Limits...
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.

Show MeSH

Related in: MedlinePlus

PCR analysis of the plasmids found in tetracycline resistant mutants. PCR with primers G11 and G12 was carried out in order to amplify the selection cartridge of pGBG1 in tetracycline resistant mutants obtained in Brucella. The black arrow indicates the position of the PCR fragment obtained from pGBG1/TcR mutants due to insertions encountered in B. ovis BOC22 and B. pinnipedialis B2/94 strains (Lanes 3, 7, 8, 9, 10, 12, 13, 14 and 15). Mutants due to deletions and point mutations (Lanes 2, 4, 5, 6, 11, 16, 17 and 18) produced PCR products of the same size or smaller than the vector pGBG1 (Lane 1). M: Molecular Weight Marker 1 Kb plus DNA Ladder.1. Lane 19. Negative control (PCR reaction mixture without DNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2266754&req=5

Figure 2: PCR analysis of the plasmids found in tetracycline resistant mutants. PCR with primers G11 and G12 was carried out in order to amplify the selection cartridge of pGBG1 in tetracycline resistant mutants obtained in Brucella. The black arrow indicates the position of the PCR fragment obtained from pGBG1/TcR mutants due to insertions encountered in B. ovis BOC22 and B. pinnipedialis B2/94 strains (Lanes 3, 7, 8, 9, 10, 12, 13, 14 and 15). Mutants due to deletions and point mutations (Lanes 2, 4, 5, 6, 11, 16, 17 and 18) produced PCR products of the same size or smaller than the vector pGBG1 (Lane 1). M: Molecular Weight Marker 1 Kb plus DNA Ladder.1. Lane 19. Negative control (PCR reaction mixture without DNA).

Mentions: The frequencies of occurrence of tetracycline resistant colonies relative to chloramphenicol resistant CFU's ranged from 10-7 to 10-9, depending on the strain (Table 1). To determine whether the appearance of tetracycline resistant colonies was due either to transposition into pGBG1 of a Brucella transposon or to any other mutational event, selected colonies from each independent experiment were analysed by PCR using the primers G11 and G12 (Fig. 1, Table 2), which amplified the selection cartridge of pGBG1 plasmid as a 1.2 kb fragment. Some of the tetracycline resistant isolates produced a PCR amplification fragment of around 2.0 kb. The size of these DNA fragments represented an increase of approximately 0.8 kb relative to the size of the target region of pGBG1, which was consistent with the integration of a copy of IS711 into the repressor controlling the tetracycline resistance gene of pGBG1. From the four Brucella strains in which the transposon trap pGBG1 was used, only B. ovis BOC22 and B. pinnipedialis B2/94 produced tetracycline resistant plasmids containing insertions (Fig. 2). No tetracycline resistant colonies containing insertions in the experiments that used B. abortus or B. melitensis as hosts were obtained. Some tetracycline resistant colonies were obtained with these two donors. PCR analysis of the corresponding plasmids in these cases, (and in some colonies from B. ovis and B. pinnipedialis as well) could not be explained by integration events. These resistant mutants probably resulted from point mutations (when the size of target region was apparently unchanged) or from deletions (when the size of target region decreased) in the pGBG1 selection cartridge (Fig. 1).


Demonstration of IS711 transposition in Brucella ovis and Brucella pinnipedialis.

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

PCR analysis of the plasmids found in tetracycline resistant mutants. PCR with primers G11 and G12 was carried out in order to amplify the selection cartridge of pGBG1 in tetracycline resistant mutants obtained in Brucella. The black arrow indicates the position of the PCR fragment obtained from pGBG1/TcR mutants due to insertions encountered in B. ovis BOC22 and B. pinnipedialis B2/94 strains (Lanes 3, 7, 8, 9, 10, 12, 13, 14 and 15). Mutants due to deletions and point mutations (Lanes 2, 4, 5, 6, 11, 16, 17 and 18) produced PCR products of the same size or smaller than the vector pGBG1 (Lane 1). M: Molecular Weight Marker 1 Kb plus DNA Ladder.1. Lane 19. Negative control (PCR reaction mixture without DNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PCR analysis of the plasmids found in tetracycline resistant mutants. PCR with primers G11 and G12 was carried out in order to amplify the selection cartridge of pGBG1 in tetracycline resistant mutants obtained in Brucella. The black arrow indicates the position of the PCR fragment obtained from pGBG1/TcR mutants due to insertions encountered in B. ovis BOC22 and B. pinnipedialis B2/94 strains (Lanes 3, 7, 8, 9, 10, 12, 13, 14 and 15). Mutants due to deletions and point mutations (Lanes 2, 4, 5, 6, 11, 16, 17 and 18) produced PCR products of the same size or smaller than the vector pGBG1 (Lane 1). M: Molecular Weight Marker 1 Kb plus DNA Ladder.1. Lane 19. Negative control (PCR reaction mixture without DNA).
Mentions: The frequencies of occurrence of tetracycline resistant colonies relative to chloramphenicol resistant CFU's ranged from 10-7 to 10-9, depending on the strain (Table 1). To determine whether the appearance of tetracycline resistant colonies was due either to transposition into pGBG1 of a Brucella transposon or to any other mutational event, selected colonies from each independent experiment were analysed by PCR using the primers G11 and G12 (Fig. 1, Table 2), which amplified the selection cartridge of pGBG1 plasmid as a 1.2 kb fragment. Some of the tetracycline resistant isolates produced a PCR amplification fragment of around 2.0 kb. The size of these DNA fragments represented an increase of approximately 0.8 kb relative to the size of the target region of pGBG1, which was consistent with the integration of a copy of IS711 into the repressor controlling the tetracycline resistance gene of pGBG1. From the four Brucella strains in which the transposon trap pGBG1 was used, only B. ovis BOC22 and B. pinnipedialis B2/94 produced tetracycline resistant plasmids containing insertions (Fig. 2). No tetracycline resistant colonies containing insertions in the experiments that used B. abortus or B. melitensis as hosts were obtained. Some tetracycline resistant colonies were obtained with these two donors. PCR analysis of the corresponding plasmids in these cases, (and in some colonies from B. ovis and B. pinnipedialis as well) could not be explained by integration events. These resistant mutants probably resulted from point mutations (when the size of target region was apparently unchanged) or from deletions (when the size of target region decreased) in the pGBG1 selection cartridge (Fig. 1).

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