A novel plasmid entry exclusion system in pKPC_UVA01, a promiscuous conjugative plasmid carrying the bla KPC carbapenemase gene

Conjugative plasmids are the principal mediator in the emergence and spread of antibiotic resistance genes in Enterobacterales. Plasmid entry-exclusion (EEX) systems can restrict their transfer into the recipient bacteria carrying closely related plasmids. In this study, we have identified and characterized a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, responsible for widespread dissemination of the bla KPC carbapenemase gene among Enterobacterales in the United States. The identified eex gene in the recipient strain of different Enterobacterales species inhibits the conjugation transfer of pKPC_UVA01 plasmids at a range of 200-400 fold, and this inhibition was found to be a dose-dependent function of the EEX protein in recipient cells. The C-terminus truncated version of eex or eex with an early termination codon at the C-terminus region alleviates inhibition of conjugative transfer. Unlike the strict specificity of plasmid exclusion by the known EEX protein, the newly identified EEX in the recipient strain can inhibit the transfer of IncP and IncN plasmids. The eex gene from the plasmid pKPC_UVA01 is not required for conjugative transfer but is essential in the donor bacteria for entry exclusion of this plasmid. This is a novel function of a single protein that is essential in both donor and recipient bacteria for entry exclusion of a plasmid. This eex gene is found to be distributed in multi-drug resistance plasmids similar to pKPC_UVA01 in different Enterobacterales species and may contribute to the stability of this plasmid type by controlling its transfer.

PixR, a Novel Activator of Conjugative Transfer of IncX4 Resistance Plasmids, Mitigates the Fitness Cost of mcr-1 Carriage in Escherichia coli

The spread of clinically relevant antibiotic resistance genes is often linked to the dissemination of epidemic plasmids. However, the underlying molecular mechanisms contributing to the successful spread of epidemic plasmids remain unclear.

A bistable orthogonal prokaryotic differentiation system underlying development of conjugative transfer competence

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that stands model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. The ICE only transfers from a small fraction of cells in a population, which we uncover here, results from a dedicated transfer competence program imposed by the ICE. Transfer competence is orthogonally maintained in individual cells in which it is activated, making them the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.

Conjugative transfer of naturally occurring plasmids in Mycolicibacterium sp.

Conjugation is considered the main horizontal gene transfer (HGT) mechanism in bacterial adaptation and evolution. In the Mycobacteriaceae family, Mycolicibacterium smegmatis has been used as the model organism for the conjugative transfer of hybrid plasmids. However, the natural conjugation process in any bacteria would involve the transfer of naturally occurring plasmids. Currently, there is a gap in this regard in relation to this abundant environmental genus of Mycobacteriaceae. Here, we performed conjugation experiments between wild Mycolicibacterium sp. strains involving naturally occurring plasmids (sizes of 21 and 274 kb), and interestingly, evidence of conjugative transfer was obtained. Thus, it is likely that conjugation occurs in Mycolicibacterium in the natural environment, representing a source of diversification and evolution in this genus of bacteria.