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

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.

Figure 1. Conjugation induces SOS in E. coli and V. cholerae.

Shaded bars: E. coli. Dotted bars: V. cholerae. Grey: values at peak of induction t₄₀-t_60min (for SXT, the peak is shown at t₂₁₀); White: values at t₂₄₀. A: conjugation rates in E. coli recA+ and recA (strains 7651 and 7713) and V. cholerae (7453); B: SOS induction in total population of recipient E. coli and V. cholerae measured by β-gal tests; C: SOS induction ratio in conjugants only, for E. coli and V. cholerae. Induction was calculated as described in Materials and Methods. Induction ratios are units at time t_x/units at time t₀.

Figure 2. PsiB alleviates SOS induction in E. coli but not in V. cholerae because of impaired interaction with RecA_Vch.

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 . No MMC was added to control cultures.

Figure 3. Conjugation induces V. cholerae integron integrase intIA expression.

Donor and recipient (7093::p4640 recA+ and ΔrecA) strains were grown until OD 0.2, mixed at a 1∶1 ratio and incubated overnight on filter. % of GFP-induced cells was measured by flow cytometry.

Figure 4. 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.

Figure 5. Conjugation triggers IntIA-dependent cassette recombination in V.cholerae superintegron.

A: model: the catB cassette moved to 2^nd position on the SI. B: cassette displacement rate, i.e Cm-R cfu over total cfu. “No plasmid” means that recipient was mixed with empty donor.