A rifamycin inactivating phosphotransferase family shared by environmental and pathogenic bacteria

Abstract

Many environmental bacteria are multidrug-resistant and represent a reservoir of ancient antibiotic resistance determinants, which have been linked to genes found in pathogens. Exploring the environmental antibiotic resistome, therefore, reveals the diversity and evolution of antibiotic resistance and also provides insight into the vulnerability of clinically used antibiotics. In this study, we describe the identification of a highly conserved regulatory motif, the rifampin (RIF) -associated element (RAE), which is found upstream of genes encoding RIF-inactivating enzymes from a diverse collection of actinomycetes. Using gene expression assays, we confirmed that the RAE is involved in RIF-responsive regulation. By using the RAE as a probe for new RIF-associated genes in several actinomycete genomes, we identified a heretofore unknown RIF resistance gene, RIF phosphotransferase (rph). The RPH enzyme is a RIF-inactivating phosphotransferase and represents a new protein family in antibiotic resistance. RPH orthologs are widespread and found in RIF-sensitive bacteria, including Bacillus cereus and the pathogen Listeria monocytogenes. Heterologous expression and in vitro enzyme assays with purified RPHs from diverse bacterial genera show that these enzymes are capable of conferring high-level resistance to a variety of clinically used rifamycin antibiotics. This work identifies a new antibiotic resistance protein family and reinforces the fact that the study of resistance in environmental organisms can serve to identify resistance elements with relevance to pathogens.

Keywords: drug inactivation; gene regulation; phosphorylation; silent resistome.

Fig. 1.

Rifamycin antibiotics.

Fig. 2.

The RAE is associated with RIF-inactivating genes. (A) The RAE is conserved upstream of various characterized RIF-inactivating genes from diverse actinobacteria. arr, RIF ADP ribosyltransferase; iri and rox, RIF monooxygenase; rgt1438, RIF glycosyltransferase; rph-Ss and rph4747, RIF phosphotransferase. The red line indicates the position of the RAE. An alignment of the RAE identified a conserved 19-nt inverted repeat boxed in gray. (B) Sequence logo from over 400 RAEs found in the nucleotide collection and whole-genome shotgun contig databases. Sequence logos were generated using WebLogo. (C) RAE gene associations.

Fig. 3.

The rph4747 gene from WAC4747 is responsible for the inactivation of RIF. Strains of WAC4747 were grown in the presence of RIF for 48 h, and conditioned media were analyzed for residual RIF activity using an RIF-sensitive indicator organism (B. subtilis). Samples were additionally analyzed using HPLC. WAC4747 and the control, WAC4747-pSET152 (empty vector), completely inactivate RIF within 48 h. The rph4747 mutant strain, WAC4747 rph4747::pSUIC, does not inactivate RIF and is comparable with the control of media and drug (Streptomyces isolation media (SIM)+RIF). Chromatograms are displayed at an absorbance of 343 nm.

Fig. 4.

The RAE is involved in gene regulation in response to RIF. (A) Quantitative RT-PCR analysis of rph4747 transcripts. WAC4747 was grown in the presence of RIF or DMSO. Data are presented as fold difference of rph4747 transcripts from three independent biological replicates, and error bars represent SDs. (B) A WAC4747 KAN resistance reporter assay. The RAE from WAC4747 was fused to a promoterless KAN resistance cassette and integrated in the genome of WAC4747. Growth on KAN-containing media were only achieved in the presence of RIF and RIF SV. Subinhibitory concentrations of antibiotics were used: AMP, 5 µg; ciprofloxacin (CIPRO), 0.5 µg; erythromycin (ERYTH), 0.125 µg; novobiocin (NOVO), 0.05 µg; RIF, 2.5 µg; RIF SV, 5 µg; streptomycin (STREPTO), 0.05 µg; vancomycin (VANCO), 0.05 µg.

Fig. 5.

Cladogram of the RPH protein family. Colored clades represent groups with over 85% bootstrap support based on maximum likelihood analysis using RAxML. Black circles indicate RPHs that are paired with the RAE. Red stars indicate RPHs selected for characterization: RPH4747, WAC4747; RPH-Bc, B. cereus ATCC 14579; RPH-Lm, L. monocytogenes str. 4b. F2365; RPH-Ss, S. sviceus ATCC 29083. The gray bar represents RPHs from bacteria of the Bacillales order. The light gray bar represents RPHs from the Listeriaceae family. A comprehensive cladogram with labels can be found in Fig. S5.