Abstract
Bacterial conjugation is one of the most important mechanisms for spread of antibiotic resistance among bacteria. We have previously demonstrated that cefotaxime (CTX) exposure up-regulates expression of Type-IV conjugation transfer genes, and that this leads to increased transfer of a blaCTX–M–1 encoding IncI1 resistance plasmid pTF2 in Escherichia coli. To elucidate the underlying mechanisms, a search for genes that are essential for the up-regulated expression of the transfer (tra) genes in the presence of CTX was undertaken. We constructed a reporter gene-fusion strain MG1655/pTF2 ΔtraF:lacZ where the promoter region of the traF-gene of the plasmid pTF2 was fused with a lacZ on the native plasmid. Random mutagenesis mediated by Tn5 transposon was carried out in the strain, and seven genes (rfaH, yhiN, waaP, waaQ, gnd, pgl, and ISEcp1) were identified where insertion prevented CTX-induced up regulation of traF. Site-specific mutagenesis was carried out, and for all seven mutants, gene deletions abolished the CTX induced up-regulation of traF, and the increased conjugation transfer of the plasmid in the presence of CTX was no longer observed. In addition, the deletion of the genes also abolished CTX induced expression of the blaCTX–M–1 gene. Our results suggested that through CTX induced induction of the identified genes, blaCTX–M–1 expression increased, which led to up-regulation of traF and plasmid transfer. These data reveal that a number of chromosomally encoded genes contribute to the antibiotic induced up-regulation of the conjugation machinery of plasmids, and such genes may be future targets to prevent antibiotic induced spread of resistance plasmids.
Highlights
Conjugation allows bacteria to transfer genetic material from one cell to another via cell to cell contact (Llosa et al, 2002)
We showed that the transfer frequency of the plasmid pTF2 in Escherichia coli MG1655 in an antibiotic free environment was increased significantly when the donor was pre-grown in broth containing cefotaxime (CTX) (Moller et al, 2017)
Our previous work have reported that the transfer genes and proteins involved in conjugation of the blaCTX−M−1 plasmid pTF2 were significantly up-regulated when E. coli MG1655/pTF2 was treated with 1/2 minimal inhibitory concentrations (MIC) (126 mg/L) concentrations of CTX during growth (Moller et al, 2017)
Summary
Conjugation allows bacteria to transfer genetic material from one cell to another via cell to cell contact (Llosa et al, 2002). It has been recognized as one of most important contributors for dissemination of antimicrobial resistance genes (Bennett, 2008). Conjugative plasmids-transfer in Gram-negative bacteria requires the expression of transfer (tra) genes involved in DNA transfer. Plasmid-conjugation is primarily mediated by Type IV secretion systems (T4SSs), which are multi-protein complexes located in the membrane of the cell and which are able to support the donor and recipient mating-pair (Frost and Koraimann, 2010).
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