The master activator of IncA/C conjugative plasmids stimulates genomic islands and multidrug resistance dissemination

Abstract

Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.

Figure 1. Regulation of IncA/C plasmids.

(A) Schematic representation of the regulatory region of IncA/C plasmids. Genes and promoters are represented by arrowed boxes and angled arrows, respectively. Repressors and activators are shown in red and green, respectively. Genes of unknown function are shown in white and the gene coding for a putative lytic transglycosylase is shown in light blue. Locus names vcrxXXX are abbreviated as XXX. The oriT locus is depicted by a blue star. (B) Effect of the deletion and the overexpression of the negative transcriptional regulator-encoding genes acr1 and acr2. Conjugation assays were carried out using as donors E. coli BW25113 Nx containing pVCR94ΔX2 (WT) or its Δacr1 and Δacr2 mutants. Complementation and overexpression assays were performed with (+) or without (−) arabinose for the expression of acr1 (pacr1) or acr2 (pacr2) from the inducible P _BAD promoter. E. coli MG1655 Rf was used as the recipient. Transfer frequencies are expressed as a number of exconjugant per recipient CFUs. The bars represent the mean and standard deviation values obtained from at least three independent experiments. Statistical analyses were performed on the logarithm value of the means using one-way ANOVA with Tukey's multiple comparison test. P-values are indicated next to the bars when comparison referred to WT or next to the brackets when comparing two bars. (C) The constitutive promoter of acr1 (P_acr1) is repressed by Acr1 and Acr2. Activity of P_acr1 was monitored from a single-copy, chromosomally integrated lacZ transcriptional fusion (P_acr1-lacZ). Colorimetric assays were carried out on LB medium supplemented with 40 µg/ml of X-Gal and induction with (+) or without (−) arabinose to express acr1, acr2 or acaCD from P _BAD on pacr1, pacr2 or pacaCD, respectively. (D) AcaC and AcaD are essential for conjugative transfer. Transfer assays were carried out using E. coli BW25113 Nx containing pVCR94ΔX2 (WT) or the mutants ΔacaC, ΔacaD or ΔacaCD. Complementation assays were performed by expressing acaC, acaD or acaCD from P _BAD on pacaC, pacaD and pacaCD, respectively. Recipient strains and statistical analyses were as described for panel B. All P-values are below 0.0001 when compared to the WT. The asterisk indicates that frequency of exconjugant formation was below the detection limit (<10⁻⁸). (E) AcaCD is the direct activator of tra gene promoters. Activity of the P_traL, P_traI, P_traF and P₁₅₂ was monitored from single-copy, chromosomally integrated lacZ transcriptional fusions. Colorimetric assays were performed as described in panel C with expression of acaCD or setCD from P _BAD on pacaCD or psetCD (pGG2B), respectively. (F) AcaD co-purifies with AcaC. Coomassie blue-stained SDS-PAGE and Western blot analysis of AcaC purified using a Ni-NTA affinity chromatography. AcaD and 6×His-tagged AcaC were co-expressed in E. coli BL21(DE3) from pacaDC ^6×His. Western blot analysis was performed using a monoclonal antibody against the 6×His-tag.

Figure 2. Molecular phylogenetic analysis of the acr1-vcr147-acaDC-acr2 locus by Maximum Likelihood method.

The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model . The tree with the highest log likelihood (−5461.6977) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.6567)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 45 nucleotide sequences (Table S2). Codon positions included were 1st+2nd+3rd+Noncoding. There were a total of 2469 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 . The background color of each leaf indicates the original host species from which each plasmid was isolated. Insertions (Ω), deletion (Δ) and frameshift (Ψ) mutations are indicated where appropriate. *, Although pYT3 shares sequence identity with the IncA/C plasmids, it lacks an IncA/C-specific replication initiation gene (repA) and contains an IncFIB replicon instead (Figure S1) .

Figure 3. In-depth analysis of the AcaCD regulon in pVCR94.

(A) Genetic organization of pVCR94ΔX adapted from Carraro et al. . The circular map of the plasmid was linearized at the start position of gene vcrx001. The locations and orientations of ORFs are indicated by arrowed boxes. Colors are coded by function as follow: white, unknown; blue, conjugative transfer; light blue, lytic transglycosylase; orange, replication; gray, antibiotic resistance; yellow, putative regulatory function; purple, recombination; green, activator; red, repressor. The origin of replication (oriV) and the origin of transfer (oriT) are also indicated. The position of the scar resulting from the deletion of the antibiotic resistance gene cluster is indicated by FRT. sul2, resistance to sulfamethoxazole. (B) Results of ChIP-exo and RNA-seq experiments on E. coli MG1655 Rf carrying pVCR94ΔacaCD with or without a single chromosomal copy of pacaDC ^3×FLAG expressing the native AcaD subunit along with a C-terminal 3×FLAG-tagged AcaC subunit induced by IPTG. The first track plots the number of ChIP-exo reads mapped as a function of the position in pVCR94ΔX (black bars). Pink dots at the top of peaks indicate a signal beyond the represented y-axis maximal value. The second track shows the position of ChIP-exo enrichment peaks found by MACS (dark gray). The asterisk at the top of the rightmost peak indicates a MACS false negative result manually incorporated in the figure. The third track is a representation of the pVCR94ΔX genes using the same color code as in panel A. The fourth track indicates the position of the AcaCD-binding motifs found by MAST within ChIP-exo peaks using the AcaCD logo shown in panel C. Green arrows, motifs on positive DNA strand; red arrows, motifs on negative DNA strand. The fifth track represents Rockhopper's predicted operons. Dark blue, operons transcribed from positive DNA strand; orange, operons transcribed from negative DNA strand. The remaining four tracks show the total RNA-seq read densities (black bars; log scale) and the genome-wide 5′-RACE signals (green and red bars respectively on the positive and negative DNA strands; linear scale) for cells harboring either pVCR94ΔacaCD or pVCR94ΔacaCD complemented with pacaDC ^3×FLAG and induced by IPTG. Pink dots at the top of 5′-RACE signals indicate a signal beyond the represented y-axis maximal value. (C) Motif sequence recognized by AcaCD in pVCR94ΔX obtained by MEME using the AcaCD-binding sequences generated from ChIP-exo experiments. (D) VAP aggregate profile showing ChIP-exo and 5′-RACE density signals centered on the AcaCD-binding motif (black box). Yellow line, density of reads mapping on the positive DNA strand (Left border); green line, density of reads mapping on the negative DNA strand (Right border). X-axis displays the distance in nucleotides from the aggregated transcription start site shown in blue (TSS). (E) Organization of vcrx059 and traI divergent promoters revealed by ChIP-exo and 5′-RACE for pVCR94ΔacaCD complemented with IPTG-induced pacaDC ^3×FLAG. The first track plots ChIP-exo read densities at single nucleotide resolution as in panel D. Dark blue, density of reads mapping on the positive DNA strand; magenta, density of reads mapping on the negative DNA strand. The second track shows the two AcaCD-binding motifs found by MAST within the ChIP-exo peak between vcrx059 (white arrows) and traI (blue arrows) genes. Motif corresponding to the positive DNA strand is represented in green and motif corresponding to the negative DNA strand is shown in red. The last track represents 5′-RACE signals as described in panel B. The exonuclease-protected regions of the vcrx059 and traI promoters are indicated by dashed lines.

Figure 4. IncA/C-dependent excision and transfer of GIs.

(A) Schematic representation of SGI1 from S. enterica Typhimurium DT104 and MGIVmi1 from V. mimicus VM573. The left and right junctions (attL and attR) within the host chromosome are indicated. SGI1 (42 596 bp, Genbank AF261825) and MGIVmi1 (16 511 bp, Genbank NZ_ACYV01000005) are integrated into the 3′ end of trmE and yicC (attL sides) in their respective hosts. ORFs with similar function are indicated by colors as follows: black, DNA recombination; orange, DNA replication; blue, conjugative transfer; yellow, regulatory function; pink, putative type III restriction-modification system; white, unknown functions; MDR, multidrug resistance locus. Green chevrons indicate the position and orientation of predicted AcaCD-binding sites (see Table 1). For clarity, ORF names S0XX were shortened as SXX for SGI1 and VMD_06XXX as XXX for MGIVmi1. (B) AcaCD induces SG1 and MGIVmi1 excision. Excision was detected by PCR on genomic DNA to specifically amplify the attB chromosomal site and the attP site resulting from the excision of the GIs in S. enterica Typhimurium or E. coli bearing SGI1 and V. mimicus bearing MGIVmi1. Integrated GIs were detected by amplification of the attL site. Assays were done in strains devoid of plasmid (−), bearing pVCR94ΔX3 (+) or only expressing acaCD (acaCD) from pacaDC ^3×FLAG for assays in E. coli or pacaCD in V. mimicus. (C) Intraspecific mobilization of both GIs was assayed using E. coli MG1655 Rf bearing pVCR94ΔX3 and SGI1 or MGIVmi1 as a donor and the otherwise isogenic strain MG1655 Nx as a recipient. Exconjugants were selected for the acquisition of either GI, pVCR94ΔX3, and for cotransfer of both. Transfer frequencies are expressed as the number of exconjugant per donor CFUs. The bars represent the mean and standard deviation values obtained from three independent experiments. The asterisk indicates that the frequency of exconjugant formation was below the detection limit (<10⁻⁸). (D) AcaCD induces the expression of the putative excision and mobilization genes of MGIVmi1. Relative mRNA levels of int (VMD_06510), 490 (VMD_06490), 480 (VMD_06480) and xis (VMD_06410) were measured by RT-qPCR assays on cDNA of V. mimicus VM573 devoid of plasmid (−) or expressing acaCD from pacaCD (+). The bars represent the mean and standard deviation values obtained from three independent experiments. Comparison between the strain expressing or not AcaCD were done using two-tailed Student's t-tests and the P-values are indicated above to the bars.

Figure 5. Model of regulation of IncA/C plasmids and interaction with genomic islands.

Expression of the master activator complex AcaCD from the promoter of acr1 (P_acr1) is directly repressed by Acr1 and Acr2 (red arrows). AcaCD directly activates the expression of the transfer genes of pVCR94, as well as the expression of the bet/exo homologous recombination system and numerous genes of unknown function (green arrows). AcaCD triggered the excision of SGI1 and MGIVmi1 by directly activating the expression of the RDF genes xis. AcaCD activates the expression of the vcrx001-like mobilization gene and other genes of unknown function in MGIVmi1. AcaCD also activates the expression of three genes coding for putative component of type IV secretion system (T4SS), as well as genes of unknown function such as S004, rep and S018 in SGI1. IncA/C plasmids likely provide additional functions for genomic islands dissemination such as oriT recognition and processing or formation of the mating pore (black hatched arrows). Genes are color-coded as in Figure 1A.