Abstract
To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of Listeria monocytogenes gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, lmo0762, is a homolog of hflX, which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits. To our knowledge, ribosome splitting has never been described as an antibiotic resistance mechanism. We thus investigated the role of lmo0762 in antibiotic resistance. First, we demonstrated that lmo0762 is an antibiotic resistance gene that confers protection against lincomycin and erythromycin, and that we renamed hflXr (hflX resistance). We show that hflXr expression is regulated by a transcription attenuation mechanism relying on the presence of alternative RNA structures and a small ORF encoding a 14 amino acid peptide containing the RLR motif, characteristic of macrolide resistance genes. We also provide evidence that HflXr is involved in ribosome recycling in presence of antibiotics. Interestingly, L. monocytogenes possesses another copy of hflX, lmo1296, that is not involved in antibiotic resistance. Phylogenetic analysis shows several events of hflXr duplication in prokaryotes and widespread presence of hflXr in Firmicutes. Overall, this study reveals the Listeria hflXr as the founding member of a family of antibiotic resistance genes. The resistance conferred by this gene is probably of importance in the environment and within microbial communities.
Highlights
To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps
We describe an antibiotic mechanism of resistance to lincomycin and erythromycin in L. monocytogenes which is mediated by Lmo0762, an HflX homolog, that we renamed HflXr, for HflX resistance
We showed that deletion of the gene renders the bacteria more sensitive to erythromycin and lincomycin, while its overexpression renders them more resistant
Summary
We performed MIC assay on a strain lacking lmo1296 (Δlmo1296), which showed no increased susceptibility to erythromycin, or any other antibiotics tested, compared with WT strain (Fig. 2A and SI Appendix, Fig. S3). We confirmed by qRT-PCR that in this strain, lmo1296 is induced by the antibiotic at a level similar to that of hflXr in the WT or Δlmo0762-cpt strains due to rli regulation (SI Appendix, Fig. S2B), and we tested the strain in a MIC assay. The analysis of genetic neighborhood of the two subfamilies showed much higher conservation for lmo1296 than for hflXr (Datasets S1 and S2) Based on these elements, it is tempting to speculate that the ancient duplication of hflX led to two proteins with specialized functions in Firmicutes, explaining why duplicates cooccur, one of which being involved in antibiotic resistance (hflXr). Such genes were found in important pathogens, such as B. cereus, Bacillus anthracis, and Clostridium difficile
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