Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Achard, A. Articles by Leclercq, R. Search for Related Content PubMed PubMed Citation Articles by Achard, A. Articles by Leclercq, R.

 Previous Article  |  Next Article 

Journal of Bacteriology, June 2007, p. 4328-4331, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.00213-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of a Small Mobilizable Transposon, MTnSag1, in Streptococcus agalactiae

Adeline Achard and Roland Leclercq^*

Service de Microbiologie and EA 2128 Interactions hôte et microorganismes des épithéliums, CHU Côte de Nacre, Université de Caen Basse-Normandie, 14033 Caen Cedex, France

Received 8 February 2007/ Accepted 26 March 2007

Top ABSTRACT TEXT REFERENCES

 
The transposon MTnSag1 from Streptococcus agalactiae carried an IS1-like transposase gene and the lnu(C) gene, which encoded a lincosamide nucleotidyltransferase. MTnSag1 could be mobilized by the conjugative transposon Tn916. An intermediate circular form of MTnSag1 and a putative origin of transfer at the 3' end of the lnu(C) gene were characterized.

Top ABSTRACT TEXT REFERENCES

 
Group B streptococcus or Streptococcus agalactiae is a component of the normal flora of human mucosa and a well-known cause of invasive infection in neonates, pregnant women, and older individuals with underlying chronic illness (4, 11). Dissemination of mobile elements largely contributes to the increasing resistance to macrolides among streptococci. Mobilizable transposons are genetic elements that are smaller than conjugative transposons, can range in size from 4.7 kb (Tn5520) to 12.7 kb (Tn4555), and have been identified only in Bacteroides spp. and Clostridium spp. (2, 14, 16, 17).

In this report, we demonstrate that MTnSag1, a 1,724-bp-long element that we previously identified in the clinical strain S. agalactiae UCN36 (accession number AY928180), is a new transposon that can be mobilized by the conjugative transposon Tn916 to S. agalactiae recipients (1).

Sequence analysis of the MTnSag1 transposase gene. The MTnSag1 element contained two open reading frames in the same orientation, ORF1 and ORF2, with sizes of 1,038 and 495 bp, respectively. ORF2, called lnu(C), encoded a lincosamide O-nucleotidyltransferase conferring resistance to lincomycin (1). ORF1 encoded a putative protein related to the protein InsA of the insertion sequence IS1 (35% identity) and to several transposases described for Clostridium spp. (33 to 42% identity). MTnSag1 had a pair of 25-bp imperfect inverted repeats at its termini. A search for motifs and domains using the NCBI Blastp and EMBOSS helix-turn-helix programs (http://www.ncbi.nlm.nih.gov/ and http://www.bioweb.pasteur.fr) revealed the presence of potential zinc finger (ZF) and helix-Turn-helix (HTH) motifs in the N-terminal region of the MTnSag1 transposase that are characteristics of IS1 transposases (8, 15) (Fig. 1). The conserved C residues were at positions 52, 55, 75, and 78. Other conserved residues, including an aromatic amino acid (F at position 82 corresponding to W at position 39 for IS1A), were identified. A putative DDE motif was also evidenced in the C terminus of the MTnSag1 transposase. This DDE motif (D-67-D-92-E) was distantly related to the DDE motif of the IS1 members (D-56/80-D-21/24-E) but was in agreement with the consensus motif of known transposases and retroviral integrases (D-50/80-D33/138-E) (15). These results lead us to classify the MTnSag1 putative transposase in the IS1 family.

View larger version (33K): [in this window] [in a new window]

FIG. 1. Sequence alignment of N termini of MTnSag1 and IS1 transposases. Putative ZF and helix-turn-helix (HTH) motifs are shown. Conserved C residues forming a putative ZF motif are underlined; a conserved aromatic residue is in bold, and the helix-turn-helix motif is boxed. The insertion sequence elements described can be found in ISfinder (http//:www-IS.biotoul.fr); the accession number of MTnSag1 in the GenBank data library is AY928180.

Transferability of MTnSag1. The transferability of the MTnSag1 transposon was tested using filter mating with S. agalactiae BM132 (resistant to rifampin and fusidic acid) or BM134 (resistant to streptomycin), Enterococcus faecalis JH2-2, and Escherichia coli K-12 AG100A (1) as recipient strains. MTnSag1 was transferable from S. agalactiae UCN36 to S. agalactiae BM134 or BM132 at a frequency of (1.6 ± 0.3) x 10^–7 transconjugants per donor cell. No transfer to E. faecalis JH2-2 and E. coli K-12 AG100A was detected (10^–9 transconjugants). Southern blot experiments revealed that six copies of MTnSag1 were present in S. agalactiae UCN36 but only one copy was present in three studied transconjugants.

To identify the insertion sites of MTnSag1, total DNA of five transconjugants was digested with AluI and RsaI and self ligated. By using inverse PCR, sequences adjacent to the left and right ends of MTnSag1 could be identified. Sequencing of the PCR products showed that transposition of MTnSag1 occurred at different sites of the host genome in regions with high AT content and generated 8-bp duplications at the target sites (Table 1).

View this table: [in this window] [in a new window]

TABLE 1. Insertion sites in S. agalactiae UCN36 and transconjugants

MTnSag1 is mobilized in S. agalactiae by the conjugative transposon Tn916. MTnSag1 lacked sequence homologous to mob and tra genes despite transfer from chromosome to chromosome, which suggested that the transposon might be mobilized by a coresident conjugative element. No plasmid could be extracted from S. agalactiae UCN36. The observation that S. agalactiae UCN36 was resistant to tetracyclines led us to suspect the presence of a Tn916-like conjugative transposon responsible for mobilization of MTnSag1. We confirmed the presence of the transposon by amplification of sequences specific for the tet(M) (resistance to tetracyclines) and int (integrase) genes of Tn916. Lincomycin resistance was not transferable from the transconjugant S. agalactiae 134TC1, devoid of Tn916 transposon or any other coresident conjugative element, to S. agalactiae BM132. Tn916 from E. faecalis JH2-2::Tn916 was introduced into S. agalactiae 134TC1 by conjugation. Using this new strain as a donor and S. agalactiae BM132 as a recipient, transconjugants were obtained at a transfer frequency equal to (3.1 ± 0.9) x 10^–8. These results showed that MTnSag1 could transfer to S. agalactiae BM132 only when Tn916 was present as a coresident conjugative element. Since previous studies (3, 12) showed that subinhibitory concentrations of tetracycline increased the conjugative transposition frequency of Tn916 by approximately 15-fold, we assessed the effect of tetracycline on the MTnSag1 frequency of transfer. Filter mating experiments performed in the presence of a subinhibitory concentration of tetracycline (1 µg/ml) did not result in a significant increase when S. agalactiae UCN36 and S. agalactiae 134TC1::Tn916 were used as donor cells. Probably, an important limiting factor for the conjugative transfer efficiency is the circularization of MTnSag1 (see below), which should be a rare event not influenced by the presence of tetracycline, explaining the lack of impact of the antibiotic on the conjugation frequency of MTnSag1.

The implication of Tn916 in mobilization of nonconjugative plasmids (7, 13) or nonconjugative transposons (5) has already been described. In these studies, mobilization of nonconjugative elements by Tn916 did not appear to be dependent on the presence of a functional mobilization gene region on the element but required only the presence of an origin of transfer.

MTnSag1 is found in a circular form. Given that the formation of an intermediate covalently closed circular form is required for transposon transfer, we tried to detect circular forms of MTnSag1 in the donor. This circular intermediate is a nonreplicating form and, as a consequence, is difficult to detect. To circumvent this problem, the intact MTnSag1 transposon was cloned in the pUC18 multicopy plasmid to form pUV15. PCR with outward-directed primers would generate a PCR product only if the left and right ends of MTnSag1 were ligated together. A PCR product with the appropriate size (966 bp) was obtained when extrachromosomal DNA from E. coli DH10B containing pUV15 was used as a template. Sequencing confirmed that the PCR product corresponded to the circular form and showed that the two ends of the transposon were separated by an 8-bp sequence that would form the coupling sequences. In experiments using pUV16, where the C terminus of the transposase had been deleted, the intermediate circular form was no longer detected. This result showed that the transposase gene of MTnSag1 was involved in excision and circularization of the transposon under the conditions of our experiment. However, the use of E. coli as a surrogate is a limitation for the demonstration that circularization happens under physiological conditions in the original host, S. agalactiae.

The MTnSag1 origin of transfer is located in the lnu(C) gene. To localize the oriT region of MTnSag1, a mobilization strategy was used. Cloning of an oriT site on a nonmobilizable plasmid introduced into a bacterial host harboring an intact conjugative element would result in transfer of the recombinant plasmid by mobilization (7).

PCR amplification of segments containing the entirety or portions of the MTnSag1 transposon was performed. The products were cloned into the nonmobilizable shuttle vector pORI23. Regions corresponding to the various PCR products are indicated in Fig. 2 and include segments ranging in size from 298 to 1,821 bp. The corresponding recombinant plasmids, designated pUV17 to pUV20, and pORI23 were introduced by electroporation into the S. agalactiae 134::Tn916 strain, which contained a single copy of Tn916 on the chromosome. The strains were then tested for their ability to mobilize recombinant plasmids in mating experiments. pUV18 and pUV20 were mobilized in E. coli BM132 at a frequency comparable to that for the positive-control pUV17 whereas pUV19 and the negative control pORI23 were not mobilized (10^–9, detection limit) (Fig. 2). These data indicate that the 3'-end region of lnu(C) is required for the mobilization of the nonmobilizable plasmid pORI23 and, therefore, contains the origin of transfer of MTnSag1.

View larger version (8K): [in this window] [in a new window]

FIG. 2. MTnSag1 oriT mobilization assays. (A) Schematic map of MTnSag1. Inverted repeats (IR) are indicated by dotted lines. (B) Cloned fragments for MTnSag1 oriT mobilization. The sizes of the fragments and the corresponding plasmid designations are indicated. The frequency of mobilization was determined by dividing the number of transconjugants that received the mobilized plasmid by the number of donor cells. Values are means of a minimum of three independent mating experiments.

Sequence comparison to the specific oriT binding site of the Tn916 integrase (6, 10) allowed us to identify a putative binding site on MTnSag1. This sequence appears to be located between nucleotides 1575 and 1635 of MTnSag1, at the 3' end of the lnu(C) gene (Fig. 3). The nucleotide sequence surrounding this site was then examined for clusters of inverted and direct repeats, which are characteristically found near plasmids and transposon transfer origins (7). The area spanning nucleotides 1488 to 1694 was found to contain four sets of direct inverted repeat sequences of 16 to 18 nucleotides (Fig. 3).

View larger version (13K): [in this window] [in a new window]

FIG. 3. Sequence analysis of the putative MTnSag1 origin of transfer. (A) Comparison of the Int-protected region of oriT from Tn916 with the MTnSag1 sequence. Upper line: nucleotides 1575 to 1635 of MTnSag1. Lower line: protected oriT sequence from Tn916. Vertical dots indicate bases in the transposon that are identical to bases of the Int-protected region. (B) Map of the putative oriT1488-1667 of MTnSag1. The putative Int binding site is indicated by a dashed line above the sequence. Inverted arrow pairs indicate inverted repeat structures.

This is the first time that an origin of transfer has been evidenced in a coding DNA region. Comparison of the nucleotidyltransferases Lnu(C), Lnu(A), Lnu(A'), and LnuA_N2 showed that most conserved domains of these proteins were clustered in the N-terminal region whereas the C terminus was more variable (data not shown). The variable 3' end of a putative lnu ancestor gene may have evolved to acquire an oriT via rearrangements or mutations.

Model for transfer of MTnSag1. We propose a model for transfer of MTnSag1 in S. agalactiae. The IS1-like transposase would mediate the excision of MTnSag1 by introducing 8-bp cuts at the ends of the directly inverted repeat sequences at the ends of the transposon. Strand exchange would then occur, resulting in excision of the transposon as a circular molecule. The circular form could then be transferred to a new S. agalactiae host. Int, the endonuclease Orf20, and other mobilization proteins encoded by Tn916 would form a DNA-protein complex (relaxosome) at sequences surrounding the nick site (10). The binding of host factors may assist in the formation of this DNA-protein complex. Tn916 would contribute in trans by additional transfer functions to establish effective mating contacts with recipient cells and provide a generalized transfer apparatus to support transfer of the nicked strand to the recipient cell. Replacement strand synthesis would occur in the donor cell, and the transferred strand would serve as a template for DNA synthesis in the recipient cell. IS1-like transposase would provoke integration of the double-stranded form by recognizing a suitable AT-rich target and promoting site-specific recombination with the joint of the circular form. Strand exchange and ligation would finally result in the insertion of MTnSag1 flanked by the direct target site duplication.

In summary, MTnSag1 is the smallest mobilizable transposon reported so far and presents an interesting case for speculation on the minimal size of a factor that can process both transposition and transfer properties and confer resistance to antibiotics. This is the first time that a mobilizable transposon has been reported in the genus Streptococcus. The acquisition of short transferable elements by group B streptococcus might be highly efficient for the spread of resistance since the vast majority of S. agalactiae isolates harbor Tn916-like elements (9).

 
We thank P. Trieu-Cuot for the gift of S. agalactiae strains BM132 and BM134 and P. Courvalin for the gift of E. faecalis JH2-2::Tn916.

We thank the Fondation pour la Recherche Médicale and Vaincre la Mucoviscidose for financial support.

 
* Corresponding author. Mailing address: CHU de Caen, Service de Microbiologie, Avenue Côte de Nacre, 14033 Caen Cedex, France. Phone: (33) 02 31 06 45 72. Fax: (33) 02 31 06 45 73. E-mail: leclercq-r{at}chu-caen.fr

Published ahead of print on 6 April 2007.

Top ABSTRACT TEXT REFERENCES

 

1
1. Achard, A., C. Villers, V. Pichereau, and R. Leclercq. 2005. New lnu(C) gene conferring resistance to lincomycin by nucleotidylation in Streptococcus agalactiae UCN36. Antimicrob. Agents Chemother. 49:2716-2719.[Abstract/Free Full Text]
2
2. Adams, V., D. Lyras, K. A. Farrow, and J. I. Rood. 2002. The clostridial mobilisable transposons. Cell. Mol. Life Sci. 59:2033-2043.[CrossRef][Medline]
3
3. Celli, J., and P. Trieu-Cuot. 1998. Circularization of Tn916 is required for expression of the transposon-encoded transfer functions: characterization of long tetracycline-inducible transcripts reading through the attachment site. Mol. Microbiol. 28:103-117.[CrossRef][Medline]
4
4. Farley, M. M. 2001. Group B streptococcal disease in nonpregnant adults. Clin. Infect. Dis. 33:556-561.[CrossRef][Medline]
5
5. Farrow, K. A., D. Lyras, and J. I. Rood. 2001. Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile. Microbiology 147:2717-2728.[Abstract/Free Full Text]
6
6. Hinerfeld, D., and G. Churchward. 2001. Specific binding of integrase to the origin of transfer (oriT) of the conjugative transposon Tn916. J. Bacteriol. 183:2947-2951.[Abstract/Free Full Text]
7
7. Jaworski, D. D., and D. B. Clewell. 1995. A functional origin of transfer (oriT) on the conjugative transposon Tn916. J. Bacteriol. 177:6644-6651.[Abstract/Free Full Text]
8
8. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol. Mol. Biol. Rev. 62:725-774.[Abstract/Free Full Text]
9
9. Poyart, C., L. Jardy, G. Quesne, P. Berche, and P. Trieu-Cuot. 2003. Genetic basis of antibiotic resistance in Streptococcus agalactiae strains isolated in a French hospital. Antimicrob. Agents Chemother. 47:794-797.[Abstract/Free Full Text]
10
10. Rocco, J. M., and G. Churchward. 2006. The integrase of the conjugative transposon Tn916 directs strand- and sequence-specific cleavage of the origin of conjugal transfer, oriT, by the endonuclease Orf20. J. Bacteriol. 188:2207-2213.[Abstract/Free Full Text]
11
11. Schuchat, A., and J. D. Wenger. 1994. Epidemiology of group B streptococcal disease. Risk factors, prevention strategies, and vaccine development. Epidemiol. Rev. 16:374-402.[Free Full Text]
12
12. Showsh, S. A., and R. E. Andrews, Jr. 1992. Tetracycline enhances Tn916-mediated conjugal transfer. Plasmid 28:213-224.[CrossRef][Medline]
13
13. Showsh, S. A., and R. E. Andrews, Jr. 1999. Analysis of the requirement for a pUB110 mob region during Tn916-dependent mobilization. Plasmid 41:179-186.[CrossRef][Medline]
14
14. Smith, C. J., and A. C. Parker. 1993. Identification of a circular intermediate in the transfer and transposition of Tn4555, a mobilizable transposon from Bacteroides spp. J. Bacteriol. 175:2682-2691.[Abstract/Free Full Text]
15
15. Ton-Hoang, B., C. Turlan, and M. Chandler. 2004. Functional domains of the IS1 transposase: analysis in vivo and in vitro. Mol. Microbiol. 53:1529-1543.[CrossRef][Medline]
16
16. Vedantam, G., T. J. Novicki, and D. W. Hecht. 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.[Abstract/Free Full Text]
17
17. Wang, J., N. B. Shoemaker, G. R. Wang, and A. A. Salyers. 2000. Characterization of a Bacteroides mobilizable transposon, NBU2, which carries a functional lincomycin resistance gene. J. Bacteriol. 182:3559-3571.[Abstract/Free Full Text]

---

Journal of Bacteriology, June 2007, p. 4328-4331, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.00213-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Lyras, D., Adams, V., Ballard, S. A., Teng, W. L., Howarth, P. M., Crellin, P. K., Bannam, T. L., Songer, J. G., Rood, J. I. (2009). tISCpe8, an IS1595-Family Lincomycin Resistance Element Located on a Conjugative Plasmid in Clostridium perfringens. J. Bacteriol. 191: 6345-6351 [Abstract] [Full Text]  
• Smyth, D. S., Robinson, D. A. (2009). Integrative and Sequence Characteristics of a Novel Genetic Element, ICE6013, in Staphylococcus aureus. J. Bacteriol. 191: 5964-5975 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Achard, A. Articles by Leclercq, R. Search for Related Content PubMed PubMed Citation Articles by Achard, A. Articles by Leclercq, R.