Transcriptome Mapping of pAR060302, a bla _CMY-2 -Positive Broad-Host-Range IncA/C Plasmid

Abstract

The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5␣ harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, bla CMY-2 , aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.

Fitness Costs of Plasmids: a Limit to Plasmid Transmission

“…Integrons are very prevalent in plasmids and are generally related to antibiotic resistance (111). Transcriptional analysis has shown that integron cassettes are among the highest-expressed plasmid genes, representing an important potential source of fitness cost to host bacteria (112)(113)(114).…”

“…To date, only a handful of studies have investigated the genetic basis of plasmid fitness costs (23,(25)(26)(27)(28) or the transcriptional effects of plasmids on the bacterial host (23,25,113,131). In our experience, the best way to investigate the molecular mechanisms underlying the fitness costs of plasmids is to first obtain a compensated clone from the parental plasmid-carrying strain (22,23).…”

Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme

“…This ParA protein has similarity to ParA of IncP plasmids, while IncA/C ParB contains both ParB and KorB domains (1). These genes are transcribed at low levels, similar to repA (21). Another putative partitioning gene, stbA, is found in a separate genetic location.…”

“…IncA/C plasmids also possess a putative toxin-antitoxin (TA) system. The TA genes are strongly transcribed, suggesting the system is functionally active in the postsegregation killing of plasmid-free progeny (21). Moreover, attempts to construct deletion mutants of the antitoxin component proved to be lethal to the cell (22).…”

Comparative Sequence Analysis of a Multidrug-Resistant Plasmid from Aeromonas hydrophila

“…Plasmids are a modular mosaic of mobile genetic elements (MGEs) which the plasmids can carry and transfer between individual bacteria of different species by lateral gene transfer (also called horizontal gene transfer) (1)(2)(3). In so doing, the plasmids can transfer these genes to the bacterial chromosome or can gain more drug resistance genes.…”