Abstract
Dissemination of multiresistance has been accelerating among pathogenic bacteria in recent decades. The broad host-range conjugative plasmids of the IncA/C family are effective vehicles of resistance determinants in Gram-negative bacteria. Although more than 150 family members have been sequenced to date, their conjugation system and other functions encoded by the conserved plasmid backbone have been poorly characterized. The key cis-acting locus, the origin of transfer (oriT), has not yet been unambiguously identified. We present evidence that IncA/C plasmids have a single oriT locus immediately upstream of the mobI gene encoding an indispensable transfer factor. The fully active oriT spans ca. 150-bp AT-rich region overlapping the promoters of mobI and contains multiple inverted and direct repeats. Within this region, the core domain of oriT with reduced but detectable transfer activity was confined to a 70-bp segment containing two inverted repeats and one copy of a 14-bp direct repeat. In addition to oriT, a second locus consisting of a 14-bp imperfect inverted repeat was also identified, which mimicked the function of oriT but which was found to be a recombination site. Recombination between two identical copies of these sites is RecA-independent, requires a plasmid-encoded recombinase and resembles the functioning of dimer-resolution systems.
Highlights
The discovery and use of antibiotics have helped to save millions of lives in recent decades[1]
OriT of several conjugative plasmids was found adjacent to genes encoding relaxosome components and oriT of IncA/C plasmids was predicted to be near traI
IncA/C plasmids are of great importance due to their efficiency in the spread of multiresistance in Enterobacteriaceae and several other families of γ-Proteobacteria
Summary
The discovery and use of antibiotics have helped to save millions of lives in recent decades[1]. The second cluster encodes three additional putative proteins involved in T4SS assembly (traFHG)[17] This region contains two ORFs encoding the FlhDC-family transcriptional activator AcaCD, which activates the expression of transfer genes[19] and, is essential for the conjugation of the IncA/C plasmids. Based on the homology of the regions surrounding oriT of SXT/R391 with the corresponding region of the IncA/C plasmid pVCR94, oriT was proposed to be within the intergenic region of vcrx[001] (mobI) and vcrx15220 This region was confirmed to carry a functional oriT, two different deletions within the predicted oriT sequence caused only a 10-fold reduction in the transfer frequency, but they did not abolish conjugation. Most conjugative plasmids possess a single oriT, dual oriT systems have been reported in the cases of ICEclc of Pseudomonas knackmussi B1324 or plasmids pAD19, 25 and R6K6, which might support the assumption
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