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.