Published Ahead of Print 14 January 2013. 10.1128/AAC.01998-12.

2013, 57(4):1603. DOI:Antimicrob. Agents Chemother. Séamus Fanning and Thamarai SchneidersShyamasree De Majumdar, Mark Veleba, Sarah Finn, pneumoniaeResistance Regulator RarA in Klebsiella Elucidating the Regulon of Multidrug

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Elucidating the Regulon of Multidrug Resistance Regulator RarA inKlebsiella pneumoniae

Shyamasree De Majumdar,a Mark Veleba,a Sarah Finn,b Séamus Fanning,b Thamarai Schneidersa

Centre for Infection and Immunity, Queen’s University Belfast, Belfast, United Kingdoma; UCD Centre for Molecular Innovation and Drug Design, School of Public Health,Physiotherapy & Population Science, University College Dublin, Dublin, Irelandb

RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant(MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also beenshown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypicrole in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken usingstrains Ecl8�rarA/pACrarA-2 (rarA-expressing construct) and Ecl8�rarA/pACYC184 (vector-only control) using bespoke mi-croarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) andKp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregu-lated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affectedgenes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding thepreviously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associ-ated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found tobe downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overex-presser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal com-pound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), mem-brane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor(sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phe-notype of K. pneumoniae.

The AraC/XylS family consists of transcriptional regulatorsinvolved in a myriad of cellular functions (1), where a sub-

set of AraC regulators, typified by the MarA/SoxS and RamAproteins, confer a multidrug resistance (MDR) phenotype viathe upregulation of the AcrAB efflux pump and downregula-tion of the OmpF porin (2–4). We recently characterized anovel AraC-type protein, RarA, (Fig. 1), which encodes a reg-ulator within the genomes of Klebsiella pneumoniae, Enterobac-ter sp. 638, Serratia proteamaculans 568, and Enterobacter clo-acae subsp. cloacae (5). When overexpressed, rarA confers anMDR phenotype which includes various unrelated classes ofantibiotics (chloramphenicol, ciprofloxacin, norfloxacin,olaquindox, tetracycline, and tigecycline). Functionally, thisrarA-associated MDR phenotype is independent of otherAraC-type regulators (MarA, RamA, SoxS, and Rob) and re-quires the presence of a functional AcrAB efflux pump (5).RarA also appears to play a role in mediating tigecycline resis-tance in Ecl8�ramA, which exhibits increased rarA transcrip-tion with concurrent increases in the expression of the effluxpump oqxAB (6). While it is clear that rarA overexpressionresults in the MDR phenotype via the increased expression ofboth the AcrAB and OqxAB efflux pumps, its wider role in thegene regulation of K. pneumoniae is not clear. Microarray stud-ies of homologous proteins, such as MarA, SoxS, and RamA,have shown that these regulators control a multitude of genesassociated with bacterial cellular metabolism and virulence (1,7, 8). As such, we hypothesized a similar role for RarA in K.pneumoniae.

Our initial work (5) demonstrated that RarA regulates the ex-pression of both acrAB and oqxAB, which suggests that the sub-

strate range of compounds due to rarA overexpression might bebroader than previously thought. Therefore, we performed phe-notype microarray (PM) experiments (Biolog) to ascertain thesubstrate range of Ecl8�rarA/pACrarA-2 compared to that ofEcl8�rarA/pACYC184 (9, 10). In this work, we describe the RarAregulon and the compounds that are affected as a result of rarAoverexpression.

MATERIALS AND METHODS

Genetic manipulation. A chromosomal deletion strategy adapted fromthe work of Merlin et al. (11) and as described in reference 5 was used tocreate the genetic deletion for the rarA (K. pneumoniae Ecl8�rarA)gene, from the parental strain K. pneumoniae Ecl8. The recombinantplasmid (pACrarA-2, estimated at 10 to 12 copies/cell using quantita-tive PCR containing rarA, was constructed as described previously (5),where either the recombinant plasmid or vector only was electropo-rated into the strain K. pneumoniae Ecl8�rarA to create K. pneumoniaeEcl8 �rarA/pACrarA-2 or �rarA/pACYC184 (Table 1) (5). Of note,

Received 27 September 2012 Returned for modification 6 November 2012Accepted 7 January 2013

Published ahead of print 14 January 2013

Address correspondence to Thamarai Schneiders, t.schneiders@qub.ac.uk.

S.D.M. and M.V. contributed equally to this work.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01998-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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levels of rarA derived from pACrarA-2 are comparable to those ob-tained in clinical isolates (38-fold) (5).

Growth curve analyses. Overnight-grown bacterial cultures in LBmedium were diluted to an A595 of 0.005, and 10-ml aliquots were dis-pensed in 50-ml tubes and grown with shaking at 200 rpm at 37°C. Bac-teria were cultured untreated or treated (with 1% and 5% SDS or 60 �g/mlclioquinol). The numbers of bacterial CFU at defined time points wereestimated by plating serial dilutions in duplicate on LB agar.

RNA extraction. (i) For microarray. Overnight cultures of strainsEcl8�rarA/pACrarA-2 and Ecl8�rarA/pACYC184 were inoculated (1/100 dilution) into LB medium and incubated at 37°C with vigorous shak-ing. Cell pellets were harvested at an optical density at 600 nm (OD600) of0.6, and RNA was extracted using the RNAeasy extraction kit (Qiagen,Hilden, Germany). All extractions were performed as biological tripli-cates. RNA was measured with a Bioanalyzer 2100 instrument (Agilent) toconfirm an RNA integrity level above 9.

(ii) For qRT-PCR. RNA for quantitative real-time reverse transcrip-tion-PCR (qRT-PCR) experiments was extracted from K. pneumoniaestrains Ecl8�rarA/pACrarA-2 and Ecl8�rarA/pACYC184 (Table 1) usingthe TRIzol extraction method (12). Briefly, cells were grown to mid-logphase (OD600 � 0.6) at 37°C with shaking and then harvested by centrif-ugation at 3,000 rpm at 4°C. The cell pellet was then resuspended inTRIzol reagent (Invitrogen, Paisley, United Kingdom) and chloroformprior to centrifugation to separate the phases. The upper phase was thenprecipitated using 3 M sodium acetate, glycogen (5 mg/ml), and 100%ethanol. The resulting pellet was washed and resuspended in 50 �l diethylpyrocarbonate (DEPC)-treated water. RNA was treated with TurboDNase to remove DNA contamination (Ambion, New York, NY).

Microarray. RNA was reverse transcribed into cDNA prior to Cy3labeling and hybridized to bespoke expression array platforms (Mycroar-ray, Ann Arbor, MI) containing probes from the K. pneumoniae MGH78578 (NC_009648.1) and Kp342 (NC_011283.1) genomes. The slideswere scanned using an Axon Genepix 4000B scanner (Molecular Devices),and data were extracted. Quantile normalization was done using theGeneSpring GX software program (Agilent, United Kingdom). Normal-ized signal intensity of the test strain was compared with that of the con-trol strain for each gene among 3 biological replicates. Pairwise compar-isons of Ecl8�rarA/pACrarA-2 versus Ecl8�rarA/pACYC184 wereperformed to generate lists of genes that were differentially expressed.Genes with a log2-fold change of �1.5 were considered to be upregulated,and those with that of �0.66 were considered to be downregulated, with aP value of �0.05.

qRT-PCR. To validate the microarray data, gene-specific primers weredesigned using the software program Primer3 (http://frodo.wi.mit.edu/)(see Table S1 in the supplemental material). cDNA was generated fromtotal RNA using the AffinityScript cDNA synthesis kit (Agilent, UnitedKingdom). qRT-PCR was performed using the Brilliant III Ultra-fastSYBR green kit (Agilent, United Kingdom) using the Stratagene Mx3005PPCR system and the Mx Pro software program. Expression levels of 16SrRNA genes were used to normalize gene expression for all samples, andrelative fold changes in expression were determined by using the vector-only control strain as the calibrator.

Biolog analyses. The phenotypic profile of Ecl8�rarA/pACrarA-2 wascompared to that of Ecl8�rarA/pACYC184 to determine growth differ-ences in various substrates using Omnilog PM11-20 phenotype microar-rays (PM) (Biolog, California) (10). Biolog PM tests are performed in96-well microplates containing different nutrients or inhibitors in whichcell respiration is measured with a redox indicator. The assay uses a tetra-zolium dye, where the reduction of this dye leads to irreversible colorchanges in the wells that can be quantified and monitored (9), with respi-ration serving as a surrogate measure for growth. The Biolog microarrayswere set up according to a protocol specified previously (10). Briefly,bacteria were grown on LB agar overnight at 37°C. Colonies were pickedwith a sterile cotton swab and resuspended in 10 ml IF-0a medium(Biolog), and cell density was adjusted to an OD600 of 0.035 using a spec-trophotometer. An aliquot of 600 �l of this suspension was added to 120ml of IF-10 medium, and the 96-well microtiter plates were inoculatedwith 100 �l/well of this final suspension. The plates were incubated for 48h in the Omnilog incubator reader, and data were analyzed using theKinetic Plot and Parametric modules of the Omnilog Phenotype Microar-ray software suite.

Electrophoretic mobility shift assay. The oqxA and acrA promoterregions were amplified and subjected to electrophoretic mobility shift

FIG 1 Genomic organization of the rarA locus. The genomic organization of the rarA-oqxABR locus is shown. The numbering scheme is based on the firstnucleotide before the ATG of rarA as position 1. rarA encodes an AraC-type transcriptional regulator that has been shown to upregulate the RND efflux pumpoqxAB. The GntR-type regulator oqxR functions as a repressor of oqxAB. The transcriptional start site of rarA, determined by 5= rapid amplification of cDNA ends(RACE) analysis (5), is labeled “TSS” and shaded. The Shine-Dalgarno sequence is shown underlined, and putative �10, �35 promoter regions determinedthrough Softberry software analysis are shown boxed and labeled accordingly.

TABLE 1 Strains used in this study

Strain Genotypea Reference

K. pneumoniae Ecl8 K. pneumoniae wild-type strain 32K. pneumoniae Ecl8Mdr1 Spontaneous MDR mutant of Ecl8 4K. pneumoniae Kp342 K. pneumoniae isolated from maize 33K. pneumoniae MGH 78578 ATCC 700721 34K. pneumoniae Ecl8�rarA rarA (KPN_02968)-deleted strain

derived from Ecl8; Kanr

5

K. pneumoniaeEcl8�rarA/pACrarA-2

Ecl8 �rarA � pACrarA-2 (wt rarAcloned into pACYC184 (BamHIHindIII); Kanr Cmr

5

K. pneumoniaeEcl8�rarA/pACYC184

Ecl8 �rarA � pACYC184 (Cmr

Tetr)5

a wt, wild type.

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assays (EMSA) with the purified RarA protein. The open reading frame ofthe rarA gene was cloned into the pGEX6P vector using a BamHI site.Purified RarA was extracted from recombinant constructs containing therarA gene using metal chelation chromatography on nickel-nitrilotriace-tate superflow agarose (Qiagen, Crawley, United Kingdom). Briefly, end-labeled (using [�-32P]ATP; PerkinElmer, Boston, MA) PCR productswere incubated with 400 nM RarA in binding buffer (125 mM Tris-Cl, 250mM KCl, 5 mM dithiothreitol [DTT], 160 ng of salmon sperm DNA, and25% glycerol). The complexes were run on 5% native polyacrylamide gelelectrophoresis (PAGE) gels for 2.5 h. The gel was then dried and exposedto the phosphor screen for image analysis. To confirm that the interac-tions between RarA and the promoter regions were specific, competitionexperiments with bovine serum albumin (BSA) as a negative control andwith cold promoter were also performed.

In vitro transcription. Purified glutathione S-transferase (GST)-tagged RarA (GST-RarA) was used in single-round transcription reac-tions in vitro as described previously (13). The DNA templates, test andcontrol, used in each reaction consisted of the promoter region. All reac-tion mixtures comprised the following: X1 IVT reaction buffer (pH 7.8)(Escherichia coli RNA polymerase holoenzyme (�70; Epicentre), 40 nM;the RNase inhibitor SUPERase In (Ambion), 500 U/ml); and test DNA(oqxA and acrA) and control DNA (gnd promoter-containing DNA), eachat 2 nM. Open complexes were allowed to form at 37°C for 15 min, whena heparin-nucleotide mixture was added to initiate transcription; the finalconcentrations were as follows: heparin, 1.2 mg/ml; nucleotides, 0.3 mMUTP and 0.96 mM ATP, CTP, and GTP, with [�-32P]UTP at 0.5 �Ci/ml(PerkinElmer Life Sciences). After 5 min, an equal volume of Ambion gelloading buffer II was added to stop the reaction; the samples were boiledfor 3 min, chilled, and fractionated by electrophoresis on a 7% polyacryl-amide– 8 M urea gel in Tris-borate-EDTA (pH 8.3).

Microarray data accession number. Raw and fully annotated mi-croarray data have been deposited with ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) under the accession number E-MEXP_3742.

RESULTSRarA regulon. Since previous studies have shown that the in-creased expression of AraC-type regulators, such as MarA or SoxS,can confer deleterious effects on bacterial growth, we sought todetermine growth defects associated with increased rarA expres-sion by determining CFU/ml measurements for Ecl8�rarA/pACrarA-2 and Ecl8�rarA/pACYC184. Our analyses show thatthe survival rates of both Ecl8 �rarA/pACrarA-2 and Ecl8�rarA/pACYC184 were comparable regardless of rarA expression levels(Fig. 2). In reviewing previous work (3, 14), we found that thetranscriptome profiles of other similar proteins, such as MarA(3), SoxS (14), and RamA (8), were determined using compar-isons between plasmid-mediated overexpression of the regula-tor and the vector-only control (2, 18). As such, our microarraydata are a pairwise comparison of K. pneumoniae Ecl8�rarA/pACrarA-2 (rarA-overexpressing strain) and K. pneumoniaeEcl8�rarA/pACYC184 (vector-only control). Genes shown tobe affected by rarA overexpression after pairwise comparisonsbetween Ecl8�rarA/pACrarA-2 and Ecl8�rarA/pACYC184 aresummarized in Table 2. As a positive indicator of our experi-mental setup, we found that rarA was upregulated 2.3-fold forthe overexpresser (Ecl8�rarA/pACrarA-2) compared to resultsfor the vector-only control. Intriguingly, the levels of oqxAB(1.32-fold) and acrAB (0.73-fold) were found to be minimallyupregulated. However, since the fold change observed in thearray experiments for both oqxAB and acrAB did not meet ourcriterion for statistical significance (P � 0.05) and/or foldchange (�1.5-fold), it was not included in Table 2. Of note,qRT-PCR analyses show that the levels of acrAB and oqxAB areupregulated 1.44-fold and 2.8-fold, respectively, in the rarA-expressing strain (Ecl8�rarA/pACrarA-2), consistent with ourprevious findings (5).

Based on our statistical and fold change cutoff values, our arraydata show that 66 genes were significantly differentially tran-

FIG 2 Growth curves (CFU/ml) of Ecl8�rarA/pACrarA-2 and Ecl8�rarA/paCYC184 in various substrates. CFU/ml of Ecl8�rarA/pACrarA-2 andEcl8�rarA/paCYC184 when grown in the presence of 1% SDS (outlinedshapes) or 5% SDS (triangles), as well as LB (filled shapes), are shown. Secondgraph shows growth (CFU/ml) of Ecl8�rarA/pACrarA-2 and Ecl8�rarA/paCYC184 in the presence of clioquinol (60 �g/ml), a substrate used in thephenotype microarray.

TABLE 2 COG analyses of differentially expressed genesa

COG classification Genesb

Energy production and conversion yfaE, nuoA, nuoB, fdoI, narJTranslation, ribosomal structure, and

biogenesiscca, rsmC

Transcription asnC, malT, ytfHDNA replication, recombination, and

repairnth, recG

Inorganic ion transport and metabolism btuC, nirDCoenzyme metabolism pdxY, ilvGCell wall/membrane biogenesis yjeP, wecG, mltC, ompFCell division and chromosome

partitioningmrdB

Carbohydrate transport and metabolism rfaDNucleotide transport and metabolism nrdBAmino acid transport and metabolism sdaB, sdaC, proW, proXPosttranslational modification, protein

turnover, and chaperonesdnaJ, dnaK, htpG, hslJ, hslV,

fklB, ppiA

General function prediction only yjgB, yraL, yaaH, lolC,KPK_1850

Unknown ychQa Genes shown to be affected by rarA overexpression after pairwise comparisonsbetween Ecl8�rarA/pACrarA-2 and Ecl8�rarA/pACYC184.b Genes that are underlined are downregulated.

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scribed, of which 42 genes were upregulated and 24 were repressed(see Table S2 in the supplemental material). The differentiallyexpressed genes identified in our transcriptome experiment weredispersed throughout the K. pneumoniae MGH genome(NC_009648.1). Accordingly, COG (clusters of orthologousgroups of proteins) analyses (15) showed that the differentiallyexpressed genes are involved in a wide range of cell functions(Table 2). Nineteen of the sixty-six differentially expressed geneswere chosen for validation through quantitative real-time PCR(Fig. 3A and B) and were found to be upregulated in the rarAoverexpresser (Ecl8�rarA/pACrarA-2) relative to levels in the vec-tor-only control (Ecl8�rarA/pACYC184).

Most of the upregulated genes belong to the category of post-translational modification and cell envelope biogenesis groups(Table 2). When rarA is overexpressed, several genes within oper-ons associated with cell wall synthesis are also affected: specificallyseen was the differential expression of the serine deaminase(sdaCB; KPN_03139_03140) operon, which is involved in serinedeamination and transport and interferes with cell wall synthesis(16), and mltC (KPK_0714), which is located in the mltC-yggXoperon and is associated with cell wall recycling during cell divi-sion and elongation. Interestingly, yggX has been associated withsuperoxide stress in E. coli (17) and is regulated by SoxS. RarAoverexpression also increases the transcription of wecG (encodingputative UDP-N-acetyl-D-mannosaminuronic acid transferase)(KPN_04295), which when deleted has been associated with theattenuation of both K. pneumoniae (18) and E. coli (19). Takentogether, these results suggest that rarA upregulation affects theexpression of genes involved cell wall synthesis, maintenance,and possibly DNA replication. In order to associate the differ-ential regulation of these cell wall-associated operons, the

sdaCB and mltC-yggX operons, with a decrease in sensitivity todetergents, we performed growth curve analyses with 1 and 5%SDS. Thus, our results demonstrate that the rarA-overexpress-ing strain Ecl8�rarA/pACrarA-2 is less susceptible to SDS-in-duced damage than the control, Ecl8�rarA/pACYC184, consis-tent with the upregulation of the sdaCB and mltC-yggX operons(Fig. 2).

Our qRT-PCR analyses (Fig. 3A and B) also support the up-regulation of several transcriptional regulators and transporters:KPN_02232 (a putative SirB-type transcriptional regulator),KPN_01053 (a TetR-type regulator), KPN_03141 (an ABC typetransporter), KPK_3440 (transport of lipoproteins to the outermembrane), and KPN_01053 (transport of vitamin B12 into thecell). Since most of these genes have not been characterized, theirexact roles in the MDR phenotype are not clear. However,KPN_01969, which is upregulated in the presence of overex-pressed RarA, encodes the electron transport system subunitRsxE, which is part of the rsxABCDEG operon. Previous work hasshown that the rsxABCDEG operon reduces SoxR levels, which inturn lowers the induction of SoxS in the absence of oxidizingagents (20).

Downregulated genes were primarily involved in the categoriesof energy production and conversion, amino acid transport andmetabolism, and transcription (Table 2). The reduction of ompFtranscription supports the MDR phenotype exhibited by the rarA-overexpressing strain and is consistent with the mode of action ofthe other regulators, such as MarA, RamA, and SoxS (21–23). Thedownregulated genes include the proWX operon, encoding thehigh-affinity glycine betaine transport system, which is involvedin bacterial survival under osmotic stress, and yebG, a SOS regulonDNA damage-inducible protein.

Of note, our previous observations, first that the rarA-medi-ated phenotype requires the presence of a functional acrAB effluxpump (5) and second that oqxAB upregulation is linked to in-creased rarA levels (5), are not reflected in the array results. How-ever, the quantitative real-time PCR results from RNA derivedfrom both Ecl8�rarA/pACrarA-2 and Ecl8�rarA/pACYC184support our initial observations (5). In order to demonstrate thatboth the oqxAB and acrAB genes are directly regulated by RarA,both EMSA and transcription in vitro (IVT) experiments wereperformed. Our results show that RarA binds the promoter re-gions of both the oqxAB and acrAB operons (Fig. 4A and B). Ad-ditionally, transcription in vitro experiments also show that RarAactivates the expression of both acrAB and oqxAB (1.3-fold and2.4-fold, respectively) compared to results for the no-protein con-trol. Thus, we conclude from our experiments that RarA is anactivator of both acrAB and oqxAB.

Thus far, our results demonstrate that RarA affects the expres-sion of genes involved in various cellular roles. Like MarA, SoxS,Rob, and RamA, RarA impacts on the expression of acrAB andompF. However, the effect on acrAB levels has been modest despitethe rarA-associated MDR phenotype being entirely dependent onthe presence of a functional AcrAB efflux pump. Since the upregu-lation of two efflux pump genes, namely, acrAB and oqxAB, in-creases the substrate range of the rarA-associated phenotype, weperformed Biolog phenotype assays to determine the substraterange affected by RarA overexpression.

Biolog phenotype analyses. Biolog PM tests are performed in96-well microplates containing different nutrients or inhibitors inwhich cell respiration is measured with a redox indicator. The

FIG 3 Quantitative RT-PCR results showing log2-fold change in expres-sion of upregulated genes (A) or downregulated genes (B). All qRT-PCRexperiments were performed as outlined in Materials and Methods. Foldchange values were generated after normalizing to 16S levels for bothstrains and calibrating Ecl8�rarA/pACrarA-2 expression levels againstthose of Ecl8�rarA/pACYC184.

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assay uses a tetrazolium dye, where the reduction of this dye leadsto irreversible color changes in the wells that can be quantified andmonitored (9). As with the microarray, we tested K. pneumoniaeEcl8�rarA/pACrarA-2 (rarA-overexpressing strain) and K. pneu-

moniae Ecl8�rarA/pACYC184 (vector-only control), using thePM11-20 chemical sensitivity assay plates. The overexpression ofrarA resulted in enhanced growth in the presence of antimicrobi-als, such as beta-lactams (narrow-, expanded-, and broad-spec-trum cephalosporins and fluoroquinolones, such as ciprofloxacinand norfloxacin), fungicides (such as clioquinol), and disinfectants,such as 8-hydroxyquinoline (Table 3), a topical skin disinfectantfrom the antibacterial alkoxyquinolines (24–26). The increasedgrowth of Ecl8�rarA/pACrarA-2 compared to the vector-only con-trol Ecl8�rarA/pACYC184 in the presence of ciprofloxacin and nor-floxacin is consistent with previous work (5). However, the increasedgrowth of the rarA-overexpressing strain Ecl8�rarA/pACrarA-2 wasalso observed with compounds not previously associated with thesubstrate range of AcrAB (20) (Fig. 5). Specifically, Ecl8�rarA/pACrarA-2 (rarA overexpresser) exhibited growth in the presence ofthe benzophenanthridine alkaloid sanguinine, the fungicide clio-quinol, and the alkoxyquinolone, hydroxyquinoline, which also pos-sess antibacterial properties. In order to demonstrate that rarA over-expression results in enhanced growth in the presence of clioquinol,we performed sensitivity assays that support the enhanced growth ofEcl8�rarA/pACrarA-2 (50 �g/ml) in clioquinol compared to that ofthe vector-only control strain Ecl8�rarA/pACYC184 (data notshown).

DISCUSSION

RarA is a newly identified AraC-type regulator that is associatedwith the multidrug resistance phenotype which includes the gly-cycline tigecycline (5, 6). Given its role in conferring MDR, wesought to determine the transcriptome profile of a rarA-overex-pressing strain in comparison to that of the vector-only control.Our results demonstrate that rarA overexpression differentiallyaffects the expression of at least 66 genes: 42 upregulated and 24

FIG 4 EMSA and in vitro transcription using the purified RarA protein. Re-sults of EMSA (A) or IVT (B) experiments determining binding of RarA topromoter regions of the oqxA and acrA efflux pumps are shown. (A) Lanes 1and 4, 2 nM labeled DNA; lanes 2 and 5, 2 nM labeled DNA plus 400 nM RarA;lanes 3 and 6, 2 nM labeled DNA plus 200 nM cold DNA plus 400 nM RarA. C,DNA-protein complex; F, free DNA. (B) In panel i, the IVT figure showsdifferences in signal between promoter region DNA of each efflux pump (acrAand oqxA) without (-) or with (�) RarA present. Control and protein resultsare visible on the bottom row. In panel ii, the graph shows fold increases insignal of RarA-DNA complexes relative to that of no-protein controls.

TABLE 3 Substrates in which Ecl8�rarA/pACrarA-2 exhibits enhanced growth

Substrate type Function Substratea

Acriflavine DNA damage ProflavineAlkaloid Cytokinesis SanguinarineAlkaloid Imidazoline binding site agonist HarmaneCephalosporin, narrow spectrum Cell wall CephalothinCephalosporin, expanded spectrum Cell wall Cefoxitin, cefuroximeCephalosporin, broad spectrum Cell wall Cefoperazone, cefotaxime, ceftriaxone, moxalactamDiamide Thiol cross-linking agent DiamideFluoroquinolone DNA damage Ciprofloxacin, enoxacin, lomefloxacin, norfloxacin, ofloxacinLactam Cell wall Amoxicillin, azlocillin, carbenicillin, cloxacillin, penicillin G,

phenethicillin, piperacillinLincosamide Protein synthesis LincomycinMacrolide Protein synthesis JosamycinMinonucleoside Protein synthesis PuromycinNitrofuran DNA damage FuraltadonePhenothiazine Antihistamine PromethazinePolymyxin Membrane Polymyxin BQuinoline Disinfectant 8-Hydroxyquinoline, clioquinol, chloroxineQuinolone DNA damage Nalidixic acid, oxolinic acid, pipemidic acidSulfenamide Antifungal DichlofluanidTetracycline Protein synthesis MinocyclineOther Antibacterial Amitriptyline, cobalt chloride,

Chelator 1,10-Phenanthroline, 2,2=-dipyridylCholinergic antagonist PridinolDisinfectant AlexidineMembrane Dodine, domiphen bromide

a Novel substrates not previously associated with rarA overexpression are shown in bold.

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downregulated genes. The regulon size is comparable to that pre-viously reported for other AraC regulators, such as marA, soxS,and rob, which have been linked to a core regulon of around 40genes (27).

Our transcriptome profiling has indicated that several genesinvolved in a variety of cellular processes (Table 2) are up- ordownregulated as a result of rarA overexpression. Our array datasuggest that other previously uncharacterized transporters, suchas sdaCB and lolC (Fig. 3A; see Table S2A in the supplementalmaterial), are upregulated by increased rarA levels. Although ourprofiling experiment did not support the increased transcriptionof acrAB and oqxAB in the presence of RarA, subsequent EMSAand transcription in vitro experiments demonstrate that acrABand oqxAB are both directly activated by RarA. Like MarA, SoxS,Rob, and RamA, RarA affects multiple transport-related proteinsthat are or have not been directly associated with multidrug resis-tance, which supports the broad phenotypes that the increasedexpression of RarA-like proteins can confer. Since RarA-associ-ated expression is linked to increased levels of both acrAB andoqxAB efflux pumps, it suggests that the net effect of this com-bined expression might affect a broader substrate range of com-pounds. Accordingly, our Biolog experiments notably show sig-nificant differences between the rarA-complemented and vector-only control strains (Table 3) in the presence of antimicrobialcompounds, supporting the role of rarA in conferring reducedsusceptibility to multiple antibiotics (e.g., beta-lactams, cephalo-sporins, fluoroquinolones, quinolones, and macrolides) and

other previously reported antibacterial compounds, such as 8-hy-droxyquinoline, chloroxine, and clioquinol.

Interestingly, a recent study has shown that increased expres-sion of nitric oxide (NO) synthase results in decreased suscepti-bility to compounds such as clioquinol due to endogenous NO(28). Our array and qRT-PCR results demonstrate that increasedrarA expression results in the downregulation of both KPN_02212narJ (nitrate reductase) and KPK_3485 nirD (nitrite reductase),which are involved in the reduction of nitrate or nitrite to ammo-nia. Generally, nitrite metabolism can occur through either theassimilatory or dissimilatory nitrate reduction pathway, where thedissimilatory nitrate reduction includes two different processes:denitrification and dissimilatory nitrate reduction to ammonium.Denitrification is respiration in which nitrate or nitrite is reducedas a terminal electron acceptor under low oxygen or anoxic con-ditions. As a consequence, gaseous nitrogen compounds (N2, NO,and N2O) are produced (29). Thus, the increased growth of therarA overexpresser (Ecl8�rarA/pACrarA-2) in the presence of5-chloro-7-iodo-8-hydroxyquinoline (clioquinol) is highly sug-gestive of elevated production of endogenous NO. Further geneticstudies are ongoing to confirm this link to RarA.

Comparisons of regulons controlled by MarA and SoxS haveshown considerable overlap in the type of affected genes (30).Interestingly, a comparison of the RarA and MarA regulons doesnot show much redundancy. While this may be due to the com-parisons being drawn against the MarA regulon in E. coli as op-posed to K. pneumoniae, previous comparisons have shown that

FIG 5 Biolog phenotype data in the presence of antimicrobial compounds. This image, derived from Omnilog kinetic plot data, shows growth of rarAoverexpresser Ecl8�rarA/pACrarA-2 (gray) and vector-only control Ecl8�rarA/pACYC184 (black) over 48 h on a Biolog phenotype microarray plate. Biolog PMtests are performed in 96-well microplates containing different nutrients or inhibitors in which cell respiration is measured with a redox indicator. (a) Clioquinol(58 �g/ml); (b) norfloxacin (0.6 �g/ml); (c) 8-hydroxyquinoline (733 �g/ml); (d) chloroxine (28 �g/ml).

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genes such as fumC, sodA, and zwf are coregulated by the MarAand SoxS proteins from the E. coli (31) and Salmonella (14) sys-tems. In this case, only yfaE and acrAB were found to be com-monly upregulated with the MarA regulon described for E. coli(3). Similarly, for the downregulated genes, only ompF was foundto be commonly repressed. Therefore, we surmise that with theexception of a few genes, RarA has a regulon distinct from that ofMarA. There are several points that are yet to be addressed, how-ever: first, whether RarA recognizes the “marbox-like” bindingsequence, and second, which of these genes, with the exception ofacrAB and oqxAB, identified in this study are directly regulated byRarA. Given that RarA, like MarA, SoxS, and Rob, controls acrABand ompF, it is likely that it recognizes the “marbox-like” se-quence. Since clinically multidrug-resistant Klebsiella and Entero-bacter isolates have been associated with increased rarA transcrip-tion (5), it is essential that we determine the precise contributionof rarA activity to antimicrobial resistance. These studies demon-strate that RarA has a distinct regulon which contributes to themultidrug resistance phenotype in K. pneumoniae.

ACKNOWLEDGMENTS

This work was funded by MRC New Investigator Grant G0601199 and aPfizer grant (R5708CII) to T.S., studentship support for M.V. by the De-partment for Employment and Learning (Northern Ireland), and supportfor S.D.M. from a Pfizer grant (R5708CII).

REFERENCES1. Martin RG, Rosner JL. 2001. The AraC transcriptional activators. Curr.

Opin. Microbiol. 4:132–137.2. Alekshun MN, Levy SB. 1999. The mar regulon: multiple resistance to

antibiotics and other toxic chemicals. Trends Microbiol. 7:410 – 413.3. Barbosa TM, Levy SB. 2000. Differential expression of over 60 chromo-

somal genes in Escherichia coli by constitutive expression of MarA. J. Bac-teriol. 182:3467–3474.

4. George AM, Hall RM, Stokes HW. 1995. Multidrug resistance in Kleb-siella pneumoniae: a novel gene, ramA, confers a multidrug resistance phe-notype in Escherichia coli. Microbiology 141(Part 8):1909 –1920.

5. Veleba M, Higgins PG, Gonzalez G, Seifert H, Schneiders T. 2012.Characterization of RarA, a novel AraC family multidrug resistance regu-lator in Klebsiella pneumoniae. Antimicrob. Agents Chemother. 56:4450 –4458.

6. Veleba M, Schneiders T. 2012. Tigecycline resistance can occur indepen-dently of the ramA gene in Klebsiella pneumoniae. Antimicrob. AgentsChemother. 56:4466 – 4467.

7. Casaz P, Garrity-Ryan LK, McKenney D, Jackson C, Levy SB, TanakaSK, Alekshun MN. 2006. MarA, SoxS and Rob function as virulencefactors in an Escherichia coli murine model of ascending pyelonephritis.Microbiology 152:3643–3650.

8. Bailey AM, Ivens A, Kingsley R, Cottell JL, Wain J, Piddock LJ. 2010.RamA, a member of the AraC/XylS family, influences both virulence andefflux in Salmonella enterica serovar Typhimurium. J. Bacteriol. 192:1607–1616.

9. Bochner BR, Gadzinski P, Panomitros E. 2001. Phenotype microarraysfor high-throughput phenotypic testing and assay of gene function. Ge-nome Res. 11:1246 –1255.

10. O’Regan E, Quinn T, Pages JM, McCusker M, Piddock L, Fanning S.2009. Multiple regulatory pathways associated with high-level ciprofloxa-cin and multidrug resistance in Salmonella enterica serovar enteritidis:involvement of RamA and other global regulators. Antimicrob. AgentsChemother. 53:1080 –1087.

11. Merlin C, McAteer S, Masters M. 2002. Tools for characterization ofEscherichia coli genes of unknown function. J. Bacteriol. 184:4573– 4581.

12. Rosenblum R, Khan E, Gonzalez G, Hasan R, Schneiders T. 2011.Genetic regulation of the ramA locus and its expression in clinical isolatesof Klebsiella pneumoniae. Int. J. Antimicrob. Agents 38:39 – 45.

13. Schneiders T, Levy SB. 2006. MarA-mediated transcriptional repressionof the rob promoter. J. Biol. Chem. 281:10049 –10055.

14. Pomposiello PJ, Demple B. 2000. Identification of SoxS-regulated genesin Salmonella enterica serovar Typhimurium. J. Bacteriol. 182:23–29.

15. Tatusov RL, Galperin MY, Natale DA, Koonin EV. 2000. The COGdatabase: a tool for genome-scale analysis of protein functions and evolu-tion. Nucleic Acids Res. 28:33–36.

16. Zhang X, El-Hajj ZW, Newman E. 2010. Deficiency in L-serine deami-nase interferes with one-carbon metabolism and cell wall synthesis inEscherichia coli K-12. J. Bacteriol. 192:5515–5525.

17. Pomposiello PJ, Koutsolioutsou A, Carrasco D, Demple B. 2003.SoxRS-regulated expression and genetic analysis of the yggX gene of Esch-erichia coli. J. Bacteriol. 185:6624 – 6632.

18. Lawlor MS, Hsu J, Rick PD, Miller VL. 2005. Identification of Klebsiellapneumoniae virulence determinants using an intranasal infection model.Mol. Microbiol. 58:1054 –1073.

19. Ramos-Morales F, Prieto AI, Beuzon CR, Holden DW, Casadesus J.2003. Role for Salmonella enterica enterobacterial common antigen in bileresistance and virulence. J. Bacteriol. 185:5328 –5332.

20. Koo MS, Lee JH, Rah SY, Yeo WS, Lee JW, Lee KL, Koh YS, Kang SO,Roe JH. 2003. A reducing system of the superoxide sensor SoxR in Esch-erichia coli. EMBO J. 22:2614 –2622.

21. Cohen SP, McMurry LM, Levy SB. 1988. marA locus causes decreasedexpression of OmpF porin in multiple-antibiotic-resistant (Mar) mutantsof Escherichia coli. J. Bacteriol. 170:5416 –5422.

22. Zheng J, Tian F, Cui S, Song J, Zhao S, Brown EW, Meng J. 2011.Differential gene expression by RamA in ciprofloxacin-resistant Salmo-nella typhimurium. PLoS One 6:e22161. doi:10.1371/journal.pone.0022161.

23. Li Z, Demple B. 1994. SoxS, an activator of superoxide stress genes inEscherichia coli. Purification and interaction with DNA. J. Biol. Chem.269:18371–18377.

24. Chevalier J, Bredin J, Mahamoud A, Mallea M, Barbe J, Pages JM. 2004.Inhibitors of antibiotic efflux in resistant Enterobacter aerogenes and Kleb-siella pneumoniae strains. Antimicrob. Agents Chemother. 48:1043–1046.

25. Okide GB, Adikwu MU, Esimone CO. 2000. Antimicrobial activities ofsome amino derivatives of 5,7-dibromo-2-methyl-8-hydroxyquinoline.Biol. Pharm. Bull. 23:257–258.

26. Jeon JH, Lee CH, Lee HS. 2009. Antimicrobial activities of 2-methyl-8-hydroxyquinoline and its derivatives against human intestinal bacteria. J.Korean Soc. Appl. Biol. Chem. 52:202–205.

27. Wall ME, Markowitz DA, Rosner JL, Martin RG. 2009. Model of tran-scriptional activation by MarA in Escherichia coli. PLoS Comput. Biol.5:e1000614. doi:10.1371/journal.pcbi.1000614.

28. Gusarov I, Shatalin K, Starodubtseva M, Nudler E. 2009. Endogenousnitric oxide protects bacteria against a wide spectrum of antibiotics. Sci-ence 325:1380 –1384.

29. Ye RW, Averill BA, Tiedje JM. 1994. Denitrification: production andconsumption of nitric oxide. Appl. Environ. Microbiol. 60:1053–1058.

30. White DG, McDermott AMNPF. 2005. Frontiers in antimicrobial resis-tance. ASM Press, Washington, DC.

31. Martin RG, Gillette WK, Rhee S, Rosner JL. 1999. Structural require-ments for marbox function in transcriptional activation of mar/sox/robregulon promoters in Escherichia coli: sequence, orientation and spatialrelationship to the core promoter. Mol. Microbiol. 34:431– 441.

32. Forage RG, Lin EC. 1982. DHA system mediating aerobic and anaerobicdissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J. Bacteriol.151:591–599.

33. Fouts DE, Tyler HL, DeBoy RT, Daugherty S, Ren Q, Badger JH,Durkin AS, Huot H, Shrivastava S, Kothari S, Dodson RJ, MohamoudY, Khouri H, Roesch LF, Krogfelt KA, Struve C, Triplett EW, MetheBA. 2008. Complete genome sequence of the N2-fixing broad host rangeendophyte Klebsiella pneumoniae 342 and virulence predictions verified inmice. PLoS Genet. 4:e1000141. doi:10.1371/journal.pgen.1000141.

34. McClelland M, Florea L, Sanderson K, Clifton SW, Parkhill J, ChurcherC, Dougan G, Wilson RK, Miller W. 2000. Comparison of the Escherichiacoli K-12 genome with sampled genomes of a Klebsiella pneumoniae andthree Salmonella enterica serovars, Typhimurium, Typhi and Paratyphi.Nucleic Acids Res. 28:4974 – 4986.

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