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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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PsiB alleviates SOS induction in E. coli but not in V. cholerae because of impaired interaction with RecAVch.β-gal tests showing SOS induction following MMC treatment. A: E. coli MG1655 sfiA::lacZ. B: V. cholerae recN::lacZ. Overnight cultures of E. coli 7651 and V. cholerae 7453 were diluted 100× in LB containing 0.2% arabinose and grown until OD∼0.5. SOS was induced for 1h with 0.2µg/ml MMC and β-gal tests were performed as described [61]. No MMC was added to control cultures.
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pgen-1001165-g002: PsiB alleviates SOS induction in E. coli but not in V. cholerae because of impaired interaction with RecAVch.β-gal tests showing SOS induction following MMC treatment. A: E. coli MG1655 sfiA::lacZ. B: V. cholerae recN::lacZ. Overnight cultures of E. coli 7651 and V. cholerae 7453 were diluted 100× in LB containing 0.2% arabinose and grown until OD∼0.5. SOS was induced for 1h with 0.2µg/ml MMC and β-gal tests were performed as described [61]. No MMC was added to control cultures.

Mentions: We observed a strong induction of SOS by the same 3 plasmids in V. cholerae (Figure 1C), suggesting that the psiB gene is either not expressed in V. cholerae or that its product is not active in this species (R64drd and R100-1 do not replicate in V. cholerae, thus no activity per conjugant could be calculated). Moreover, SOS induction is continuously high for R64drd and R100-1 plasmids after ∼60 min, whereas SOS induction declined after 60 min for RP4, R6Kdrd and R388, as mentioned above. We were unable to delete psiB from R64drd and thus could not check if in its absence SOS induction would be restored in E. coli. The reason for the unsuccessful cloning attempts could be the presence of several genes (such as ssb coding the single strand binding protein, anti-restriction gene ardA, or flm/hok) in the same region where ORFs and regulatory regions overlap [9], [11], [39]–[41], such that deletion of psiB could have unpredicted consequences on plasmid transfer and replication. Instead, psiB from R64drd was cloned and over-expressed from a pBAD plasmid, under the control of the arabinose inducible promoter. SOS induction after mitomycin C (MMC) treatment was measured in E. coli sfiA::lacZ and V. cholerae recN::lacZ containing either empty pBAD or pBAD-PsiB+ plasmids. As previously published [31], MMC treatment induced SOS in E. coli and V. cholerae (Figure 2). SOS induction was strongly reduced in E. coli when PsiB was expressed from pBAD (6 fold induction instead of 11.6 fold, Figure 2A) whereas SOS induction was insensitive to PsiB expression in V. cholerae (∼60 fold induction with and without PsiB over-expression, Figure 2B). These results show that the psiBR64drd (and presumably the psiBR100-1 which presents 85% identity to psiBR64) is expressed during conjugation in E. coli and inhibits the SOS response, whereas in V. cholerae, psiBR64drd/R100-1 has no or very little anti-SOS activity, allowing R64drd and R100-1 transfer to induce SOS. The fact that R64 and R100-1 are narrow host range enterobacterial plasmids [40], [42] and do not replicate in V. cholerae, can explain the continuous induction we observe. Entering plasmid DNA is not replicated and new rounds of conjugation can carry on, resulting in continuous re-induction of SOS.


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)

PsiB alleviates SOS induction in E. coli but not in V. cholerae because of impaired interaction with RecAVch.β-gal tests showing SOS induction following MMC treatment. A: E. coli MG1655 sfiA::lacZ. B: V. cholerae recN::lacZ. Overnight cultures of E. coli 7651 and V. cholerae 7453 were diluted 100× in LB containing 0.2% arabinose and grown until OD∼0.5. SOS was induced for 1h with 0.2µg/ml MMC and β-gal tests were performed as described [61]. No MMC was added to control cultures.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958807&req=5

pgen-1001165-g002: PsiB alleviates SOS induction in E. coli but not in V. cholerae because of impaired interaction with RecAVch.β-gal tests showing SOS induction following MMC treatment. A: E. coli MG1655 sfiA::lacZ. B: V. cholerae recN::lacZ. Overnight cultures of E. coli 7651 and V. cholerae 7453 were diluted 100× in LB containing 0.2% arabinose and grown until OD∼0.5. SOS was induced for 1h with 0.2µg/ml MMC and β-gal tests were performed as described [61]. No MMC was added to control cultures.
Mentions: We observed a strong induction of SOS by the same 3 plasmids in V. cholerae (Figure 1C), suggesting that the psiB gene is either not expressed in V. cholerae or that its product is not active in this species (R64drd and R100-1 do not replicate in V. cholerae, thus no activity per conjugant could be calculated). Moreover, SOS induction is continuously high for R64drd and R100-1 plasmids after ∼60 min, whereas SOS induction declined after 60 min for RP4, R6Kdrd and R388, as mentioned above. We were unable to delete psiB from R64drd and thus could not check if in its absence SOS induction would be restored in E. coli. The reason for the unsuccessful cloning attempts could be the presence of several genes (such as ssb coding the single strand binding protein, anti-restriction gene ardA, or flm/hok) in the same region where ORFs and regulatory regions overlap [9], [11], [39]–[41], such that deletion of psiB could have unpredicted consequences on plasmid transfer and replication. Instead, psiB from R64drd was cloned and over-expressed from a pBAD plasmid, under the control of the arabinose inducible promoter. SOS induction after mitomycin C (MMC) treatment was measured in E. coli sfiA::lacZ and V. cholerae recN::lacZ containing either empty pBAD or pBAD-PsiB+ plasmids. As previously published [31], MMC treatment induced SOS in E. coli and V. cholerae (Figure 2). SOS induction was strongly reduced in E. coli when PsiB was expressed from pBAD (6 fold induction instead of 11.6 fold, Figure 2A) whereas SOS induction was insensitive to PsiB expression in V. cholerae (∼60 fold induction with and without PsiB over-expression, Figure 2B). These results show that the psiBR64drd (and presumably the psiBR100-1 which presents 85% identity to psiBR64) is expressed during conjugation in E. coli and inhibits the SOS response, whereas in V. cholerae, psiBR64drd/R100-1 has no or very little anti-SOS activity, allowing R64drd and R100-1 transfer to induce SOS. The fact that R64 and R100-1 are narrow host range enterobacterial plasmids [40], [42] and do not replicate in V. cholerae, can explain the continuous induction we observe. Entering plasmid DNA is not replicated and new rounds of conjugation can carry on, resulting in continuous re-induction of SOS.

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