 of pRRI2 and ORF2 of pRAM4 (Ogata et al., 1996). ORF2, a putative plasmid recombination/mobilization protein (192 amino acids), shows greatest amino acid identity (34% over a 144-amino-acid overlap) with a putative plasmid recombination protein from the plasmid pFL1 of Flavobacterium spp. KP1 (Asiuchi et al., 1999). ORF2 also shows 30% amino acid identity with the mobilization protein BmpH of the B. fragilis transposon Tn5520 (Vedantam et al., 1999). Lower amino acid identities (23–25%) over 172- to 177-amino-acid overlaps are also observed with a number of mobilization/recombination proteins, including PreT of Bacillus spp. plasmid pTB53 (Accession No. D14852), Mob of Bacillus subtilis plasmid pTB19 (Oskam et al., 1991), and Pre of the S. aureus plasmids pUB110, pSK41, and pSV41 (Bashkirov et al., 1986; Berg et al., 1998). Immediately upstream of the putative plasmid recombination/mobilization protein are two stem–loop structures (Fig. 1) that might play a role in the regulation of gene expression. Plasmid recombination proteins are normally associated with the recombination site (RSA) and examination of the sequence upstream of ORF2 (2130–2148) revealed a recombination site RSA-like sequence, which contains a short (7 bp) inverted repeat and shows 45% homology with RSA sequences from pFL1 of Flavobacterium spp. KP1 (Asiuchi et al., 1999), pOM1 of Butyrivibrio fibrisolvens (Hefford et al., 1997), and pT181 of S. aureus (Kahn and Novick, 1983). RSA sequences are characteristic of plasmids that demonstrate a rolling-circle mechanism of replication (Hefford et al., 1997). pRRI2 contains a unique HindIII restriction site (1632–1637) and when pRRI2 was cut with HindIII and ligated with the E. coli plasmid vector pHG165, the resulting plasmid was capable of replication in E. coli but could not replicate when transferred by electroporation into B. uniformis (Thomson et al., 1992). The authors con-
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FIG. 1. Putative regulatory region upstream of the putative plasmid recombination/mobilization protein gene (ORF2) of pRRI2. The two stem loops have free energies (⌬G) of ⫺15.4 and ⫺16.6 kcal/mol, respectively. The start codon of the putative plasmid recombination/mobilization protein gene (ORF2) is underlined.
cluded that the HindIII site must fall within a site necessary for plasmid replication or maintenance in Bacteroides spp. and the sequence data confirm that the HindIII site falls within a gene involved in plasmid replication, rep. When pRRI2 was cut with EcoRI* (Gardner et al., 1982) and ligated with pHG165 plus an erythromycin/clindamycin resistance marker to form pRRI207, this construct was capable of replication in both E. coli and several Bacteroides species. This indicates that EcoRI* cleavage did not destroy functions necessary for replication in Bacteroides spp. (Thomson et al., 1992). Consistent with this, EcoRI* does not cut within rep, but within ORF2, the putative plasmid recombination/mobilization protein. pRRI207 was mobilizable from E. coli to E. coli or Bacteroides spp. recipients using the helper plasmid pRK2013 (which acts on the pHG165 section of the plasmid), but was not mobilizable by the IncP helper plasmid, which has been shown previously to mobilize a number of other Bacteroides spp. plasmids (Pheulpin et al., 1988; Shoemaker et al., 1986). It is possible that an intact ORF2 might extend the mobilization potential of constructs based on pRRI2. Restriction analysis of pRRI2 reveals unique ClaI (0), NcoI (368), and StuI (623) sites that fall outside both ORFs and
OriR. These sites may be used to construct other shuttle vectors able to replicate and perhaps be mobilized by native plasmids in the Bacteroides/Prevotella group. Another consequence of the disruption of ORF2 in pRRI2 is that the putative ORF2 promoter is placed immediately upstream of a cloning site. This promoter may therefore have contributed to expression of xylanase activity from P. ruminicola inserts cloned into this site in the Bacteroides/Prevotella shuttle vector pRH3, which contains the complete sequence of pRRI2 (Daniel et al., 1995). One of the major stumbling blocks in further molecular analysis of Prevotella spp. is the lack of systems for the genetic manipulation of these bacteria in the laboratory. An important step toward solving this problem is the further development of Prevotella/E. coli shuttle vectors and the concomitant development of systems for the introduction of such constructs into the cells. The use of pRRI2 in the construction of the Bacteroides/Prevotella shuttle vector pRH3 (Fig. 2), despite its origin in P. ruminicola 223/M2/7, has shown that it has potential for use in the genera Prevotella and Bacteroides. The complete nucleotide sequence of this plasmid offers the potential to develop more widely applicable shuttle vector systems for Prevotella spp., for
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FIG. 2. Plasmid map of pRRI2 and pRH3, a shuttle vector derived from pRRI2. pRRI2 was cut with EcoRI* and inserted into pBluescript SK⫹ (Stratagene) at the EcoRI site. The tet(Q) gene from a Bacteroides spp. isolate (Stevens et al., 1990) was inserted at the SstI site to give the plasmid pRH2. pRH2 was cleaved with SalI and religated to form the vector pRH3. Xylanase genes of P. ruminicola were inserted using the unique KpnI and SalI sites and were expressed, presumably making use of the putative plasmid recombination/mobilization protein gene promoter (Daniel et al., 1995).
example, by restoring plasmid mobilization. Conjugal transfer has proved, so far, to be the only means for delivering plasmids into certain Prevotella species [e.g., P. bryantii B14 (Gardner et al., 1996], and the ability to mobilize constructs with a wider range of helper elements is likely to be crucial. ACKNOWLEDGMENTS We acknowledge the support of the Scottish Executive Rural Affairs Department and the UK Food Standards Agency. Saleem Patel was
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