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Demonstration of IS711 transposition in Brucella ovis and Brucella pinnipedialis.

Ocampo-Sosa AA, García-Lobo JM - BMC Microbiol. (2008)

Bottom Line: The strains B. melitensis 16 M, B. abortus RB51, B. ovis BOC22 (field strain) and B. pinnipedialis B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (TcR).Four different copies of IS711 were found to transpose to the same target sequence in the plasmid pGBG1.This demonstrated that IS711 are active in vivo, specially in Brucella species with a high number of IS711 copies as B. ovis and B. pinnipedialis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biología Molecular, Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC, CSIC-Universidad de Cantabria-IDICAN, Santander, Spain. alainocampo@hotmail.com

ABSTRACT

Background: The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.

Results: In this study we obtained evidence of transposition of IS711 from the B. ovis and B. pinnipedialis chromosomes by using the "transposon trap" plasmid pGBG1. This plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. The strains B. melitensis 16 M, B. abortus RB51, B. ovis BOC22 (field strain) and B. pinnipedialis B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (TcR). TcR mutants due to IS711 transposition were only detected in B. ovis and B. pinnipedialis strains.

Conclusion: Four different copies of IS711 were found to transpose to the same target sequence in the plasmid pGBG1. This demonstrated that IS711 are active in vivo, specially in Brucella species with a high number of IS711 copies as B. ovis and B. pinnipedialis.

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Diversity of restriction profiles and nucleotide sequence of IS711 copies recovered in IS711 insertions in pGBG1. A) PCR products from several pGBG1/TcR mutants digested with HaeIII. Lanes 1 to 6 contain insertions obtained in B. pinnipedialis B2/94. Lanes 7–10 insertions obtained in B. ovis BOC22 Lane 11, PCR product from pGBG1. M: Molecular size marker (1 Kb DNA ladder, Invitrogen). Letters under the picture indicate the different restriction patterns obtained. In B) the nucleotide sequences of the IS711 copies transposed into cI lambda repressor gene were compared with IS711A from B. ovis (GenBank Accession number: M94960). Only the variable regions and the HaeIII sites are shown. Identical nucleotides are represented by dots. Numerals in the top indicate the positions where nucleotide changes occur. Nucleotide substitutions are boldface and shaded. The HaeIII restriction sites are indicated within open boxes.
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Figure 3: Diversity of restriction profiles and nucleotide sequence of IS711 copies recovered in IS711 insertions in pGBG1. A) PCR products from several pGBG1/TcR mutants digested with HaeIII. Lanes 1 to 6 contain insertions obtained in B. pinnipedialis B2/94. Lanes 7–10 insertions obtained in B. ovis BOC22 Lane 11, PCR product from pGBG1. M: Molecular size marker (1 Kb DNA ladder, Invitrogen). Letters under the picture indicate the different restriction patterns obtained. In B) the nucleotide sequences of the IS711 copies transposed into cI lambda repressor gene were compared with IS711A from B. ovis (GenBank Accession number: M94960). Only the variable regions and the HaeIII sites are shown. Identical nucleotides are represented by dots. Numerals in the top indicate the positions where nucleotide changes occur. Nucleotide substitutions are boldface and shaded. The HaeIII restriction sites are indicated within open boxes.

Mentions: As a first step towards identification of the DNA inserted into the tetracycline resistant insertional mutants, the PCR products were digested with the restriction enzyme HaeIII. Six different restriction patterns were observed, four in B. ovis BOC22 (C, D, E and F) and two in B. pinnipedialis B2/94 (A and B, Fig. 3A).


Demonstration of IS711 transposition in Brucella ovis and Brucella pinnipedialis.

Ocampo-Sosa AA, García-Lobo JM - BMC Microbiol. (2008)

Diversity of restriction profiles and nucleotide sequence of IS711 copies recovered in IS711 insertions in pGBG1. A) PCR products from several pGBG1/TcR mutants digested with HaeIII. Lanes 1 to 6 contain insertions obtained in B. pinnipedialis B2/94. Lanes 7–10 insertions obtained in B. ovis BOC22 Lane 11, PCR product from pGBG1. M: Molecular size marker (1 Kb DNA ladder, Invitrogen). Letters under the picture indicate the different restriction patterns obtained. In B) the nucleotide sequences of the IS711 copies transposed into cI lambda repressor gene were compared with IS711A from B. ovis (GenBank Accession number: M94960). Only the variable regions and the HaeIII sites are shown. Identical nucleotides are represented by dots. Numerals in the top indicate the positions where nucleotide changes occur. Nucleotide substitutions are boldface and shaded. The HaeIII restriction sites are indicated within open boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266754&req=5

Figure 3: Diversity of restriction profiles and nucleotide sequence of IS711 copies recovered in IS711 insertions in pGBG1. A) PCR products from several pGBG1/TcR mutants digested with HaeIII. Lanes 1 to 6 contain insertions obtained in B. pinnipedialis B2/94. Lanes 7–10 insertions obtained in B. ovis BOC22 Lane 11, PCR product from pGBG1. M: Molecular size marker (1 Kb DNA ladder, Invitrogen). Letters under the picture indicate the different restriction patterns obtained. In B) the nucleotide sequences of the IS711 copies transposed into cI lambda repressor gene were compared with IS711A from B. ovis (GenBank Accession number: M94960). Only the variable regions and the HaeIII sites are shown. Identical nucleotides are represented by dots. Numerals in the top indicate the positions where nucleotide changes occur. Nucleotide substitutions are boldface and shaded. The HaeIII restriction sites are indicated within open boxes.
Mentions: As a first step towards identification of the DNA inserted into the tetracycline resistant insertional mutants, the PCR products were digested with the restriction enzyme HaeIII. Six different restriction patterns were observed, four in B. ovis BOC22 (C, D, E and F) and two in B. pinnipedialis B2/94 (A and B, Fig. 3A).

Bottom Line: The strains B. melitensis 16 M, B. abortus RB51, B. ovis BOC22 (field strain) and B. pinnipedialis B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (TcR).Four different copies of IS711 were found to transpose to the same target sequence in the plasmid pGBG1.This demonstrated that IS711 are active in vivo, specially in Brucella species with a high number of IS711 copies as B. ovis and B. pinnipedialis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biología Molecular, Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC, CSIC-Universidad de Cantabria-IDICAN, Santander, Spain. alainocampo@hotmail.com

ABSTRACT

Background: The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.

Results: In this study we obtained evidence of transposition of IS711 from the B. ovis and B. pinnipedialis chromosomes by using the "transposon trap" plasmid pGBG1. This plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. The strains B. melitensis 16 M, B. abortus RB51, B. ovis BOC22 (field strain) and B. pinnipedialis B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (TcR). TcR mutants due to IS711 transposition were only detected in B. ovis and B. pinnipedialis strains.

Conclusion: Four different copies of IS711 were found to transpose to the same target sequence in the plasmid pGBG1. This demonstrated that IS711 are active in vivo, specially in Brucella species with a high number of IS711 copies as B. ovis and B. pinnipedialis.

Show MeSH
Related in: MedlinePlus