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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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Position of IS711 copies in the chromosomes of the published Brucella genomes. The two blocks contain the large and the small chromosomes respectively. Key to the species: S, B. suis; M, B. melitensis; A9, B abortus 9_941; A8, B abortus 2308; O, B. ovis. Common copies numbered from 1 to 6 are marked on the top. xs, xiii, x-08 and xa are IS711 species specific copies. The inverted segment of B. abortus chromosome II is marked in grey.
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Figure 5: Position of IS711 copies in the chromosomes of the published Brucella genomes. The two blocks contain the large and the small chromosomes respectively. Key to the species: S, B. suis; M, B. melitensis; A9, B abortus 9_941; A8, B abortus 2308; O, B. ovis. Common copies numbered from 1 to 6 are marked on the top. xs, xiii, x-08 and xa are IS711 species specific copies. The inverted segment of B. abortus chromosome II is marked in grey.

Mentions: The genome of B. ovis was known to contain a high copy number of IS711 estimated by hybridisation in around 30 copies [8]. The genome sequence of this species has been recently deposited in GenBank (Accesion Numbers CP000708 and CP000709). Genome sequence analysis revealed the presence of 38 complete copies of IS711 in the B. ovis genome, 25 and 13 in each chromosome respectively. Upon analysis of the genomic location of the IS711 copies we could determine that the position of six out of the seven copies was conserved in all the Brucella species. Four of the conserved copies were found in the large chromosome and two in the small chromosome. The common copies will be numbered from 1 to 6 plus a species or strain identifier (Fig. 5). When the nucleotide sequence of individual IS copies was analyzed it was evident that some sequence divergence existed among them. This indicated that IS sequence had drifted along with the other chromosomal genes in Brucella. At the same time this sequence variation allowed us to interpret the sequence identity between IS copies in the same organism as an indicator of recent transposition. In B. abortus the extra copy 711_xa was found to be identical to 711_1a. In B. abortus 2308 there is an additional IS711 copy found next to 711_3a. This copy was also identical to 711_1a. These observations strongly suggest that IS711_1a could be active and also show the strong preference of IS711 to transpose to some sites on the genome. On the other hand the IS711 copy found in the wboA gene of the B. abortus vaccine strain RB51 was found to be identical to 711_a6. This finding was rather unexpected, since 711_a6 lacks the first five nucleotides from the left IR, which would usually result in loss of the transposition ability. Insertion of this copy of IS711 into the wboA gene could also be explained by a different mobility mechanism other than transposition.


Demonstration of IS711 transposition in Brucella ovis and Brucella pinnipedialis.

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

Position of IS711 copies in the chromosomes of the published Brucella genomes. The two blocks contain the large and the small chromosomes respectively. Key to the species: S, B. suis; M, B. melitensis; A9, B abortus 9_941; A8, B abortus 2308; O, B. ovis. Common copies numbered from 1 to 6 are marked on the top. xs, xiii, x-08 and xa are IS711 species specific copies. The inverted segment of B. abortus chromosome II is marked in grey.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Position of IS711 copies in the chromosomes of the published Brucella genomes. The two blocks contain the large and the small chromosomes respectively. Key to the species: S, B. suis; M, B. melitensis; A9, B abortus 9_941; A8, B abortus 2308; O, B. ovis. Common copies numbered from 1 to 6 are marked on the top. xs, xiii, x-08 and xa are IS711 species specific copies. The inverted segment of B. abortus chromosome II is marked in grey.
Mentions: The genome of B. ovis was known to contain a high copy number of IS711 estimated by hybridisation in around 30 copies [8]. The genome sequence of this species has been recently deposited in GenBank (Accesion Numbers CP000708 and CP000709). Genome sequence analysis revealed the presence of 38 complete copies of IS711 in the B. ovis genome, 25 and 13 in each chromosome respectively. Upon analysis of the genomic location of the IS711 copies we could determine that the position of six out of the seven copies was conserved in all the Brucella species. Four of the conserved copies were found in the large chromosome and two in the small chromosome. The common copies will be numbered from 1 to 6 plus a species or strain identifier (Fig. 5). When the nucleotide sequence of individual IS copies was analyzed it was evident that some sequence divergence existed among them. This indicated that IS sequence had drifted along with the other chromosomal genes in Brucella. At the same time this sequence variation allowed us to interpret the sequence identity between IS copies in the same organism as an indicator of recent transposition. In B. abortus the extra copy 711_xa was found to be identical to 711_1a. In B. abortus 2308 there is an additional IS711 copy found next to 711_3a. This copy was also identical to 711_1a. These observations strongly suggest that IS711_1a could be active and also show the strong preference of IS711 to transpose to some sites on the genome. On the other hand the IS711 copy found in the wboA gene of the B. abortus vaccine strain RB51 was found to be identical to 711_a6. This finding was rather unexpected, since 711_a6 lacks the first five nucleotides from the left IR, which would usually result in loss of the transposition ability. Insertion of this copy of IS711 into the wboA gene could also be explained by a different mobility mechanism other than transposition.

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