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Sequence analysis of plasmid pTS1 isolated from oral spirochetes
Plasmid 51 (2004) 61–65 www.elsevier.com/locate/yplas
Short Communication
Sequence analysis of plasmid pTS1 isolated from oral spirochetes Sarita Chauhan* and Howard K. Kuramitsu Department of Oral Biology, State University of New York, Buffalo, NY, USA Received 7 August 2003, revised 29 October 2003
Abstract The naturally occurring plasmid pTS1, identified previously in several species of oral spirochetes, has now been completely sequenced. Analysis of the four open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Treponema denticola; Spirochetes; rep; mob; Conjugation; Periodontitis; Iterons
Oral spirochetes represent the predominant group of organisms associated with the common human disease periodontitis (Loesche and Laughon, 1982). Many of these organisms have not yet been cultivated in the laboratory but a number of these have now been characterized (Olsen et al., 2000). Plasmids have been identified in some strains of oral spirochetes including Treponema denticola and Treponema socranskii (Chan et al., 1996). However, the role of these plasmids in the physiology of these organisms has yet to be demonstrated. Two plasmids, pTD1 and pTS1 have been identified in some, but not all strains of *
Corresponding author. Present address: E320/274, Information and Computing Technologies, DuPont Central Research and Development, Wilmington, DE 19880-0320, USA. Fax: 1-302-695-9873. E-mail addresses: [email protected] (S. Chauhan), kuramits@buffalo.edu (H.K. Kuramitsu).
T. denticola. The former 2.67 kb plasmid has been sequenced and a putative replication region identified (MacDougall et al., 1992). However, plasmid pTS1 has not yet been similarly analyzed. The present communication reports the complete sequence of this cryptic plasmid. Plasmid pTS1 was isolated from T. denticola U9b, kindly provided by E. Chan, Montreal University, Canada. A Qiagen Maxiprep plasmid purification kit was used for isolating pTS1. Consistent with the published restriction map of pTS1 (Chan et al., 1996) four fragments (Fig. 1) were generated following HindIII digestion. The largest HindIII fragment (H4 in Fig. 2) was about 1.9 kb in size and could not be cloned into pBluescript II SK(+) even after multiple attempts. The smaller three HindIII fragments (H1, H2, and H3) were easily cloned into the HindIII site of the above plasmid. Not consistent with the published map mentioned above, two BamHI fragments were
0147-619X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2003.11.002
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S. Chauhan, H.K. Kuramitsu / Plasmid 51 (2004) 61–65
obtained upon digesting pTS1 (Fig. 1) instead of a single fragment. Both of these (B1 and B2, Fig. 2) were successfully cloned into the BamHI site of
Fig. 1. Map of T. denticola plasmid pTS1. The four open reading frames rep (ORF1), mob (ORF2), ORF3 and ORF4 are shown. TD represents the location of a 70 bp region identical to a region in pTD1. Inverted repeats (IR1 and IR2) and repeat elements (RE1 and RE2) are shown as vertical arrows. Key restriction sites and positions in base pairs are also shown. B, BamHI; E, EcoRI; H, HindIII; Ps, PstI; Pv, PvuII.
pBluescript II SK(+) and sequenced along with H1, H2, and H3. In addition, some sequence was obtained by amplifying pTS1 directly as a template using primers based on the sequence of B2 fragment. The sequencing strategy is graphically presented in Fig. 2. We were unable to clone pTS1 linearized with either EcoRI or PstI into pBluescript II SK(+). The pTS1 sequences was submitted to GenBank and assigned accession number AF112856. The complete nucleotide sequence of pTS1 revealed a size of 3.7 kb, slightly smaller than that estimated from earlier electrophoretic analysis (Caudry et al., 1995). Four putative open-reading frames, ORF1, ORF2, ORF3, and ORF4 (Fig. 1), were identified on pTS1. All four ORFs had the same orientation. No significant ORFs were identified on the reverse strand of pTS1. ORF1 (start 217 bp; stop 1662 bp) encoded 481 amino acids and exhibited 45% identity to a hypothetical protein expressed from plasmid pJDB23 isolated from Selenomonas ruminantium. Low levels of homology were also detected with the replication protein gene (sequence kindly provided by Francis L. Macrina, Virginia Commonwealth University, Richmond, VA, USA) found on plasmid pYH420, a shuttle vector with its rep ORF derived from Porphyromonas asaccharolytica ATCC 27067 (Yoshimoto et al., 1997). No significant blast hits
Fig. 2. Strategy for pTS1 sequencing B1 and B2 are BamHI fragments, and H1, H2, and H3 are HindIII fragments that were cloned in pBluescript II SK(+) and sequenced using T3 and T7 vector-borne primers. H4 could not be cloned in pBluescript II SK(+) after several attempts. TS1-primer is a fragment obtained by direct sequencing of pTS1 by primer walk.
S. Chauhan, H.K. Kuramitsu / Plasmid 51 (2004) 61–65
were obtained with the two ORFs identified in plasmid pTD1 (MacDougall et al., 1992) suggesting that the ORF1 of pTS1 is not closely related to pTD1 rep. However, the Clustal W alignment in Fig. 3A shows that there are at least some residues on pTD1 rep that are conserved with pTS1 ORF1 and other rep ORFs. These residues, which are shown in black in Fig. 3A may be important for function of the replication proteins. Taken together, the above information suggested the possibility that the ORF1 corresponded to the rep gene of plasmid pTS1. This was confirmed by cloning a DNA fragment containing this putative gene into an Escherichia coli plasmid to produce a shuttle plasmid, pKMR4PE, capable of replicating in T. denticola 33520 (Chi et al., 1999) as well as in Treponema phagedenis (Chi and Kuramitsu, unpublished results). A 70 bp nucleotide sequence downstream of the rep ORF (TD, 1722–1791 bp, Fig. 1) was found to be 98% identical to a region on pTD1 downstream of rep ORF. Such a high level of identity was surprising because rep open reading frames from pTS1 and pTD1 had no significant homologies at the amino acid level except for a few conserved residues. Presence of the sequence TD on H4 might be responsible for incompatibility with cloning into pBluescript II SK(+) because the fragment B2 (Fig. 2) which overlaps with most of the H4 fragment but lacks the TD sequence was easily cloned in the above plasmid. Many plasmid replicons consist of two essential functional units: an origin of replication and an adjacent replication initiator protein, the Rep protein. Iterons consist of arrays of 20 bp repeats and bind a plasmid-specific replication initiator protein in replication origins of many bacterial plasmids (Chattoraj, 2000). The pTS1 sequence upstream of rep had two identical 22 bp regions RE1 and RE2, which overlapped with inverted repeats IR1 and IR2, respectively (Fig. 3B). We predict that these iteron-like features of pTS1 might constitute the replication origin. Blast analysis of ORF2 (start 2126 bp; stop 3094 bp; Fig. 1) resulted in multiple hits for the mob family of proteins. Clustal W (Thompson et al., 1994) alignments of ORFs from the 10 most homologous sequences revealed that these mob
63
proteins are most conserved in the N-terminal region corresponding to 201 amino acids from the start of the pTS1 putative mob gene. While there was no significant homology among rep ORFs of pTS1 and pTD1 using Blast, the mob ORF of pTS1 was 37% identical to the ORF B of the T. denticola plasmid pTD1 (MacDougall et al., 1992). This suggests that both T. denticola genes may code for mob genes and that plasmids pTD1 and pTS1 can be conjugated between different organisms. The observation that a plasmid with properties identical to pTS1 was also isolated from another oral spirochete T. socranskii (Chan et al., 1996) is compatible with in vivo gene transfer between oral spirochetes. ORF3 (start 3144 bp; stop 3455 bp; Fig. 1) had significant homology to an ORF of unknown function (E-score 7e 13 ) on the chromosome of T. denticola strain 35405. ORF3 appears to be conserved among many bacteria, although a function has not been assigned to the putative protein product. It was interesting to note that ORF3 mapped close to the nucleic acid modification genes on the chromosomes of several pathogenic bacteria, including T. denticola strain 35405, Salmonella typhi CT18, Salmonella paratyphi, Salmonella typhimurium LT2, and Mannheimia haemolytica. ORF4 (start 3421 bp; stop 38 bp, Fig. 1) did not have significant homology with any known proteins. Interestingly ORF4 had Blast hits with very low level of homologies to the genes adjacent to ORF3 homologs in the above mentioned Salmonella species. The conservation of spatial proximity of ORF3 and ORF4 homologs in the plasmid pTS1 and several human pathogens mentioned above suggests that ORF3 and ORF4 may be functionally related. Additionally, the proximity of ORF3 to DNA modification enzymes in several organisms and, in at least one case, to DNA/RNA helicases suggests that ORF3 and ORF4 may be involved in the replication of pTS1. ERGO suite of bioinformatics tools http://www.integratedgenomics.com was found to be very useful for the above analysis. The identification of the rep gene of plasmid pTS1 has already proven valuable in constructing shuttle plasmids for characterizing genes from unculturable treponemes such as T. pallidum expressed in T. denticola (Chi et al., 1999). Very
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Fig. 3. (A) Clustal W alignment of rep ORFs of pTS1 (481 aa), pJDB23,1 pS23,2 pYH420,3 and pTD1.4 [1 Attwood and Brooker (1992) (303 aa). 2 EMBL/Genbank M86247 (303 aa). 3 Francis Macrina (Personal communication) (530 aa). 4 MacDougall et al. (1992) (336 aa)]. (B) Nucleotide sequence of the region upstream of rep ORF. The rep start is shown as underlined ATG. Two repetitive elements are shown in bold and two inverted repeats, IR1 and IR2, are shown with arrows.
S. Chauhan, H.K. Kuramitsu / Plasmid 51 (2004) 61–65
recently it has also been demonstrated that the pTS1 rep gene exhibits significant homology with a gene present on a plasmid isolated from a nontreponemal oral bacterium P. asacchrolytica (Fig. 3A).The identification of a putative mob region on the treponemal plasmids further suggests that transfer of these plasmids may occur within the oral cavity. Therefore, it may be possible to construct conjugation systems based upon these plasmids for transfer into treponemes which cannot be transformed following electroporation.
Acknowledgments This investigation was supported in part by NIH Grant DE09821. The authors are grateful to Gail K. Donaldson (for valuable discussion) and Pat Pulcher (for help in proof reading), both from DuPont Central Research and Development.
References Attwood, G.T., Brooker, J.D., 1992. Complete nucleotide sequence of Selenomonas ruminantium cryptic plasmid pJDB23 and definition of a region necessary for its replication in Escherichia coli. Plasmid 28, 123–129. Caudry, S., Litorinos, A., Gharbia, S.E., Pssara, N., Siboo, R., Keng, T., Chan, E.C., 1995. Distribution and characteriza-
65
tion of plasmids in Oral anaerobic spirochetes. Oral Microbiol. Immunol. 10, 8–12. Chan, E.C., Klitorinos, A., Gharbia, S., Caudry, S.D., Rahal, M.D., Siboo, R., 1996. Characterization of a 4.2-kb plasmid isolated from periodontopathic spirochetes. Oral Microbiol. Immunol. 11, 365–368. Chattoraj, D.K., 2000. Control of plasmid DNA replication by iterons: no longer paradoxical. Mol. Microbiol. 37, 467– 476. Chi, B., Chauhan, S., Kuramitsu, H., 1999. Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum fla A gene. Infect. Immun. 67, 3653–3656. Loesche, W.J., Laughon, B.E., 1982. Role of spirochetes in periodontal diseases. In: Mergenhagen, G.A. (Ed.), Host– Parasite Interactions in Periodontal Diseases. American Society for Microbiology, Washington, DC, pp. 67–75. MacDougall, J., Margarita, D., Saint Girons, I., 1992. Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids. J. Bacteriol. 174, 2724–2728. Olsen, I., Paster, B.J., Dewhirst, F.E., 2000. Taxonomy of spirochetes. Anaerobe 6, 39–57. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. NAR 22, 4673–4680. Yoshimoto, H., Takahasi, Y., Kato, D., Umemoto, T., 1997. Construction of a plasmid vector for transformation of Porphyromonas gingivalis. FEMS Microbiol. Lett. 152, 175–181. Communicated by George M. Weinstock
Short Communication
Sequence analysis of plasmid pTS1 isolated from oral spirochetes Sarita Chauhan* and Howard K. Kuramitsu Department of Oral Biology, State University of New York, Buffalo, NY, USA Received 7 August 2003, revised 29 October 2003
Abstract The naturally occurring plasmid pTS1, identified previously in several species of oral spirochetes, has now been completely sequenced. Analysis of the four open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Treponema denticola; Spirochetes; rep; mob; Conjugation; Periodontitis; Iterons
Oral spirochetes represent the predominant group of organisms associated with the common human disease periodontitis (Loesche and Laughon, 1982). Many of these organisms have not yet been cultivated in the laboratory but a number of these have now been characterized (Olsen et al., 2000). Plasmids have been identified in some strains of oral spirochetes including Treponema denticola and Treponema socranskii (Chan et al., 1996). However, the role of these plasmids in the physiology of these organisms has yet to be demonstrated. Two plasmids, pTD1 and pTS1 have been identified in some, but not all strains of *
Corresponding author. Present address: E320/274, Information and Computing Technologies, DuPont Central Research and Development, Wilmington, DE 19880-0320, USA. Fax: 1-302-695-9873. E-mail addresses: [email protected] (S. Chauhan), kuramits@buffalo.edu (H.K. Kuramitsu).
T. denticola. The former 2.67 kb plasmid has been sequenced and a putative replication region identified (MacDougall et al., 1992). However, plasmid pTS1 has not yet been similarly analyzed. The present communication reports the complete sequence of this cryptic plasmid. Plasmid pTS1 was isolated from T. denticola U9b, kindly provided by E. Chan, Montreal University, Canada. A Qiagen Maxiprep plasmid purification kit was used for isolating pTS1. Consistent with the published restriction map of pTS1 (Chan et al., 1996) four fragments (Fig. 1) were generated following HindIII digestion. The largest HindIII fragment (H4 in Fig. 2) was about 1.9 kb in size and could not be cloned into pBluescript II SK(+) even after multiple attempts. The smaller three HindIII fragments (H1, H2, and H3) were easily cloned into the HindIII site of the above plasmid. Not consistent with the published map mentioned above, two BamHI fragments were
0147-619X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2003.11.002
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S. Chauhan, H.K. Kuramitsu / Plasmid 51 (2004) 61–65
obtained upon digesting pTS1 (Fig. 1) instead of a single fragment. Both of these (B1 and B2, Fig. 2) were successfully cloned into the BamHI site of
Fig. 1. Map of T. denticola plasmid pTS1. The four open reading frames rep (ORF1), mob (ORF2), ORF3 and ORF4 are shown. TD represents the location of a 70 bp region identical to a region in pTD1. Inverted repeats (IR1 and IR2) and repeat elements (RE1 and RE2) are shown as vertical arrows. Key restriction sites and positions in base pairs are also shown. B, BamHI; E, EcoRI; H, HindIII; Ps, PstI; Pv, PvuII.
pBluescript II SK(+) and sequenced along with H1, H2, and H3. In addition, some sequence was obtained by amplifying pTS1 directly as a template using primers based on the sequence of B2 fragment. The sequencing strategy is graphically presented in Fig. 2. We were unable to clone pTS1 linearized with either EcoRI or PstI into pBluescript II SK(+). The pTS1 sequences was submitted to GenBank and assigned accession number AF112856. The complete nucleotide sequence of pTS1 revealed a size of 3.7 kb, slightly smaller than that estimated from earlier electrophoretic analysis (Caudry et al., 1995). Four putative open-reading frames, ORF1, ORF2, ORF3, and ORF4 (Fig. 1), were identified on pTS1. All four ORFs had the same orientation. No significant ORFs were identified on the reverse strand of pTS1. ORF1 (start 217 bp; stop 1662 bp) encoded 481 amino acids and exhibited 45% identity to a hypothetical protein expressed from plasmid pJDB23 isolated from Selenomonas ruminantium. Low levels of homology were also detected with the replication protein gene (sequence kindly provided by Francis L. Macrina, Virginia Commonwealth University, Richmond, VA, USA) found on plasmid pYH420, a shuttle vector with its rep ORF derived from Porphyromonas asaccharolytica ATCC 27067 (Yoshimoto et al., 1997). No significant blast hits
Fig. 2. Strategy for pTS1 sequencing B1 and B2 are BamHI fragments, and H1, H2, and H3 are HindIII fragments that were cloned in pBluescript II SK(+) and sequenced using T3 and T7 vector-borne primers. H4 could not be cloned in pBluescript II SK(+) after several attempts. TS1-primer is a fragment obtained by direct sequencing of pTS1 by primer walk.
S. Chauhan, H.K. Kuramitsu / Plasmid 51 (2004) 61–65
were obtained with the two ORFs identified in plasmid pTD1 (MacDougall et al., 1992) suggesting that the ORF1 of pTS1 is not closely related to pTD1 rep. However, the Clustal W alignment in Fig. 3A shows that there are at least some residues on pTD1 rep that are conserved with pTS1 ORF1 and other rep ORFs. These residues, which are shown in black in Fig. 3A may be important for function of the replication proteins. Taken together, the above information suggested the possibility that the ORF1 corresponded to the rep gene of plasmid pTS1. This was confirmed by cloning a DNA fragment containing this putative gene into an Escherichia coli plasmid to produce a shuttle plasmid, pKMR4PE, capable of replicating in T. denticola 33520 (Chi et al., 1999) as well as in Treponema phagedenis (Chi and Kuramitsu, unpublished results). A 70 bp nucleotide sequence downstream of the rep ORF (TD, 1722–1791 bp, Fig. 1) was found to be 98% identical to a region on pTD1 downstream of rep ORF. Such a high level of identity was surprising because rep open reading frames from pTS1 and pTD1 had no significant homologies at the amino acid level except for a few conserved residues. Presence of the sequence TD on H4 might be responsible for incompatibility with cloning into pBluescript II SK(+) because the fragment B2 (Fig. 2) which overlaps with most of the H4 fragment but lacks the TD sequence was easily cloned in the above plasmid. Many plasmid replicons consist of two essential functional units: an origin of replication and an adjacent replication initiator protein, the Rep protein. Iterons consist of arrays of 20 bp repeats and bind a plasmid-specific replication initiator protein in replication origins of many bacterial plasmids (Chattoraj, 2000). The pTS1 sequence upstream of rep had two identical 22 bp regions RE1 and RE2, which overlapped with inverted repeats IR1 and IR2, respectively (Fig. 3B). We predict that these iteron-like features of pTS1 might constitute the replication origin. Blast analysis of ORF2 (start 2126 bp; stop 3094 bp; Fig. 1) resulted in multiple hits for the mob family of proteins. Clustal W (Thompson et al., 1994) alignments of ORFs from the 10 most homologous sequences revealed that these mob
63
proteins are most conserved in the N-terminal region corresponding to 201 amino acids from the start of the pTS1 putative mob gene. While there was no significant homology among rep ORFs of pTS1 and pTD1 using Blast, the mob ORF of pTS1 was 37% identical to the ORF B of the T. denticola plasmid pTD1 (MacDougall et al., 1992). This suggests that both T. denticola genes may code for mob genes and that plasmids pTD1 and pTS1 can be conjugated between different organisms. The observation that a plasmid with properties identical to pTS1 was also isolated from another oral spirochete T. socranskii (Chan et al., 1996) is compatible with in vivo gene transfer between oral spirochetes. ORF3 (start 3144 bp; stop 3455 bp; Fig. 1) had significant homology to an ORF of unknown function (E-score 7e 13 ) on the chromosome of T. denticola strain 35405. ORF3 appears to be conserved among many bacteria, although a function has not been assigned to the putative protein product. It was interesting to note that ORF3 mapped close to the nucleic acid modification genes on the chromosomes of several pathogenic bacteria, including T. denticola strain 35405, Salmonella typhi CT18, Salmonella paratyphi, Salmonella typhimurium LT2, and Mannheimia haemolytica. ORF4 (start 3421 bp; stop 38 bp, Fig. 1) did not have significant homology with any known proteins. Interestingly ORF4 had Blast hits with very low level of homologies to the genes adjacent to ORF3 homologs in the above mentioned Salmonella species. The conservation of spatial proximity of ORF3 and ORF4 homologs in the plasmid pTS1 and several human pathogens mentioned above suggests that ORF3 and ORF4 may be functionally related. Additionally, the proximity of ORF3 to DNA modification enzymes in several organisms and, in at least one case, to DNA/RNA helicases suggests that ORF3 and ORF4 may be involved in the replication of pTS1. ERGO suite of bioinformatics tools http://www.integratedgenomics.com was found to be very useful for the above analysis. The identification of the rep gene of plasmid pTS1 has already proven valuable in constructing shuttle plasmids for characterizing genes from unculturable treponemes such as T. pallidum expressed in T. denticola (Chi et al., 1999). Very
64
S. Chauhan, H.K. Kuramitsu / Plasmid 51 (2004) 61–65
Fig. 3. (A) Clustal W alignment of rep ORFs of pTS1 (481 aa), pJDB23,1 pS23,2 pYH420,3 and pTD1.4 [1 Attwood and Brooker (1992) (303 aa). 2 EMBL/Genbank M86247 (303 aa). 3 Francis Macrina (Personal communication) (530 aa). 4 MacDougall et al. (1992) (336 aa)]. (B) Nucleotide sequence of the region upstream of rep ORF. The rep start is shown as underlined ATG. Two repetitive elements are shown in bold and two inverted repeats, IR1 and IR2, are shown with arrows.
S. Chauhan, H.K. Kuramitsu / Plasmid 51 (2004) 61–65
recently it has also been demonstrated that the pTS1 rep gene exhibits significant homology with a gene present on a plasmid isolated from a nontreponemal oral bacterium P. asacchrolytica (Fig. 3A).The identification of a putative mob region on the treponemal plasmids further suggests that transfer of these plasmids may occur within the oral cavity. Therefore, it may be possible to construct conjugation systems based upon these plasmids for transfer into treponemes which cannot be transformed following electroporation.
Acknowledgments This investigation was supported in part by NIH Grant DE09821. The authors are grateful to Gail K. Donaldson (for valuable discussion) and Pat Pulcher (for help in proof reading), both from DuPont Central Research and Development.
References Attwood, G.T., Brooker, J.D., 1992. Complete nucleotide sequence of Selenomonas ruminantium cryptic plasmid pJDB23 and definition of a region necessary for its replication in Escherichia coli. Plasmid 28, 123–129. Caudry, S., Litorinos, A., Gharbia, S.E., Pssara, N., Siboo, R., Keng, T., Chan, E.C., 1995. Distribution and characteriza-
65
tion of plasmids in Oral anaerobic spirochetes. Oral Microbiol. Immunol. 10, 8–12. Chan, E.C., Klitorinos, A., Gharbia, S., Caudry, S.D., Rahal, M.D., Siboo, R., 1996. Characterization of a 4.2-kb plasmid isolated from periodontopathic spirochetes. Oral Microbiol. Immunol. 11, 365–368. Chattoraj, D.K., 2000. Control of plasmid DNA replication by iterons: no longer paradoxical. Mol. Microbiol. 37, 467– 476. Chi, B., Chauhan, S., Kuramitsu, H., 1999. Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum fla A gene. Infect. Immun. 67, 3653–3656. Loesche, W.J., Laughon, B.E., 1982. Role of spirochetes in periodontal diseases. In: Mergenhagen, G.A. (Ed.), Host– Parasite Interactions in Periodontal Diseases. American Society for Microbiology, Washington, DC, pp. 67–75. MacDougall, J., Margarita, D., Saint Girons, I., 1992. Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids. J. Bacteriol. 174, 2724–2728. Olsen, I., Paster, B.J., Dewhirst, F.E., 2000. Taxonomy of spirochetes. Anaerobe 6, 39–57. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. NAR 22, 4673–4680. Yoshimoto, H., Takahasi, Y., Kato, D., Umemoto, T., 1997. Construction of a plasmid vector for transformation of Porphyromonas gingivalis. FEMS Microbiol. Lett. 152, 175–181. Communicated by George M. Weinstock