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Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation.

Baharoglu Z, Bikard D, Mazel D - PLoS Genet. (2010)

Bottom Line: We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements.Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell.Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité Plasticité du Génome Bactérien, Département Génomes et Génétique, Paris, France.

ABSTRACT
Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

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Conjugation increases IntI1-dependent cassette excision rate in E. coli.A: experimental setup. 7949 strain contains plasmid p7755 carrying intI1 under the control of its natural LexA-regulated promoter. B: cassette excision rate was calculated by counting recombined cfu (Dap+) over total cfu. “No plasmid” means that recipient 7949 p7755 was mixed with empty donor. “No integrase” means that recipient 7949 without p7755 was conjugated with donor containing a conjugative plasmid.
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pgen-1001165-g004: Conjugation increases IntI1-dependent cassette excision rate in E. coli.A: experimental setup. 7949 strain contains plasmid p7755 carrying intI1 under the control of its natural LexA-regulated promoter. B: cassette excision rate was calculated by counting recombined cfu (Dap+) over total cfu. “No plasmid” means that recipient 7949 p7755 was mixed with empty donor. “No integrase” means that recipient 7949 without p7755 was conjugated with donor containing a conjugative plasmid.

Mentions: We have shown that conjugation induces SOS in the recipient bacteria and flow cytometry analysis clearly shows that the integron integrase is induced during conjugation in V. cholerae. In a first set of experiments, we wanted to test if the SOS induction leads to a higher activity of the integrase promoter in E. coli, using the class 1 integrase IntI1. We developed an experimental strategy in an E. coli strain that contains an insertion in the dapA gene (7949). This strain is unable to synthesize DAP (2,6-diaminopimelic acid), and as a result is not viable without DAP supplemented in the medium. The insertion in dapA is flanked by two specific recombination sites, attI and attC. Integrase expression causes site-specific recombination and excision of the synthetic cassette, restoring a functional dapA gene and allowing the strain to grow on DAP-free medium (Figure 4A). We transformed in this dapA- strain a multi-copy plasmid (p7755) carrying the intI1 gene under the control of its natural SOS regulated promoter. The recombination rate due to integrase expression is calculated as the ratio of the number of cells growing in the absence of DAP over the total number of cells. Figure 4B shows the cassette excision rate in E. coli 7949 p7755 after conjugation with different conjugative plasmids. In the absence of a conjugative plasmid in the donor cell, the spontaneous excision rate is about 10−5, which reflects the stringency of the intI promoter. Conjugation with R6Kdrd and R388 increases excision rate to 10−3 and 10−2 respectively, whereas conjugation with R64drd does not increase significantly beyond the basal recombination level. RP4 yields an intermediate level of DAP+ cells, which is compatible with its intermediate SOS induction level in E. coli. These results are consistent with SOS induction results in E. coli, and as expected, there is a correlation between SOS induction and integrase induced cassette recombination. To confirm that cassette recombination is due to integrase expression, we performed the same experiment in strain 7949 lacking the integrase carrying plasmid p7755, and no cassette excision was observed (<10−8). We conclude that conjugation with psiB deficient plasmids in E. coli induces the expression of the integrase from the intI1 promoter, and thus triggers cassette recombination.


Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation.

Baharoglu Z, Bikard D, Mazel D - PLoS Genet. (2010)

Conjugation increases IntI1-dependent cassette excision rate in E. coli.A: experimental setup. 7949 strain contains plasmid p7755 carrying intI1 under the control of its natural LexA-regulated promoter. B: cassette excision rate was calculated by counting recombined cfu (Dap+) over total cfu. “No plasmid” means that recipient 7949 p7755 was mixed with empty donor. “No integrase” means that recipient 7949 without p7755 was conjugated with donor containing a conjugative plasmid.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958807&req=5

pgen-1001165-g004: Conjugation increases IntI1-dependent cassette excision rate in E. coli.A: experimental setup. 7949 strain contains plasmid p7755 carrying intI1 under the control of its natural LexA-regulated promoter. B: cassette excision rate was calculated by counting recombined cfu (Dap+) over total cfu. “No plasmid” means that recipient 7949 p7755 was mixed with empty donor. “No integrase” means that recipient 7949 without p7755 was conjugated with donor containing a conjugative plasmid.
Mentions: We have shown that conjugation induces SOS in the recipient bacteria and flow cytometry analysis clearly shows that the integron integrase is induced during conjugation in V. cholerae. In a first set of experiments, we wanted to test if the SOS induction leads to a higher activity of the integrase promoter in E. coli, using the class 1 integrase IntI1. We developed an experimental strategy in an E. coli strain that contains an insertion in the dapA gene (7949). This strain is unable to synthesize DAP (2,6-diaminopimelic acid), and as a result is not viable without DAP supplemented in the medium. The insertion in dapA is flanked by two specific recombination sites, attI and attC. Integrase expression causes site-specific recombination and excision of the synthetic cassette, restoring a functional dapA gene and allowing the strain to grow on DAP-free medium (Figure 4A). We transformed in this dapA- strain a multi-copy plasmid (p7755) carrying the intI1 gene under the control of its natural SOS regulated promoter. The recombination rate due to integrase expression is calculated as the ratio of the number of cells growing in the absence of DAP over the total number of cells. Figure 4B shows the cassette excision rate in E. coli 7949 p7755 after conjugation with different conjugative plasmids. In the absence of a conjugative plasmid in the donor cell, the spontaneous excision rate is about 10−5, which reflects the stringency of the intI promoter. Conjugation with R6Kdrd and R388 increases excision rate to 10−3 and 10−2 respectively, whereas conjugation with R64drd does not increase significantly beyond the basal recombination level. RP4 yields an intermediate level of DAP+ cells, which is compatible with its intermediate SOS induction level in E. coli. These results are consistent with SOS induction results in E. coli, and as expected, there is a correlation between SOS induction and integrase induced cassette recombination. To confirm that cassette recombination is due to integrase expression, we performed the same experiment in strain 7949 lacking the integrase carrying plasmid p7755, and no cassette excision was observed (<10−8). We conclude that conjugation with psiB deficient plasmids in E. coli induces the expression of the integrase from the intI1 promoter, and thus triggers cassette recombination.

Bottom Line: We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements.Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell.Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité Plasticité du Génome Bactérien, Département Génomes et Génétique, Paris, France.

ABSTRACT
Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

Show MeSH
Related in: MedlinePlus