A Rifamycin Inactivating Phosphotransferase Family Shared by Environmental and Pathogenic Bacteria

A rifamycin inactivating phosphotransferase family shared by environmental and pathogenic bacteria

Peter Spanogiannopoulos, Nicholas Waglechner, Kalinka Koteva, and Gerard D. Wright1

Michael G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada L8S 3Z5

Edited by Richard Losick, Harvard University, Cambridge, MA, and approved March 27, 2014 (received for review February 6, 2014)

Many environmental bacteria are multidrug-resistant and repre- rifamycins are broad-spectrum antibiotics that inhibit prokaryotic sent a reservoir of ancient antibiotic resistance determinants, RNA polymerase (RNAP) by binding to a highly conserved re- which have been linked to genes found in pathogens. Exploring gion within the β-subunit (encoded by the gene rpoB), blocking the environmental antibiotic resistome, therefore, reveals the the exit tunnel from the growing RNA nucleotide chain, and diversity and evolution of antibiotic resistance and also provides thus, sterically hindering the extension of nascent mRNA tran- insight into the vulnerability of clinically used antibiotics. In this scripts (13). Recently, synthetic exploration of the rifamycin study, we describe the identification of a highly conserved regula- scaffold has generated a number of new analogs designed to tory motif, the rifampin (RIF) -associated element (RAE), which is treat a much broader repertoire of bacterial infections. These found upstream of genes encoding RIF-inactivating enzymes from drugs include rifaximin, which is administered for the treatment a diverse collection of actinomycetes. Using gene expression assays, of travelers’ diarrhea and irritable bowel syndrome (14, 15), we confirmed that the RAE is involved in RIF-responsive regulation. rifalazil, used for chlamydial infections (16), and a number of By using the RAE as a probe for new RIF-associated genes in several additional compounds (reviewed in ref. 12). This recent exten- actinomycete genomes, we identified a heretofore unknown RIF sion of the rifamycin family of antibiotics will likely apply pres- resistance gene, RIF phosphotransferase (rph). The RPH enzyme is sure for the selection of an expanded repertoire of rifamycin a RIF-inactivating phosphotransferase and represents a new protein resistance determinants (17). family in antibiotic resistance. RPH orthologs are widespread and In mycobacteria, point mutations of the drug target are the

found in RIF-sensitive bacteria, including Bacillus cereus and the most prevalent RIF resistance mechanism (18). Rifamycin re- MICROBIOLOGY −8 −9 pathogen Listeria monocytogenes. Heterologous expression and sistance develops quite rapidly (frequency of 10 –10 per in vitro enzyme assays with purified RPHs from diverse bacterial bacterium per cell division), where single amino acid changes in genera show that these enzymes are capable of conferring high- the β-subunit significantly decrease binding of the antibiotic to its level resistance to a variety of clinically used rifamycin antibiotics. target (19). In other microbes, diverse RIF-inactivating mecha- This work identifies a new antibiotic resistance protein family and nisms have been described from both pathogenic and non- reinforces the fact that the study of resistance in environmental pathogenic bacteria. Three RIF group transfer mechanisms of organisms can serve to identify resistance elements with relevance resistance have been described: glycosylation, ADP ribosylation, to pathogens. and phosphorylation (17). These resistance enzymes chemically modify the ansa polyketide bridge of RIF, attenuating its affinity drug inactivation | phosphorylation | gene regulation | silent resistome for the β-subunit of RNAP. In addition, the decomposition of RIF initiated by an RIF monooxygenase has also been described ur control of infectious disease is increasingly challenged Obecause of the emergence and dissemination of antibiotic Significance resistance (1). This problem is exacerbated because of a dwin- dling number of new antimicrobials entering the clinic (2). The Environmental microorganisms are a source of diverse antibi- majority of clinically used antibiotics originates from micro- otic resistance determinants. With the appropriate selection organisms that reside in the environment (3). Recent studies pressure, these resistance genes can be mobilized to clinically have established that antibiotic resistance is ancient, and con- relevant pathogens. Identifying and characterizing elements temporary environmental bacteria are multidrug-resistant (4–6). of the environmental antibiotic resistome provides an early There is also growing evidence that environmental bacteria, in- warning of what we may expect to encounter in the clinic. We cluding antibiotic producers, are the progenitors of antibiotic-re- uncover a conserved genetic element associated with various sistant determinants from pathogenic bacteria (7–9). Exploring rifamycin antibiotic-inactivating mechanisms. This element led and characterizing the antibiotic resistome, the collection of all to the identification of a new resistance gene and associated antibiotic resistance genes and their precursors from the global enzyme responsible for inactivating rifamycin antibiotics by microbiota, will facilitate our ability to anticipate and counter phosphorylation. Cryptic orthologous genes are also found in pathogenic bacteria but remain susceptible to the drug. This antibiotic resistance before its emergence into the clinic (10). With study reveals a new antibiotic resistance protein family and the this knowledge, we may begin to focus efforts on circumventing unexpected prevalence of a silent rifamycin resistome among these determinants by strategic drug development, resistance in- pathogenic bacteria. hibitor combinations, and prudent administration programs.

Rifamycin B, the initial member of the rifamycin family of Author contributions: P.S. and G.D.W. designed research; P.S., N.W., and K.K. performed antibiotics, was first described in the late 1950s and produced research; P.S. and G.D.W. analyzed data; P.S. and N.W. performed bioinformatic/phylo- from the soil actinomycete Amycolatopsis mediterranei (11). Al- genetic analysis; and P.S. and G.D.W. wrote the paper. though the parent natural product has modest antibiotic activity, The authors declare no conflict of interest. semisynthetic derivatives of this antibiotic family have experi- This article is a PNAS Direct Submission. enced great success in the clinic. The most well-known is ri- Data deposition: The DNA sequence reported in this paper has been deposited in the NCBI fampin (RIF), introduced into the clinic almost 50 y ago (Fig. 1). database (accession no. KJ151292). RIF continues to be a frontline treatment of tuberculosis and 1To whom correspondence should be addressed. E-mail: [email protected]. other mycobacterial infections and increasingly, is as a fallback This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. therapy for drug-resistant Staphylococcal infections (12). The 1073/pnas.1402358111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1402358111 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 (Fig. 2C). This survey revealed that the RAE is highly conserved across many actinobacterial genomes and has coevolved with a repertoire of genes, including RIF-inactivating genes and several other genes that are presumably expressed in response to RIF.

The rph Gene Is Responsible for Phosphorylating RIF. Anumberof actinomycetes capable of inactivating RIF by phosphorylation have been described; however, the gene responsible for this phenotype, rph, has yet to be identified (22, 23). The strong correlation of the RAE with genes encoding RIF-inactivating enzymes led us to apply this link to identify the rph gene. In Fig. 1. Rifamycin antibiotics. a previous phenotypic screen, we identified a collection of RIF-phosphorylating soil actinomycetes, and thus, we selected

(20, 21). Despite the fact that RIF phosphorylation has been known for 20 y, the gene encoding the RIF phosphotransferase (rph) has yet to be identified (22). Here, we describe the discovery of a conserved upstream nucleotide element associated with various genes encoding RIF- inactivating enzymes from actinomycetes. By exploiting this conservation, we successfully identified the heretofore unrecognized rph gene. Through a combination of phylogenetics, heterologous protein expression, and in vitro enzyme assays, we establish that RPH orthologs are prevalent in both environmental and pathogenic bacteria, conferring high-level and broad resistance against clinically used rifamycin antibiotics. This effort highlights the power of screening genome data for noncoding elements in the discovery of new resistance elements. Results A Conserved Upstream Nucleotide Element Is Associated with RIF- Inactivating Genes from Actinomycetes. A diverse collection of RIF-inactivating mechanisms has been described from various pathogenic and nonpathogenic actinomycetes. We recently reported the identification and characterization of a RIF- inactivating glycosyltransferase (rgt1438) from a screen of soil actinomycetes (23). Examination of the intergenic DNA sequence upstream of rgt1438 identified a 19-bp inverted repeat motif (9-bp repeats separated by a single nucleotide spacer) (Fig. 2A). We hypothesized that this element could be involved in gene regulation. The conservation of this putative RIF-associated element (RAE) was examined by scanning the upstream intergenic regions from various characterized genes encoding RIF-inactivating enzymes (20, 21, 23, 24). This collection in- cluded three mechanisms of RIF inactivation (glycosylation, ADP ribosylation, and monooxygenation) from four divergent genera of actinomycetes (Streptomyces, Mycobacteria, Rhodo- coccus, and Nocardia) (Fig. 2A). The RAE was conserved upstream of all these genes with near-perfect nucleotide identity (Fig. 2A). A BLASTn search of the RAE was conducted from intergenic regions of the nucleotide collection and whole- genome shotgun contigs from the National Center of Bio- technology Information (25). The RAE was found over 400 times and predominantly associated with actinobacterial genomes (Dataset S1). The RAE was not associated with rifamycin-producing bacteria. A sequence logo with this RAE collection shows the remarkable conservation of this element (Fig. 2B). To determine if there is a correlation between the RAE and specific genes, we compiled a list of available divergent ORFs flanking each RAE and loosely binned orthologous proteins (Dataset S2). The RAE was strongly associated with ORFs resembling pre- Fig. 2. The RAE is associated with RIF-inactivating genes. (A)TheRAEis viously characterized RIF-inactivating enzymes (Fig. 2C). The conserved upstream of various characterized RIF-inactivating genes from diverse actinobacteria. arr, RIF ADP ribosyltransferase; iri and rox,RIF RAE is observed accompanying an ORF encoding a RIF ADP monooxygenase; rgt1438, RIF glycosyltransferase; rph-Ss and rph4747,RIF ribosyltransferase (Arr), RIF glycosyltransferase (Rgt), or RIF phosphotransferase. The red line indicates the position of the RAE. An monooxygenase (Iri/Rox) enzyme at a frequency of 0.33. Further- alignment of the RAE identified a conserved 19-nt inverted repeat boxed in more, the RAE is strongly associated with a number of unchar- gray. (B) Sequence logo from over 400 RAEs found in the nucleotide col- acterized ORFs. The RAE is found upstream of ORFs annotated lection and whole-genome shotgun contig databases. Sequence logos were as phosphoenolpyruvate (PEP) synthases and two distinct helicases generated using WebLogo. (C) RAE gene associations.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402358111 Spanogiannopoulos et al. Downloaded by guest on October 2, 2021 one of these strains for additional investigation (23). The draft Table 1. Rifamycin MIC determinations of various rph genes genome of WAC4747 (most closely resembling Streptomyces expressed in E. coli viridochromogenes based on 16S rRNA analysis) was deter- MIC (μg/mL) mined. We also biochemically screened our in-house collection of genome-sequenced actinomycetes for their ability to phosphor- Construct*/IPTG RIF Rifamycin SV Rifabutin Rifaximin ylate RIF and identified Streptomyces sviceus ATCC 29083 as a candidate (Fig. S1). Putative rph genes were identified by probing pET22b the genome sequences of WAC4747 and S. sviceus for the RAE − 864 8 16 and examining associated downstream genes. The RAE was not + 864 8 16 found to be flanking any genes resembling known antibiotic pET22b-rph4747 phosphotransferases (26). The RAE was found on two contigs − 512 512 512 64 within the WAC4747 genome: on a 3-kb contig upstream of an +>512 >512 512 512 ORF encoding a putative PEP synthase (ORF1) and an 8.5-kb pET28b contig upstream of a putative helicase (ORF2) (Table S1). − 864 8 16 Similarly, the RAE was found upstream of predicted PEP syn- + 864 8 16 thase and helicase ORFs within the S. sviceus genome in addition pET28b-rph-Ss to a candidate RIF monooxygenase (Table S1). PEP synthase − 512 >512 512 512 and helicase proteins have not been previously associated with +>512 >512 512 512 antibiotic resistance. Helicases harness the energy of ATP hy- pET19Tb drolysis for separation of nucleotide strands, whereas PEP syn- − 864 8 8 thases catalyze the conversion of ATP and pyruvate to PEP, + 864 8 8 AMP, and Pi (27, 28). Orthologous genes of ORF1 and ORF2 pET19Tb-rph-Bc are conserved in other actinomycete genomes and colocalized − 128 256 64 64 with the RAE (Fig. 2C). +>512 >512 512 512 We individually disrupted ORF1 and ORF2 from WAC4747 pET19Tb-rph-Lm and evaluated the ability of the mutant strains to phosphorylate − 128 128 32 32 RIF. Analysis of conditioned media from WAC4747 grown in +>512 >512 512 512 the presence of RIF showed complete inactivation of RIF within 48 h (Fig. 3). In contrast, a mutant strain of WAC4747 IPTG, isopropyl β-D-1-thiogalactopyranoside. MICROBIOLOGY with a disrupted ORF1 (putative PEP synthase) failed to in- *E. coli Rosetta(DE3)pLysS host. activate RIF (Fig. 3). Disruption of ORF2 (putative helicase) in WAC4747 had no effect on RIF inactivation. These findings suggest that ORF1 (named rph4747) encodes the RPH enzyme. This result suggests, similar to other actinomycetes, that WAC4747 Inactivation of the orthologous putative PEP synthase gene from and S. sviceus harbor multiple RIF resistance mechanisms (21, 23). S. sviceus (rph-Ss) generated identical results (Fig. S2). Mutant We assessed the ability of the rph gene to contribute to RIF strains of WAC4747 and S. sviceus with a disrupted rph gene resistance by expression in a heterologous host. Introduction of displayed a modest twofold decrease in RIF minimum inhibitory rph4747 or rph-Ss into RIF-sensitive Escherichia coli conferred concentrations (MICs) compared with the WT strains (Table S2). a 64-fold increase in RIF MIC compared with the control (Table 1). E. coli-expressing rph displayed high-level resistance up to or over 512 μg/mL to a panel of natural product and clinically used semisynthetic rifamycin antibiotics (Table 1). This result highlights the ability of rph to confer broad high-level resistance to rifamycin antibiotics.

RAE Responds to RIF. Because of the strong association of the RAE and genes encoding RIF-inactivating enzymes, we hypothesized that this DNA motif is likely involved in gene expression in response to RIF. Changes in transcript levels of rph4747 expression were monitored using quantitative RT-PCR with growth of WAC4747 in the presence or absence of RIF. In the presence of RIF, the rph4747 transcript was up-regulated 63- and 91-fold after 1 and 2 h of growth, respectively, compared with the control, DMSO (Fig. 4A). This finding confirms that genes paired with the RAE are up-regulated in response to RIF. To validate the role of the RAE in RIF-specific gene regulation, we used a kanamycin (KAN) resistance reporter assay. The reporter was constructed by fusing the intergenic DNA upstream of the rph4747 gene ahead of a promoter-less KAN resistance cassette followed by integration into the genome of WAC4747, creating the strain WAC4747-PRAE/neo-pSET152. This strain was not KAN-resistant; showing gene expression downstream Fig. 3. The rph4747 gene from WAC4747 is responsible for the inactivation of the RAE is tightly regulated. However, exposure to sub- of RIF. Strains of WAC4747 were grown in the presence of RIF for 48 h, and inhibitory concentrations of rifamycin antibiotics (RIF and conditioned media were analyzed for residual RIF activity using an RIF-sensitive indicator organism (B. subtilis). Samples were additionally analyzed the parent natural product rifamycin SV) induced growth on using HPLC. WAC4747 and the control, WAC4747-pSET152 (empty vector), KAN-containing media, confirming that RAE is involved in completely inactivate RIF within 48 h. The rph4747 mutant strain, WAC4747 rifamycin-responsive gene regulation (Fig. 4B). A number of rph4747::pSUIC, does not inactivate RIF and is comparable with the control diverse antibiotics, from natural and synthetic origins, inhibiting of media and drug (Streptomyces isolation media (SIM)+RIF). Chromato- various biological targets failed to induce KAN resistance, con- grams are displayed at an absorbance of 343 nm. firming that gene regulation from the RAE is rifamycin-specific.

Spanogiannopoulos et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 We performed a phylogenetic analysis of the RPH protein family to explore the relationship of the sequences in this family (Dataset S3). The RPH cladogram is shown in Fig. 5. We can identify several clades with high bootstrap support that corre- spond to well-defined taxonomic groups, even if resolution within and between these groups remains unclear (Fig. S5). RPH proteins are widely distributed, predominately in Gram- positive bacteria. Although many RPH orthologs are found within Actinobacteria, the majority is associated with Bacilli and Clostridia. We examined the intergenic DNA sequence upstream of RPH ORFs and discovered that the majority of ORFs from Actinobacteria is associated with the RAE (Fig. 5). This finding is consistent with our previous survey of the RAE and gene associations (Fig. 2C). The RAE was not associated with RPHs outside Actinobacteria. It is, therefore, uncertain whether these enzymes possess RPH activity. A previous biochemical screen by Dabbs et al. (34) showed Fig. 4. The RAE is involved in gene regulation in response to RIF. (A) that a number of bacteria from the genus Bacillus inactivated Quantitative RT-PCR analysis of rph4747 transcripts. WAC4747 was grown in RIF by phosphorylation. This report was intriguing consid- the presence of RIF or DMSO. Data are presented as fold difference of ering that Bacillus spp. are highly susceptible to RIF (35). Our rph4747 transcripts from three independent biological replicates, and error phylogenetic analysis of the RPH protein family revealed bars represent SDs. (B) A WAC4747 KAN resistance reporter assay. The RAE from WAC4747 was fused to a promoterless KAN resistance cassette and orthologous enzymes from a number of Bacilli. This list also integrated in the genome of WAC4747. Growth on KAN-containing media encompassed a number of pathogenic bacteria, including Bacillus were only achieved in the presence of RIF and RIF SV. Subinhibitory con- cereus, Bacillus anthracis,andListeria monocytogenes.These centrations of antibiotics were used: AMP, 5 μg; ciprofloxacin (CIPRO), 0.5 μg; pathogens have been previously reported to be highly susceptible erythromycin (ERYTH), 0.125 μg; novobiocin (NOVO), 0.05 μg; RIF, 2.5 μg; RIF to RIF (36, 37). Indeed, this finding was confirmed by per- SV, 5 μg; streptomycin (STREPTO), 0.05 μg; vancomycin (VANCO), 0.05 μg. forming RIF susceptibility testing with B. cereus and L. monocytogenes, which showed a RIF MIC of 62.5 ng/mL. RPH orthologs from B. cereus (RPH-Bc) and L. monocytogenes (RPH- Additionally, a reporter construct was made where the RAE was Lm) displayed 55% and 52% identity and 74% and 71% simi- replaced with a random sequence of nucleotides. This construct larity, respectively, compared with RPH4747. We selected these failed to respond to RIF (Fig. S3) and established that the RAE two RPH orthologs as representatives from our phylogenetic nucleotide sequence is critical for gene expression. analysis and evaluated their ability to confer RIF resistance when In some instances, antibiotic resistance gene expression is expressed in a heterologous host. E. coli expressing rph-Bc or dependent on the antibiotic successfully binding and inhibiting its target (29, 30). We generated RIF-resistant rpoB mutants of WAC4747-PRAE/neo-pSET152 to determine if inhibition of RNAP is a prerequisite for RAE-mediated gene induction. This mutant strain gained KAN resistance in response to RIF (Fig. S3), indicating that gene expression mediated by the RAE is independent of RNAP inhibition.

Diversity of the RPH Protein Family. A BLASTp analysis of the RPH enzyme reveals that it is related to enzymes annotated as PEP synthases (EC 2.7.9.2). The mechanism of PEP synthase proceeds through a phosphoenzyme intermediate, where the β-phosphate of ATP is transferred to a histidine (His) residue, which in turn, is transferredtopyruvate(31).PEPsyn- thase is composed of three domains: the N-terminal domain is responsible for nucleotide binding, the C-terminal domain binds pyruvate, and the central segment contains the catalytic His domain. A global alignment of RPH4747 and PEP synthase from E. coli indicates low overall amino acid similarity but relatively high similarity near the N terminus. A search of the Conserved Domain Database (32) with RPH4747 showed that the N terminus consists of an ATP binding domain, similar to PEP synthase. In contrast to PEP synthase, the catalytic His domain of RPH is located at the C terminus (Fig. S4). The central region of RPH4747 shows no similarity to any previously characterized proteins. Therefore, RPH4747 shares some similarities with PEP synthase, but the overall architectures of these two enzymes Fig. 5. Cladogram of the RPH protein family. Colored clades represent differ. A search of the RPH using the Conserved Domain Ar- groups with over 85% bootstrap support based on maximum likelihood chitecture Retrieval Tool (33) showed that this domain archi- analysis using RAxML. Black circles indicate RPHs that are paired with the RAE. Red stars indicate RPHs selected for characterization: RPH4747, tecture is found in over 1,600 proteins and primarily restricted to WAC4747; RPH-Bc, B. cereus ATCC 14579; RPH-Lm, L. monocytogenes str. 4b. bacterial genomes. Although the majority of these enzymes is F2365; RPH-Ss, S. sviceus ATCC 29083. The gray bar represents RPHs from annotated as PEP synthases (or PEP binding proteins), there is bacteria of the Bacillales order. The light gray bar represents RPHs from the no biochemical evidence confirming this activity, and therefore, Listeriaceae family. A comprehensive cladogram with labels can be found in it represents an unexplored protein family. Fig. S5.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402358111 Spanogiannopoulos et al. Downloaded by guest on October 2, 2021 rph-Lm exhibited a 64-fold increase in RIF MIC and also con- essential for modulating gene expression. Such inverted re- ferred high-level resistance to a variety of rifamycin analogs peat DNA motifs have long been linked to gene regulation and (Table 1). This result confirms that RPH orthologs are found in often interact with protein regulators. Additional work is re- RIF-sensitive pathogenic bacteria and will confer high-level quired to reveal the details of the mechanism of induction as- and broad rifamycin resistance when expressed in a hetero- sociated with this RAE and determine if any additional logous host. regulators are involved. For over two decades, a number of actinomycetes has been Biochemical Characterization of RPH Activity. Recombinant RPH reported to inactivate RIF by phosphorylation; however, the enzymes were overexpressed from E. coli and purified for char- gene encoding the RPH was not known. Our observation of the acterization. Initially, RPH activity was monitored using a TLC strong correlation of the RAE with various actinomycete genes assay, which confirmed that ATP served as a cosubstrate (Fig. known to encode RIF-inactivating enzymes suggested a common S6). RIF-phosphate product from various RPHs was purified regulatory mechanism and an entry point to discover the rph and analyzed using high-resolution MS and NMR spectroscopy gene. Indeed, we successfully identified and validated that a gene (Fig. S6). This analysis confirmed that RPHs share the same predicted to encode a PEP synthase was, in fact, the rph. The regiospecificity and phosphorylate RIF at the hydroxyl group RPH enzyme does not show similarity to any previously identi- attached to the C21 of the ansa-chain, consistent with precedent fied antibiotic phosphotransferases. Instead, RPH is related to using whole-cell inactivation (Fig. 1) (22). the multidomain gluconeogenic enzyme PEP synthase. Although Liquid chromatography electrospray ionization MS coupled RPH and PEP synthase differ in overall domain architectures with multiple reaction monitoring were used to quantify RPH (Fig. S4), RPH enzymes have been routinely annotated as PEP activity. Steady-state kinetics were performed for three RPH synthases from sequenced genomes. Therefore, RPH represents enzymes from diverse bacterial hosts; RPH-Ss from S. sviceus, a previously unexplored and misannotated protein family. Ki- RPH-Bc from B. cereus, and RPH-Lm from L. monocytogenes. netic analysis and site-directed mutagenesis confirmed that the The gene encoding RPH-Ss is associated with the RAE, whereas RPH enzyme is mechanistically related to PEP synthase and the genes encoding RPH-Bc and RPH-Lm are not. All RPHs does not use pyruvate as a substrate. A Conserved Domain Ar- showed comparable kinetic constants regardless of the host chitecture Retrieval Tool search has shown that this protein source and RAE association (Table S3). RPHs exhibited very low family is widely distributed across bacteria and that these enzymes Km values for RIF within the high-nanomolar range, indicating are likely not involved in gluconeogenesis and may have diverse a high affinity for RIF. The Km values for RIF only varied twofold functions, including antibiotic resistance. Additional work is re-

among the different RPH enzymes. Similarly, all RPHs displayed quired to decipher their substrates and functions. MICROBIOLOGY comparable kcat constants (catalytic rate constant). RPH orthologs from actinomycetes are colocalized with the Although PEP synthase and RPH share aspects of function and RAE. However, the majority of RPHs is found in other Gram- amino acid structure, it is unclear whether these two protein positive genera (Fig. 5). Interestingly, many of these Gram pos- families have similar catalytic features. In addition to the liquid itives have an RIF-sensitive phenotype. Of particular concern are chromatography electrospray ionization MS assay, we further the orthologs of rph that are found within the genomes of patho- monitored RPH-Bc activity by measuring the generation of free Pi. gens, such as B. anthracis, B. cereus ,andL. monocytogenes.Het- Steady-state kinetic constants generated using this orthogonal erologous expression and in vitro enzyme assays confirmed they assay were analogous to values obtained with the MS method are bona fide RPHs and not PEP synthases and confer high-level described above (Table S3), confirming stoichiometric production and broad-spectrum resistance with equivalent competence to their of RIF-phosphate and Pi by RPH-Bc. With this assay, RPHs were Streptomycete counterparts. This unexpected diversity and preva- also assayed for PEP synthase activity. All RPHs failed to phos- lence of rph genes in a number of bacterial genera are troubling phorylate pyruvate and showed no PEP synthase activity. given the renewed interest in expanding the application of these A sequence alignment of the region surrounding the catalytic antibiotics outside their use in mycobacterial infection (12, 14–16). His residue from PEP synthase and structurally and mecha- Antibiotic resistance genes that do not normally confer re- nistically related enzymes identified a conserved motif T-X-X- sistance for their native host but are proficient at conferring G-G-X-X-X-H; the conserved His is required for formation of resistance when expressed in an alternate host are called silent a phosphoenzyme intermediate (Fig. S7). A similar motif was resistance genes (40). The majority of rph genes is not from found in the RPH family of enzymes fewer than 50 aa away actinomycetes and not associated with the RAE. Therefore, from the C terminus of the protein. An His-827-Ala mutant of these bacteria cannot respond to RIF and activate gene ex- RPH-Bc failed to confer RIF resistance in E. coli, and the en- pression accordingly. We speculate that these nonactinomycete zyme had no in vitro enzymatic activity (Fig. S7). Together, these rph genes are evolutionary related to rphs from actinomycetes results suggest that RPH is mechanistically related to PEP but have an alternate function in their native host, which is not synthase, likely using a phosphohistidine intermediate during associated with antibiotic resistance. Their ability to bind and catalysis, despite having architectural differences with the phosphorylate rifamycins is possibly caused by structural simi- metabolic enzyme. larities with their natural substrates. Silent resistance genes have, thus far, been largely underappreciated, likely because of their Discussion inability to confer an antibiotic resistance phenotype in their The genetic reservoirs of antibiotic resistance and their regula- native host. Recently, functional metagenomics have revealed tion are poorly understood. We and others have shown that numerous novel resistance genes, and continued research in this nonpathogenic environmental bacteria harbor an extensive and area is likely to uncover additional genes (41–43). ancient resistome that is the likely source of resistance elements Antibiotic resistance mediated by enzymes is of particular circulating in pathogens today (4–6, 9). The regulation of re- clinical concern because of their potential to become mobilized sistance genes by target antibiotics has been shown in only a few and distributed horizontally between diverse species of bacteria. cases (for example, the induction of the efflux protein TetA by Examples of this mobilization are prevalent for many families of tetracycline binding to TetR and the complex regulation of antibiotics, and this trend has begun to emerge with RIF-inac- β-lactam resistance by these antibiotics in Staphylococcus aureus) tivating enzymes. The Arr enzyme, which is responsible for the (38, 39). In this study, we characterize a new family of rifamycin inactivation of RIF by ADP ribosylation, was first described from resistance enzymes along with a conserved RIF-associated reg- the normally benign Mycobacterium smegmatis; however, the ulatory element, the RAE, which is shared among genes gene encoding this enzyme is now mobile and found on trans- encoding various RIF-inactivating enzymes from actinomycetes. missible plasmids and integrons carrying multiple antibiotic re- The RAE is composed of a 19-nt inverted repeat that is sistance determinants (24, 44, 45). These mobile elements are now

Spanogiannopoulos et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 found in a variety of pathogens. The dissemination of Arr and above. The helicase gene from WAC4747 was amplified using primers potentially other RIF-inactivating enzymes in the near future hel4747-part-F and hel4747-part-R. Conjugations were performed as pre- threatens the efficacy of the entire rifamycin family of antibiotics. viously described (23). A list of primers can be found in Table S4. This work has established that RPH enzymes do not dis- criminate between various rifamycins and effectively inactivate RIF Inactivation Assay. Fresh spores were used to inoculate 5 mL SIM media (5). natural product and semisynthetic derivatives of this family. Cultures were grown for 4 d at 30 °C with shaking at 250 rpm; 0.5 mL culture Understanding the molecular interactions between RPH and was then used to inoculate 4.5 mL fresh SIM containing RIF at 20 μg/mL and rifamycin substrates will provide the basis for generating rifa- represented the zero time point. At 48 h, conditioned media samples were mycin analogs that are not susceptible to these enzymes. The taken and analyzed for residual RIF activity using the Kirby–Bauer disk dif- possibility of resistance-proof rifamycins has been exemplified fusion assay with Bacillus subtilis as the RIF-sensitive indicator microorgan- with the recent development of C25 carbamate rifamycin deriv- ism. Additionally, supernatant samples were extracted with an equal volume atives that retain activity against Arr-containing bacteria (46). of methanol, clarified by centrifugation, and analyzed using a Waters e2695 HPLC system equipped with an XSelect CSH C18 5 μM column (4.6 × 100 mm) Materials and Methods with the following method: linear gradient of 37–55% (vol/vol) acetonitrile Additional details are available in SI Text. in water with 0.05% TFA at 1 mL/min over 12 min.

Inactivation of the rph Gene from WAC4747 and S. sviceus. Disruption of the ACKNOWLEDGMENTS. We thank Andrew G. McArthur for assistance with rph gene was accomplished by insertional inactivation using a previously the RPH phylogenetic analysis and Christine King for help with sequencing of the WAC4747 genome. We thank Michael Fischbach for providing Streptomyces described strategy (23). A partial fragment (∼1,000 bp) of the rph4747 gene sviceus ATCC 20983 and Lori Burrows for the gift of Listeria monocytogenes from WAC4747 was amplified by PCR using primers rph4747-part-F and str. 4b. F2365. We also thank Georgina Cox and Grace Yim for helpful rph4747-part-R and cloned into pSUIC (a suicide version of pSET152 that discussions. This research was funded by Canadian Institutes of Health lacks attP and int). The rph-Ss gene from S. sviceus ATCC 29083 was ampli- Research Grant MT-13536 and a Canada Research Chair in Antibiotic fied using primers rph-Ss-part-F and rph-Ss-part-R and cloned as described Biochemistry (G.D.W.).

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