Cassette recruitment in the chromosomal Integron of Vibrio cholerae

Abstract

Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.

Figure 1.

The integron. (A) The integron system in Vibrio cholerae. V. cholerae sedentary chromosomal integron is located on the second chromosome close to the termination site, Ter2. The four components of the integron stable platform are shown: the integrase expressing gene, intIA, the two promoters, P_C and P_int and the attIA recombination site (red triangle). The variable cassette array contains a large number of cassettes, which are represented by small arrows. Their expression level is reflected by the colour intensity of each arrow. Only the first cassettes of the array are expressed, and the subsequent ones can be seen as a low-cost cassette reservoir. Upon expression of the integrase (grey forms) cassette shuffling can occur through cassette excision (attC × attC) and integration in the first position in the array (attIA × attC). (B) Integron cassette insertion in an attI site. Recombination between the double-stranded attI site (bold red lines) and a single-stranded bottom attC site (green lines) ending a cassette is shown. Since we do not exactly know the nature of the cassettes (ss or ds), the top strand of the attC site is represented as a dotted line. The synaptic complex comprises both att sites bound by four integrase monomers (grey ovals). One strand from each att site is cleaved and transferred to form an atypical Holliday junction (aHJ). aHJ resolution implies a replication step. The origin of replication is represented by a grey circle and the newly synthesized leading and lagging strands by dashed lines. Both products are represented: the initial substrate resulting from the top strand replication, and the reactional product containing the inserted cassette and resulting from the bottom strand replication.

Figure 2.

Cassette recruitment in Vibrio cholerae SCI during horizontal gene transfer. (A) Experimental setup of the cassette insertion assay. The pSW23T::attC_aadA7 suicide vector is delivered to N16961 V. cholerae recipient strains containing an integrase expressing vector or the sole endogenous integrase, and the SCI. The delivery occurs by two horizontal gene transfer processes: conjugation from the β2163 donor or natural transformation. As pSW23T cannot replicate in V. cholerae recipient strains, recombinant clones can be selected on appropriate Cm containing plates to evaluate the recombination frequency (see also Results and Materials and Methods). The attC_aadA7 site carried by the suicide vector is represented by a green triangle and the attIA site on the V. cholerae SCI by a red triangle. (B) Frequency of insertion of the pSW23T::attC_aadA7suicide vector into the attIA site. The recombination frequencies were calculated in N16961 V. cholerae wt, ΔintIA and lexA(ind-) strains. Results correspond to recombination frequencies that were normalized after analysis of PCR reactions (Materials and Methods). +IntIA: recipient strains transformed with the pBAD43 integrase expressing vector; -IntIA: control strains transformed with the empty pBAD43 vector. * correspond to the limits of detection. Values represent the mean of at least three independent experiments and error bars correspond to average deviations from the mean.

Figure 3.

Effect of the RecA protein on attIA × attC recombination in Vibrio cholerae SCI. (A) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7 suicide vector into the chromosomic attIA site. N16961 recipient strains transformed with the pBAD43 IntIA expressing vector were used (left panel). The recombination rates were calculated in N16961 V. cholerae wt and in the corresponding recA mutant (ΔrecA) and ectopic complemented (ΔrecA-att_tn7::P_LAC-recA_Vch and ΔrecA-att_tn7::P_LAC-recA_Ec) strains (right panel). (B) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7suicide vector into the attIA site located on plasmid. N16961 recipient strains transformed with both pBAD43 IntIA expressing vector and pSU38Δ::attIA vector were used (left panel). The recombination rates were calculated in N16961 V. cholerae wt and in the corresponding recA mutant strains (ΔrecA, right panel). For both (A) and (B), results correspond to recombination frequencies that were normalized after analysis of PCR reactions (Materials and Methods). +IntIA: recipient strains transformed with the pBAD43 integrase expressing vector; -IntIA: control strains transformed with the empty pBAD43 vector. * correspond to the limits of detection. Values represent the mean of at least three independent experiments and error bars correspond to average deviations from the mean.

Figure 4.

Effect of the RecA protein on attC × attC recombination in Vibrio cholerae. (A) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7 suicide vector into VCR sites of the SCI. N16961 recipient strains deleted for the attIA site (ΔattIA strains) and transformed with the pBAD43 IntIA expressing vector were used (left panel). The recombination rates were calculated in N16961 V. cholerae ΔattIA and in the corresponding recA mutant strain (ΔattIA ΔrecA, right panel). (B) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7suicide vector into the attC_aadA1 site located on the pBAD43 plasmid. N16961 recipient strains deleted for the attIA site (ΔattIA strains) and transformed with the IntIA expressing and attC_aadA1 containing pBAD43 vector were used (left panel). The recombination rates were calculated in N16961 V. cholerae ΔattIA and the corresponding recA mutant strain (ΔattIA ΔrecA, right panel). For both (A) and (B), results correspond to recombination frequencies that were normalized after analysis of PCR reactions and sequencing (Materials and Methods). +IntIA: recipient strains transformed with the pBAD43 integrase expressing vector; -IntIA: control strains transformed with the empty pBAD43 vector. * correspond to the limits of detection. Values represent the mean of at least three independent experiments and error bars correspond to average deviations from the mean.

Figure 5.

Effect of the RecA protein on attI1 × attC recombination mediated by IntI1 in Vibrio cholerae. (A) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7suicide vector into the attI1 site located in the SCI platform. N16961 recipient strains with an attI1 site in place of the attIA site (ΔattIA::attI1 strains) were transformed with the pBAD43 IntI1 expressing vector (left panel). The recombination rates were calculated in N16961 V. cholerae ΔattIA::attI1 and the corresponding recA mutant strain (ΔattIA::attI1 ΔrecA, right panel). (B) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7suicide vector into the attI1 site located on a plasmid. N16961 recipient strains deleted for the attIA site (ΔattIA::attI1 strain) and transformed with both pBAD43 IntI1 expressing vector and pSU38Δ::attI1 vector were used (left panel). The recombination rates were calculated in V. cholerae ΔattIA::attI1 and the corresponding recA mutant strain (ΔattIA::attI1 ΔrecA, right panel). For both (A) and (B), results correspond to recombination frequencies that were normalized after analysis of PCR reactions (Materials and Methods). +IntIA: recipient strains transformed with the pBAD43 integrase expressing vector; -IntIA: control strains transformed with the empty pBAD43 vector. * correspond to the limits of detection. Values represent the mean of at least three independent experiments and error bars correspond to average deviations from the mean.

Figure 6.

Effect of the RecA protein on recombination reactions catalysed by IntI1 and IntIA in Escherichia coli. (A) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7suicide vector into the att sites (attI1 or attIA) located on a plasmid. MG1655 recipient strains transformed with both pBAD43 IntI expressing vector (IntI1 or IntIA) and the pSU38Δ::attI vector (attI1 or attIA) were used (left panel). The recombination rates were calculated in MG1655 and in the corresponding recA mutant (ΔrecA) and ectopic complemented (ΔrecA-att_tn7::P_LAC-recA_Vch) strains (right panels). (B) Experimental setup and frequency of insertion of the pSW23T::attC_aadA7suicide vector into the VCR_VCA0441 site located on a plasmid. MG1655 recipient strains transformed with both pBAD43 IntI expressing vector (IntI1 or IntIA) and the pSU38Δ::VCR_VCA0441 vector were used (left panel). The recombination rates were calculated in MG1655 and in the corresponding recA mutant strain (ΔrecA, right panels). For both (A) and (B), results correspond to recombination frequencies that were normalized after analysis of PCR reactions (Materials and Methods). +IntI: recipient strains transformed with the pBAD43 integrase expressing vector; -IntI: control strains transformed with the empty pBAD43 vector. * correspond to the limits of detection. Values represent the mean of at least three independent experiments and error bars correspond to average deviations from the mean.

Figure 7.

Effect of the RecA protein on attC × attI reaction when attC sites are carried by a replicative vector in Vibrio cholerae. (A) Experimental setup of the replicative assay. V. cholerae N16961 strains transformed with both pBAD18 IntIA expressing vector and the unidirectional-replicative vector, pTSC29::attC_aadA7 were used. attCaadA7 sites (green triangles) were cloned in both orientations (double arrow) in the pTSC29 vector. Since pTSC29 has a thermosensitive origin of replication, the recombination reaction is performed at 30°C and, to evaluate the recombination frequency, recombinant clones selection was performed on Cm containing plates at 42°C (see also Results and Materials and methods). The attIA site on the V. cholerae SCI is represented by a red triangle. (B) Folding of attC_aadA7 site on a replicative vector. Replicated and template strands are coloured in red and grey respectively and grey circles represent IntIA monomers. bs: bottom strand; ts: top strand; Lag st temp: Lagging strand template; Lead st temp: Leading strand template. (C) Frequency of insertion of the pTSC29::attC_aadA7unidirectional-replicative vector into the attIA site. The recombination rates were calculated in N16961 V. cholerae wt and in the corresponding recA mutant strains (ΔrecA). Under plots, the orientation of attC_aadA7 bs on lagging or leading strand template (lag st temp or lead st temp) are indicated. Results correspond to recombination frequencies that were normalized after analysis of PCR reactions (Materials and Methods). +IntI: recipient strains transformed with the pBAD18 integrase expressing vector; -IntI: control strains transformed with the empty pBAD18 vector. * correspond to the limits of detection. Values represent the mean of at least three independent experiments and error bars correspond to average deviations from the mean.

Figure 8.

Snap shot of SCI recombination during horizontal gene transfer in Vibrio cholerae. The SCI activity is represented and its connections with bacterial physiology. The steps, which involve the RecA protein, are indicated in green. HGT: horizontal gene transfer; ss: single-stranded DNA; VCR: Vibrio cholerae Repeat sequences; SCI: sedentary chromosomal integron.