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A Tn5051-like mer-containing transposon identified in a heavy metal tolerant strain Achromobacter sp. AO22.

Ng SP, Davis B, Palombo EA, Bhave M - BMC Res Notes (2009)

Bottom Line: TnAO22 is thus a new variant of Tn5051 of the Tn3 superfamily and the transposon and its associated mercury resistance system are among the few such systems reported in a soil bacterium.Achromobacter sp.AO22 can thus be exploited for applications such as in situ mercury bioremediation of contaminated sites, or the mobile unit and mer operon could be mobilized to other bacteria for similar purposes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, PO Box 218, Melbourne, Victoria 3122, Australia. shng@swin.edu.au

ABSTRACT

Background: Achromobacter sp. AO22 (formerly Alcaligenes sp. AO22), a bacterial strain isolated from a lead-contaminated industrial site in Australia, was previously found to be resistant to moderate to high levels of mercury, copper and other heavy metals. However, the nature and location of the genetic basis for mercuric ion resistance in this strain, had not been previously identified.

Findings: Achromobacter sp. AO22 contains a functional mer operon with all four essential genes (merRTPA) and shows >99% DNA sequence identity to that of Tn501. The mer operon was present on a transposon, designated TnAO22, captured by introducing a broad-host-range IncP plasmid into Achromobacter sp. AO22 and subsequently transferring it to E. coli recipients. The transposition frequency of TnAO22 was 10-2 to 10-3 per target plasmid transferred. Analysis of TnAO22 sequence revealed it belonged to the Tn21 subgroup of the Tn3 superfamily of transposons, with the transposition module having >99% identity with Tn5051 of a Pseudomonas putida strain isolated from a water sample in New York.

Conclusion: TnAO22 is thus a new variant of Tn5051 of the Tn3 superfamily and the transposon and its associated mercury resistance system are among the few such systems reported in a soil bacterium. Achromobacter sp. AO22 can thus be exploited for applications such as in situ mercury bioremediation of contaminated sites, or the mobile unit and mer operon could be mobilized to other bacteria for similar purposes.

No MeSH data available.


Related in: MedlinePlus

Genetic organisation of TnAO22. (A) Organisation of the mer operon and tnp genes. Select restriction sites are shown (E: EcoRI; N: NcoI; P: PstI). IR: inverted repeats. The solid line between the PstI site and left IR indicates a 1.1 kb section of pSV520 included in the 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 cloned into pGEM-T Easy. (B) Comparison of the res sites: Tn501 from P. aeruginosa pVS1 (Z00027), Tn21 from S. flexneri R100 (NC_002134), Tn4378 from C. metallidurans CH34 pMOL28 (NC_006525) and Tn5051 from Pseudomonas sp. (Y17719). Dots indicate nucleotides identical to those of TnAO22; dashes indicate gaps introduced to optimise identity. (C) Comparison of the putative amino acid sequences of resolvase of TnAO22 with those of Tn501 (CAA77327), Tn4378 (ABF13038) and Tn21 from S. flexneri (NP_052901) and Tn5051 (CAC14696). Arrow head indicates the presumptive serine involved in recombination. The shaded region indicates the conserved helix-turn-helix motif of resolvases.
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Figure 1: Genetic organisation of TnAO22. (A) Organisation of the mer operon and tnp genes. Select restriction sites are shown (E: EcoRI; N: NcoI; P: PstI). IR: inverted repeats. The solid line between the PstI site and left IR indicates a 1.1 kb section of pSV520 included in the 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 cloned into pGEM-T Easy. (B) Comparison of the res sites: Tn501 from P. aeruginosa pVS1 (Z00027), Tn21 from S. flexneri R100 (NC_002134), Tn4378 from C. metallidurans CH34 pMOL28 (NC_006525) and Tn5051 from Pseudomonas sp. (Y17719). Dots indicate nucleotides identical to those of TnAO22; dashes indicate gaps introduced to optimise identity. (C) Comparison of the putative amino acid sequences of resolvase of TnAO22 with those of Tn501 (CAA77327), Tn4378 (ABF13038) and Tn21 from S. flexneri (NP_052901) and Tn5051 (CAC14696). Arrow head indicates the presumptive serine involved in recombination. The shaded region indicates the conserved helix-turn-helix motif of resolvases.

Mentions: A 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 was cloned into pGEMĀ®-T Easy vector (Promega Australia) for sequencing purposes. This fragment was sequenced initially using the vector-based primers T7 (5'-GTAATACGACTCAGGGC-3') and SP6 (5'-TTTAGGTGACACAGAATC-3'). As data was generated, a further section of TnAO22 was amplified using pVS520::TnAO22 as template and the primers AO22-F (5'-GACGAATACGGGCAGCGG-3') designed 70 bp upstream of the NcoI site and VS520-R (5'-GGCGGCGGTGTGGAAGC-3') designed 100 bp into sequence of pVS520. PCR products were purified and sequenced as above, using the primers used for PCR and additional primers designed based on the emerging sequence data. DNA sequences were assembled and analyzed using the Bioedit Alignment Editor v.7.0.9 . The most closely related sequences were found using the Basic Local Alignment Search Tool (BLAST) program , multiple alignments were performed with CLUSTALW and phylogenetic and evolutionary analyses conducted using MEGA version 4 . The sequence data indicated that the 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 contained a 1.1 kb section of pVS520, followed by one end of the putative transposon, a putative mer operon, a tnpR gene, and part of tnpA (Fig. 1A). A primer designed approximately 70 bp upstream of the NcoI site using these data, in combination with a primer designed approximately 100 bp into pVS520, gave a 2.8 kb PCR product from pVS520::TnAO22 templates isolated from E. coli cells. The sequences of the 6.7 kb PstI-NcoI fragment and this PCR product were assembled and showed TnAO22 had a length of 8230 bp (GenBank number EU696790). It was inserted 173 bp downstream of the truncated Tn1 in pVS520 (in pVS520::TnAO22), equivalent to position 10614 of RP1 (BN000925), and had resulted in 5 bp duplications (TCTAT) of target sequence in the flanking region of pVS520 (data not shown), the latter being a characteristic of Tn3 family [12]. TnAO22 was bounded by 38 bp imperfect IRs differing by only 1 bp (Fig. 2), the IR adjacent to mer operon being identical to that of Tn21 at the tnpA end. The IRs were highly similar to those of the ancestral Tn501 except its EcoRI sites and contained conserved sequences recognized by the Tn21 transposase [12]. The TnAO22 insertion site in pVS520 (equivalent to a region between Tn1 and oriV in RP1 or its derivatives) appears to be a hot spot for insertions, as reported for several Tn5041-type elements [24]. Nine ORFs were identified within TnAO22, the first seven closest to the IR from insertion point containing sequences homologous to the mer operon, including a merR that terminated within the adjacent IR and merTPADEurf2 transcribed divergently, and the two other ORFs being similar to tnpR and tnpA genes and separated from the mer ORFs by a 131 bp sequence similar to the res site (Fig. 1A).


A Tn5051-like mer-containing transposon identified in a heavy metal tolerant strain Achromobacter sp. AO22.

Ng SP, Davis B, Palombo EA, Bhave M - BMC Res Notes (2009)

Genetic organisation of TnAO22. (A) Organisation of the mer operon and tnp genes. Select restriction sites are shown (E: EcoRI; N: NcoI; P: PstI). IR: inverted repeats. The solid line between the PstI site and left IR indicates a 1.1 kb section of pSV520 included in the 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 cloned into pGEM-T Easy. (B) Comparison of the res sites: Tn501 from P. aeruginosa pVS1 (Z00027), Tn21 from S. flexneri R100 (NC_002134), Tn4378 from C. metallidurans CH34 pMOL28 (NC_006525) and Tn5051 from Pseudomonas sp. (Y17719). Dots indicate nucleotides identical to those of TnAO22; dashes indicate gaps introduced to optimise identity. (C) Comparison of the putative amino acid sequences of resolvase of TnAO22 with those of Tn501 (CAA77327), Tn4378 (ABF13038) and Tn21 from S. flexneri (NP_052901) and Tn5051 (CAC14696). Arrow head indicates the presumptive serine involved in recombination. The shaded region indicates the conserved helix-turn-helix motif of resolvases.
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Related In: Results  -  Collection

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Figure 1: Genetic organisation of TnAO22. (A) Organisation of the mer operon and tnp genes. Select restriction sites are shown (E: EcoRI; N: NcoI; P: PstI). IR: inverted repeats. The solid line between the PstI site and left IR indicates a 1.1 kb section of pSV520 included in the 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 cloned into pGEM-T Easy. (B) Comparison of the res sites: Tn501 from P. aeruginosa pVS1 (Z00027), Tn21 from S. flexneri R100 (NC_002134), Tn4378 from C. metallidurans CH34 pMOL28 (NC_006525) and Tn5051 from Pseudomonas sp. (Y17719). Dots indicate nucleotides identical to those of TnAO22; dashes indicate gaps introduced to optimise identity. (C) Comparison of the putative amino acid sequences of resolvase of TnAO22 with those of Tn501 (CAA77327), Tn4378 (ABF13038) and Tn21 from S. flexneri (NP_052901) and Tn5051 (CAC14696). Arrow head indicates the presumptive serine involved in recombination. The shaded region indicates the conserved helix-turn-helix motif of resolvases.
Mentions: A 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 was cloned into pGEMĀ®-T Easy vector (Promega Australia) for sequencing purposes. This fragment was sequenced initially using the vector-based primers T7 (5'-GTAATACGACTCAGGGC-3') and SP6 (5'-TTTAGGTGACACAGAATC-3'). As data was generated, a further section of TnAO22 was amplified using pVS520::TnAO22 as template and the primers AO22-F (5'-GACGAATACGGGCAGCGG-3') designed 70 bp upstream of the NcoI site and VS520-R (5'-GGCGGCGGTGTGGAAGC-3') designed 100 bp into sequence of pVS520. PCR products were purified and sequenced as above, using the primers used for PCR and additional primers designed based on the emerging sequence data. DNA sequences were assembled and analyzed using the Bioedit Alignment Editor v.7.0.9 . The most closely related sequences were found using the Basic Local Alignment Search Tool (BLAST) program , multiple alignments were performed with CLUSTALW and phylogenetic and evolutionary analyses conducted using MEGA version 4 . The sequence data indicated that the 6.7 kb PstI-NcoI fragment of pVS520::TnAO22 contained a 1.1 kb section of pVS520, followed by one end of the putative transposon, a putative mer operon, a tnpR gene, and part of tnpA (Fig. 1A). A primer designed approximately 70 bp upstream of the NcoI site using these data, in combination with a primer designed approximately 100 bp into pVS520, gave a 2.8 kb PCR product from pVS520::TnAO22 templates isolated from E. coli cells. The sequences of the 6.7 kb PstI-NcoI fragment and this PCR product were assembled and showed TnAO22 had a length of 8230 bp (GenBank number EU696790). It was inserted 173 bp downstream of the truncated Tn1 in pVS520 (in pVS520::TnAO22), equivalent to position 10614 of RP1 (BN000925), and had resulted in 5 bp duplications (TCTAT) of target sequence in the flanking region of pVS520 (data not shown), the latter being a characteristic of Tn3 family [12]. TnAO22 was bounded by 38 bp imperfect IRs differing by only 1 bp (Fig. 2), the IR adjacent to mer operon being identical to that of Tn21 at the tnpA end. The IRs were highly similar to those of the ancestral Tn501 except its EcoRI sites and contained conserved sequences recognized by the Tn21 transposase [12]. The TnAO22 insertion site in pVS520 (equivalent to a region between Tn1 and oriV in RP1 or its derivatives) appears to be a hot spot for insertions, as reported for several Tn5041-type elements [24]. Nine ORFs were identified within TnAO22, the first seven closest to the IR from insertion point containing sequences homologous to the mer operon, including a merR that terminated within the adjacent IR and merTPADEurf2 transcribed divergently, and the two other ORFs being similar to tnpR and tnpA genes and separated from the mer ORFs by a 131 bp sequence similar to the res site (Fig. 1A).

Bottom Line: TnAO22 is thus a new variant of Tn5051 of the Tn3 superfamily and the transposon and its associated mercury resistance system are among the few such systems reported in a soil bacterium.Achromobacter sp.AO22 can thus be exploited for applications such as in situ mercury bioremediation of contaminated sites, or the mobile unit and mer operon could be mobilized to other bacteria for similar purposes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, PO Box 218, Melbourne, Victoria 3122, Australia. shng@swin.edu.au

ABSTRACT

Background: Achromobacter sp. AO22 (formerly Alcaligenes sp. AO22), a bacterial strain isolated from a lead-contaminated industrial site in Australia, was previously found to be resistant to moderate to high levels of mercury, copper and other heavy metals. However, the nature and location of the genetic basis for mercuric ion resistance in this strain, had not been previously identified.

Findings: Achromobacter sp. AO22 contains a functional mer operon with all four essential genes (merRTPA) and shows >99% DNA sequence identity to that of Tn501. The mer operon was present on a transposon, designated TnAO22, captured by introducing a broad-host-range IncP plasmid into Achromobacter sp. AO22 and subsequently transferring it to E. coli recipients. The transposition frequency of TnAO22 was 10-2 to 10-3 per target plasmid transferred. Analysis of TnAO22 sequence revealed it belonged to the Tn21 subgroup of the Tn3 superfamily of transposons, with the transposition module having >99% identity with Tn5051 of a Pseudomonas putida strain isolated from a water sample in New York.

Conclusion: TnAO22 is thus a new variant of Tn5051 of the Tn3 superfamily and the transposon and its associated mercury resistance system are among the few such systems reported in a soil bacterium. Achromobacter sp. AO22 can thus be exploited for applications such as in situ mercury bioremediation of contaminated sites, or the mobile unit and mer operon could be mobilized to other bacteria for similar purposes.

No MeSH data available.


Related in: MedlinePlus