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Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te / j p ro t

Low abundance of respiratory nitrate reductase is

essential for Escherichia coli in resistance toaminoglycoside and cephalosporin

Yan Ma1, Chang Guo1, Hui Li, Xuan-xian Peng⁎

Center for Proteomics, State Key Laboratory of Bio-Control, MOE Key Lab Aquat Food Safety, School of Life Sciences, Sun Yat-sen University,University City, Guangzhou 510006, People's Republic of China

A R T I C L E I N F O

⁎ Corresponding author at: School of Life ScienTel.: +86 13580548832.

E-mail addresses: pxuanx@sysu.edu.cn, w1 The first two authors have equally contri

1874-3919/$ – see front matter © 2013 Elseviehttp://dx.doi.org/10.1016/j.jprot.2013.05.019

A B S T R A C T

Article history:Received 27 September 2012Accepted 16 May 2013Available online 24 May 2013

In the present study, we have characterized low abundance of NarG and NarH, twocomponents of respiratory nitrate reductase (Nar), in streptomycin (SM)-, gentamicine(GEN)-, ceftazidime (CAZ)-, tetracycline (TET)- and nalidixic acid (NA)-resistant Escherichia colistrains using native/SDS-PAGE based proteomics. We validate the finding using Westernblotting and native/SDS-PAGE upon narG and narH deletion mutants. However, furtherfunctional evidence indicates that loss of narG and narH results in two types of growthbehaviors, higher and lower than control, in these antibiotic-resistant E. coli strains.Specifically, SM-, GEN- and CAZ-resistant bacteria grow faster, whereas NA- and TET-resistant E. coli strains grow slower. Our data indicate that low abundance of respiratory Nar isessential for E. coli in resistance to aminoglycoside and cephalosporin antibiotics. Meanwhile,the results show that differential mechanisms exist in different antibiotic-resistant bacteria.The reasonwhy the reversal growths are detected inNA- and TET-resistant E. coli strainswaitsinvestigation. Our findings serve to propose novel strategies for controlling of aminoglycoside-and cephalosporin-resistant E. coli strains through elevation of respiratory Nar activity.

Biological significanceOur data indicate that low abundance of respiratory Nar is essential for E. coli in resistance toaminoglycoside and cephalosporin antibiotics. Meanwhile, the results show that differentialmechanisms exist in different antibiotic-resistant bacteria. Our findings serve to proposenovel strategies for controlling of aminoglycoside- and cephalosporin-resistant E. coli strainsthrough elevation of respiratory Nar activity.

© 2013 Elsevier B.V. All rights reserved.

Keywords:Respiratory nitrate reductaseE. coliProteomicsAntibiotic resistanceNative-SDS/PAGEProtein–protein interactions

1. Introduction

The increasing incidence of antibiotic-resistant bacteria hasposed a serious threat to human health and aquaculture todayand thus has become a scientific issueworldwide [1,2]. A line ofevidence has indicated that four mechanisms contribute to the

ces, Sun Yat-sen Univers

angpeng@xmu.edu.cn (Xbuted.

r B.V. All rights reserved

bacterial resistance, including modification or hydrolysis ofenzymes, modification of drug targets, activation of effluxpump systems, and reduce of outer membrane permeability[3,4]. Despite these progresses over past sixty years, ourunderstanding of the resistant mechanisms is still incomplete,which should be one of the major causes resulting in

ity, University City, Guangzhou 510006, People's Republic of China.

.-X. Peng).

.

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antibiotic-resistant bacteria out of control. Thus, further inves-tigation of bacterial resistant strategies to antibiotics may benecessary for combating antibiotic-resistant bacteria.

The recently developed technologies are expected to facili-tate the study of bacterial antibiotic-resistant mechanisms. Forexample, protein–protein interactions, being important for themajority of biological functions including antibiotic resistance,may be potentiatedusinghighly throughput screeningmethodsof 2-D native PAGE (N-PAGE) and 2-D blue native PAGE(BN-PAGE) based proteomics [5–7]. The two methods havebeen proven especially useful for investigation of native proteincomplexes in the context of proteomics. Comparatively,N-PAGE based on proteomics may offer more information onlabile protein complexes containing metabolic enzymes thanBN-PAGE based proteomics [6,8]. Using the two approaches,researchers have investigatedmembrane and outermembraneprotein complexes in Escherichia coli and identified a series ofhomogeneous and heterogeneous complexes. The identifiedmembrane-related protein complexesmay contain cytoplasmicand periplasmic proteins since approximately 30–40% E. coliproteinsmay function in the membrane of the cell envelope, inwhich there are soluble proteins tethered to the inner and outermembranes through hydrophobic patches, lipid moieties, orcharge interactions or in membrane protein complexes [6–8].On the other hand, recent reports have indicated thatmetabolicenzymes are related to antibiotic resistance [9,10], and theregulation of metabolism elevates susceptibility of bacterialpersisters to antibiotics [11]. We have also shown that Na(+)NQR complex is essential for Vibrio alginolyticus in resistance tobalofloxacin using N-PAGE based proteomics coupled withfunctional validation [12]. We hypothesized that the compara-tive N-PAGE approach might provide metabolic-related proteincomplexes that contributed to antibiotic resistance for furtherfunctional investigation since many of metabolic enzymes aredetected in membrane protein fraction [13]. In the presentstudy, thus, N-PAGE based proteomics is used to identify ametabolic protein complex in membrane fraction of E. coli K12BW25113 and then the role of the complex in bacterial antibioticresistance is further investigated.

Herewepresent a comparative analysis ofmembraneproteincomplexes in E. coli responsible to five antibiotics, streptomycin(SM), gentamicine (GEN), ceftazidime (CAZ), tetracycline (TET)and nalidixic acid (NA). The five represent four classes ofantibiotics containing aminoglycoside (SM, GEN), β-lactam(CAZ), tetracyclines (TET) and fluoroquinolone (NA) that areused in clinic. We are interested in identifying NarG and NarH,two components of respiratory nitrate reductase (Nar) in line.Nar is membrane-bound heterotrimeric enzymes that catalyzethe reduction of nitrate, coupled to the generation of aproton-motive force across the cytoplasmic membrane duringanaerobic respiration. The protein complex is validated usingFar-Western blotting and the N-PAGE analysis of narG and narHmutants. We further reveal low abundance of NarG and NarH inSM-, GEN-, CAZ-, TET- and NA-resistant E. coli strains (SM-R,GEN-R, CAZ-R, TET-R and NA-R, respectively). At last, we reportthe functional investigation using narG- and narH-deletedmutants. Survival capabilities are higher in SM-R, GEN-R andCAZ-R, but lower in TET-R and NA-R compared with control.These findings indicate that decrease of Nar level elevatesbacterial resistance to SM, GEN, CAZ and susceptibility to NA,

TET. These results may have significant implications in under-standing bacterial antibiotic-resistant mechanisms based onmetabolic regulation due to the importance of the proteincomplex in bacterial energy metabolism.

2. Materials and methods

2.1. Bacterial strains and culture conditions

The bacterial strain, E. coliK12 BW25113, and itsmutants, ΔnarGand ΔnarH, used in the present study were kindly provided byNBRP (NIG, Japan): E. coli. The antimicrobial agents SM, GEN,CAZ, TET and NA were purchased from a commercial source(Shanghai SangonBiological Engineering Technology&ServicesCo. Ltd. China). SM-, GEN-, CAZ-, TET- and NA-resistant strains(SM-R, GEN-R, CAZ-R, TET-R and NA-R, respectively) wereselected by the use of ten sequential propagations in LBmedium with 1/2 MIC of these drugs of the original strain(control) as describedpreviously [14]. The resultingMICswere atleast four fold higher in SM-R, GEN-R, CAZ-R, TET-R and NA-Rthan control, indicating that they were antibiotic-resistantstrains. These bacteria were cultured overnight in LB mediumat 37 °C in a shaker bath as seed. Fresh overnight cultures wereinoculated into LBmedium. The bacteria were cultured at 37 °Cand grown to an OD600 nm of 0.6.

2.2. Extraction of membrane proteins

Extraction of E. coli membrane proteins was performed asdescribed previously [8]. In brief, bacterial cells were harvestedby centrifugation at 4000g for 15 min at 4 °C. Followed bywashing three times, the cells were resuspended in 5 mL of50 mM, pH 7.4 Tris–HCl buffer and then disrupted by intermit-tent ultrasonic treatment of 5.0 s once for a total of 40 min atintervals of 9.9 s on ice. Supernatants were collected bycentrifugation at 5000g for 20 min. The supernatants werefurther centrifuged at 100,000g for 1 h at 4 °C in a BeckmanCoulter L-100XP centrifuge using a SW41TiRotor. The collectingpellets were resuspended in 50 mM, pH 7.4 Tris–HCl buffer fordetermination of concentrations of the proteins using theBradford method. The membrane fraction was validated to befree of cytoplasmic proteins throughmeasurement of superox-ide dismutase (SOD) activity as described previously [13].

2.3. Two-dimensional native/SDS-PAGE

Two-dimensional native/SDS-PAGE was performed accordingto a procedure described previously [8]. In brief, the membraneprotein samples were diluted into 1:4 using buffer containing100 mM Tris–HCl, 50% glycerol, 0.2% bromophenol blue and0.5% Triton X-100 and kept at 4 °C overnight or 80 °C for lateruse. Triton X-100 was added since it is commonly used tosolubilize membrane proteins, especially inner membraneproteins. The samples were directly electrophoresed in a1 mm thick and 8% discontinuous native polyacrylamide slabgel without SDS and beta-mercaptoethanol at 4 °C withconstant voltage of 80 V for running gels until the tracking dyereached the bottom of the gel. After the electrophoreses, thegels were visualized by reverse staining with imidazole–zinc

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sulfate and the visualized gel strips containing protein bandswere excised from the native gels. The gel strips were soaked inequilibration buffer containing 0.06 M Tris–HCl, pH 6.8, 5%beta-mercaptoethanol (v/v), 10% glycerol and 2% SDS, on agentle shaker for 30 min and then switched to a second-dimensional SDS-PAGE. The second dimension SDS-PAGE wascarried out in 1.2 mm thick and 10% discontinued polyacryl-amide slab gels at room temperature with constant voltage of80 V 40 min and 120 V 2.5 h. Protein spots were visualized bycolloidal Coomassie brilliant blue (cCBB) staining method andthese gels were scanned in an AGFA white-light scanner at aresolution of 600 by 200 μm. The obtained raw images wereprocessed using the 2-D software Melanie 5.0. Followingbackground subtraction and spot detection, the gel patternswere matched to each other by visual comparison.

2.4. In-gel protein digestion and tandem massspectrometric analysis

In brief, protein spots separated by N-PAGE were finelyexcised and washed three times with 100 μL of 50 mMammonium bicarbonate/50% acetonitrile (ACN) for 30 min atambient temperature. After vacuum drying, gel pieces werereduced with fresh solution of 10 mM DTT in 100 mMammonium bicarbonate at 57 °C for 45 min, and subsequentlythe gel pieces were alkylated with 55 mM iodoacetamide in100 mM ammonium bicarbonate solution at room temperaturefor 45 min in the dark. After washing with 100 mM ammoniumbicarbonate and dehydrated with acetonitrile, the gelspieces were dried again. In-gel digestion was performed using12.5 ng/μL sequencing grade-iron-deprivation porcine trypsin(Promega, Madison, WI) in 25 mM ammonium bicarbonate at37 °C for 12–15 h. The peptidemixtureswere extracted from thegel pieces with 20 mM NH4HCO3/50% (v/v) acetonitrile(containing 5% formic acid). Finally, the extracts werevacuum-dried and redissolved in 0.1% trifluoroacetic acid inwater. Protein samples were sequenced by a fuzzy logicfeedback control system (Reflex III MALDI-TOF system, Bruker)equipped with delayed ion extraction. The sample solution(30–100 ppm) with equivalent matrix solution (alpha-cyano-4-hydroxycinnamic acid) was applied onto the MALDITOF-target using alpha cyano-4-hydroxycinnamic acid (HCCA)as a MALDI matrix for peptide mapping and was prepared forMALDI-TOF/MS analysis. MALDI-TOF spectra were calibratedusing trypsin autodigestion peptide signals and matrix ionsignals. FdoG and FdnG were further identified using MALDITOF/TOF. For MS/MS spectra, the 5 most abundant precursorions per sample were selected for subsequent fragmentationand 1000–1200 Da laser shots were accumulated per precursorion. The criterion for precursor selectionwas aminimumS/N of50. All MALDI analyses were performed by a fuzzy logicfeedback control system (Reflex III MALDI-TOF system, Bruker)equipped with delayed ion extraction. Both the MS and MS/MSdata were interpreted and processed by using Flexanalysis3.0 (Bruker Daltonics), and then the obtained MS and MS/MSspectra per spot were combined and submitted toMASCOT search engine (V2.3, Matrix Science, London, U.K.) byBiotools 3.1 (Bruker Daltonics) and searched with the followingparameters: the NCBI in SwissProt (http://www.matrixscience.com), one missed cleavage site, carbamidomethyl as fixed

modification of cysteine, oxidation of methionine as a variablemodification, and tolerance of 100 ppm for MS and 0.6 Da forMS/MS. Known contaminant ions (keratin) were excluded.

2.5. Bioinformatics analysis

The protein subcellular locations were determined by ProgramPSORTb version 2.0 (http://www.psort.org/psortb/) [15]. DAVID(http://david.abcc.ncifcrf.gov/list.jsp) was used to analyze pro-tein list for pathway enrichment [16]. Names of 119 proteinswere uploaded into the server. The set of identifier was officialgene symbol and the E. coli genome was selected as functionalannotation background. A total of 76 proteins were enriched inKEGG pathways of E. coli K-12 genome (Supplementary Table 1).Processes were selected based on P values smaller than 0.05.Seventeen enriched pathways with P value smaller than 0.05and their corresponding proteins were used to further mapping(Supplementary Table 2). We used Cytoscape [17] and its pluginNetwork Analyzer [18] to visual the cross-linkage betweenproteins and pathways. The layout was manually adjusted toachieve better visualization. Protein physiological interpreta-tion was obtained by search of molecular function in EcoCyc(http://ecocyc.org/). Complex physiological interpretation wasobtained by data integration tools in DAVID (http://david.abcc.ncifcrf.gov/list.jsp). In detail, proteins within a single complexwere uploaded into the server and used for molecular functionenrichment. The set of identifier was official gene symbol andthe E. coli genome was selected as functional annotationbackground. Enriched complexes were selected. When correla-tion of altered abundance complexes to antibiotic resistancewas investigated, GO terms with P value smaller than 0.05 wereselected. P values of the selected and unselected enriched GOmolecular function terms were transformed with log(1/p) − 1and into 0, respectively, and then visualized [19].

2.6. Gene cloning, protein purification and antiserumpreparing

Two pairs of primers for genes narG and narH were designedaccording to E. coli K12 genomic sequences. For narG, the senseprimer was 5′-ACGGGATCC ATGAGTAAATTCCTG-3′ and theantisense primer was 5′-TGCCTCGAG TTTCACATCGTCATAG-3′. For narH, the sense primer was 5′-GCGGAATTC AGCGTAAAATGAAAA-3′ and the antisense primer was 5′-ATACTCGAGCGATCATGGATGCGG-3′. Standard PCR and molecular biologyprotocolswere utilized to amplify the two genes using E. coliK12genomic DNA as a template. PCR products were obtained whenthese primers were separately used. The two PCR fragmentswere directionally cloned into plasmid pET-32a digested withthe same enzyme, and then expressed in E. coli BL21. Recom-binant plasmids were detected by restriction enzyme analysisand sequencing. Sequencing was carried out by BGI, Shenzhen,Guangzhou. The overnight cultures of E. coli BL21 harboringrecombinant plasmid were diluted 1:100 (V/V) in freshLuria Broth with ampicillin (100 μg/mL), then incubated at37 °C until the optical density at an OD 600 nm of 0.6. Theprotein expressions were induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (BBI, Colorado, USA) for 3 h at37 °C after the optimization of expression conditions. Bacterialcells were harvested by centrifugation, then washed and

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resuspended in buffer D (8 M urea, 0.1 M NaH2PO4, 10 mMTris–HCl, pH 8.0). The cell suspensions were disrupted by sonicationin an ice bath (300 W, 3 × 10 min) and centrifuged (7000g for15 min at 4 °C). The supernatants containing the recombinantproteins were subsequently purified by affinity chromatogra-phy on Ni-NTA Super flow resin (Qiagen) according to the

Fig. 1 – Investigation of respiratory nitrate reductase from membrmedium using N-PAGE. A, A representative N-PAGE map, showinof Nar complex were lined. C, Enlarged sixteen proteins out of thblotting. D, Pathway enrichment analysis with DAVID (http://davand the circle represents the node and the color of the nodes corrtransformed P value of significant enriched GO molecular functioP value < 0.05 was transformedwith the formula of Log(1/P) − 1. UE, Complexes containing altered increased abundance of proteinprotein(s).

manufacturer's instructions. Non-specific proteins werewashed by buffer E (8 M urea, 0.1 M NaH2PO4, 10 mM Tris–HCl,pH 6.3). Finally, the purified recombinant proteins were elutedwith buffer F (8 Murea, 0.1 MNaH2PO4, 10 mMTris–HCl, pH 5.9)and buffer G (8 Murea, 0.1 MNaH2PO4, 10 mMTris–HCl, pH 4.5).Concentration of proteins was determined by the Bradford

ane protein fraction of E. coli K12 BW25113 cultured under LBg approximately 100 cleared protein spots. B, NarG and NarHe 100 protein spots were validated using 2-DE Westernid.abcc.ncifcrf.gov/list.jsp). Box indicates enriched pathways,esponding to the number of connection. E and F, Heatmap ofn terms with the package “gplots” in the R platform.nsupervised hierarchical clustering was applied to GO terms.(s), F, Complexes containing altered decreased abundance of

Fig. 1 (continued).

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method. The purified recombinant proteins were used forantiserum preparing (Guangzhou Chengxue Biotech. Corp.China).

2.7. Western blotting and far-Western blotting analyses

The two assays were performed as described previously [20].Rabbit antisera to NarG and NarH were used as the primaryantibodies and horseradish peroxidase (HRP)-conjugated goatanti-rabbit antibody was used as the secondary antibody(Guangzhou Chengxue Biotech. Corp. China). For 1-DE and2-DE Western blotting, the separated proteins from the 1-DEand 2-DE gels were transferred to nitrocellulose (NC) mem-branes using constant voltage of 70 V for 1 h in transfer buffer(48 mM Tris, 39 mM glycine, and 20% methanol) at 4 °C andwere stained with Ponceau S to evaluate the transferefficiency. The membranes were blocked overnight with 5%non-fat milk in Tris-buffered saline buffer containing 0.05%Tween-20 (TTBS) at 4 °C. After rinsing three times for 10 minwith TTBS buffer, the membranes were separately incubatedwith rabbit antibodies to NarG and NarH for 2 h on a gentle

shaker at room temperature. The membranes were rinsedagain, and then incubated with the secondary antibody for 2 hat the same conditions. The membranes were washed anddeveloped with dimethylaminoazobenzene (DAB) substratesystem until maximum color appearance. For Far-Westernblotting, the membranes used for the Western blotting werewashed with TTBS, and then incubated in blocking buffercontaining 2 mL of E. coli cell lysates overnight at 4 °C. Themembranes were incubated with the same primary andsecondary antibodies in the Western blotting assay and weredeveloped with DAB system as described above.

2.8. Investigation of antimicrobial susceptibility byminimal inhibitory concentration (MIC) and survivalcapability assays

MIC was performed according to National Committee forClinical Laboratory Standards. Survival capability assay wasperformed as described previously [21]. In brief, inoculums ofnarG-, narH-deleted mutants and control were separatelycultured overnight and diluted 1:1000 into 5 mL fresh LB

Fig. 2 – Validation of Nar complex in E. coli K12 BW25113.A, NarG and NarH interaction was identified by far-Westernblotting. 1, NarG and NarH location on the gel; 2, NarG wasdetected using anti-NarG; 3, the NCmembrane used in 2 wasincubated with E. coli K12 cell extraction and incubated withanti-NarG again. B, N-PAGE maps were obtained using narGand narH mutants, showing disappearance of NarG andNarH.

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medium with and without an antibiotic as tested group andcontrol, respectively, two tubes for each. Bacteria were culturedat 37 °C on a gentle shaker (150 rpm) and OD (600 nm) ofbacterial cultures was detected at 10 h. Survival capability wascharacterized by division of the OD of the cultures with theantibiotic over that of the cultures without the drug, and wastermed survival rates of bacterial strains cultured in mediumwith the drug. The experiment was repeated at least threetimes. Difference in survival rates was investigated with SPSSprogram.

3. Results

3.1. Separation of membrane proteins and detection of Narcomplex in E. coli K12 BW25113 membrane protein fractionusing N-PAGE based proteomics

N-PAGE was used to separate the membrane proteins of E. coliK12 BW25113. Reproducible N-PAGE gel maps were obtainedand good spot resolution was achieved when triplicateexperiments were carried out under identical conditions. Arepresentative map was shown in Fig. 1A. Approximately ahundred of clear protein spots were observed on each of the2-DE gels stainedwith cCBB R-250, most of whichwere locatedin the range of 25–120 kDa with minimal streaks. The streakscould be resulted from the diffusion of the same proteinsduring the first dimensional native-PAGE as reported previ-ously [6,8,22,23]. These well visualized protein spots wereexcised from 2-DE gels and analyzed by MALDI/TOF–TOFanalysis. A hundred of proteins were successfully identified(Supplementary Table 3). They represented ninety-fourunique proteins since five proteins had more than one spot.The five proteins included TnaA (spots 14, 37, 38), BamA(formerly YaeT, spots 11, 119), GlpD (spots 31, 32), TsF (spots61, 62), NuoG (spots 77, 91). In addition, two proteins, FdoGand FdnG, were identified in both spots 1 and 7. The twoproteins belonged to formate dehydrogenase system withhigh sequence similarity so that they were identified in eachof the two spots, but they could be identified by theirmolecular masses. The molecular masses of FdoG and FdnGare 113 kDa and 90 kDa, and thus spots 1 and 7 were identifiedas FdoG and FdnG, respectively, which were validated usingMALDI/TOF–MS/MS analysis (Supplementary Fig. 1). We inter-estingly found that the two components, NarG and NarH, ofNar complex were lined (Fig. 1B), which did not show in ourprevious N-PAGE to detection of E. coli DH5α membraneprotein fraction [8]. Nar complex includes four polypeptidesNarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5and 25.5 kDa, respectively [24]. NarJ and NarI did not appear inthe gel probably due to the low molecular weight. NarG andNarH were further validated in 2-DEWestern blotting (Fig. 1C).The complex is capable of reducing nitrate using normalphysiological substrates and is clearly the major respiratoryNar in E. coli since it accounts for 98% of the Nar activity whenfully induced [25]. The result provides a chance to investigatea role of the complex contributed into bacterial energymetabolism in bacterial antibiotic resistance. In addition,sixteen out of the 94 proteins were validated using Westernblotting (Fig. 1C) and these possible protein complexes andtheir identification criteria were grouped in SupplementaryTables 4, 5 and Supplementary Fig. 2. We identified thesubunits A and B of the BAM complex in two distinct proteincomplexes. The BAM complex consists of one outer mem-brane protein, BamA (formerly YaeT), and four lipoproteins,BamB, BamC, BamD and BamE (formerly YfgL, NlpB, YfiO andSmpA, respectively). The other three were not seen, whichmight be related to protein abundance, molecular weight andbinding force. Further pathway analysis was carried out andobtained 90 annotation out of the 119 proteins identified.Among the 90 annotated proteins, 76 were enriched in 16

Fig. 3 – Low abundance of Nar complex was detected in antibiotic-resistant bacteria. SM-R: streptomycin-resistant strain,GEN-R: gentamicine-resistant strain, CAZ-R: ceftazidime-resistant strain, TET-R: tetracycline-resistant strain, NA-R: nalidixicacid-resistant strain.

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pathways as shown in Fig. 1D. They are mainly involved incarbon metabolism, nitrogen metabolism, oxidative phos-phorylation. Furthermore, we investigated the relationshipsbetween protein complexes containing altered abundance ofprotein(s) and antibiotic resistance. Two and fourteen GOmolecular function terms were enriched in the complexescontaining higher and lower abundance of protein(s) thancontrol, respectively (Fig. 1E, F; Supplementary Table 6). Thetwo, 3-oxoacyl-[acyl-carrier-protein] synthase activity andfatty-acid synthase activity, were enriched only in CAZ-R,GEN-R and SM-R (Fig. 1E), whereas 4 iron, 4 sulfur clusterbinding, the most significant enriched molecular functionterm out of the fourteen, was determined in the fiveantibiotic-resistant bacteria (Fig. 1F). Nar complex belongs tothe molecular function term of 4 iron, 4 sulfur cluster binding.

3.2. Validation of Nar complex in E. coli K12 BW25113membrane proteins

Then, the Nar complex was validated using two assays. First,far-Western blotting was used to investigate the interaction ofNarH with NarG. Our results indicated that the bait proteinwas NarH when NarG was used as the prey protein (Fig. 2A).Then, the complex was further validated by N-PAGE analysisof narH and narG deletion mutants. This was based on thehypothesis that when one protein within a protein complex isabsent, the others may disappear from N-PAGE gel. Ourresults showed that loss of narG caused disappearance ofNarH, while deletion of narH resulted in loss of NarG in thesame line, meaning that NarG–NarH complex existed in theline (Fig. 2B). These results indicate that Nar complex detectedby the N-PAGE based proteomics is reliable, and thus may beused for investigation of antibiotic resistance.

3.3. Characterization of Nar complex in five antibiotic-resistant E. coli strains

In order to investigate whether abundance of Nar complexcomponents was altered to resist antibiotics, the N-PAGEbased proteomic approach was used to detect NarG and NarHlevels in SM-R, GEN-R, CAZ-R, TET-R and NA-R. Significantchanges were detected, showing that NarG and NarHdisappeared in the SM-R, CAZ-R, TET-R and decreased inGEN-R and NA-R (Fig. 3). These results not only indicate thatNar complex may play an important role in antibioticresistance, but also show that the role is related to the classesof antibiotics. In addition, we summarized the alteredabundance of proteins in Supplementary Table 7. They weremainly involved in oxidative phosphorylation and proteinsynthesis.

3.4. Functional characterization of narG and narH deletionmutants in response to five antibiotics

We next performed functional characterization of the Narcomplex proteins using narG or narH deletion mutants. Theperformance was carried out using two antimicrobial suscepti-bility assays: MIC assay and survival capability assay. Fig. 4A–Ewas a summary of MICs of ΔnarG and ΔnarH. Deletion of narG ornarH resulted in distinct elevation of MICs in medium with SMorGEN, and two-fold decrease ofMICs inmediumwithTET, andno significant changes in medium with CAZ or NA (Fig. 4A–E).The survival capability assay was also carried out to furtherinvestigate functional characterization of themutants due thatthe assay is more sensitive than MIC assay [20,26]. Thesebacteria including ΔnarI and ΔnarJ were cultured in tubes withseries of two-fold dilution of SM, GEN, CAZ, TET and NA

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concentrations from 0.625 to 5 μg/mL, 0.1953–1.1719 μg/mL,0.0781–0.3125 μg/mL, 0.78125–3.25 μg/mL, and 1.25–7.5 μg/mL,respectively. The resulting cultures were assayed by measure-ment of optical density at 600 nm and the survival capabilitywas determined by division of OD values of the cultureswith anantibiotic over those without the drug. The survival rates of themutants cultured in medium with SM, GEN or CAZ weresignificantly increased (P < 0.01) in a dose-dependent manner.However, lower survival capabilities were detected in themutants cultured in medium with TET and NA (Fig. 4F–J).Generally, the results obtained from survival capability assaywere in consistence with those obtained from MIC assay. Insummary, the results obtained from functional characteriza-tion of ΔnarG and ΔnarH were consistent with the changes of

Fig. 4 – Histogram of MIC assay by broth microdilution method anusing narG and narH deletion mutants and control E. coli K12 BWfor survival capabilities using narG, narH deletion mutants and c(I), NA (J). * P < 0.05, ** P < 0.01.

NarGandNarHat the protein abundancewhichwasdetectedby2-DE analysis of these antibiotic-resistant bacterial strains.

4. Discussion

N-PAGE based proteomics permits the study of membrane-liable protein complexes in conditions formerly inaccessibleto previous techniques [6,8,12]. Different from our previousreport, which constructed a map of E. coli DH5α membraneprotein fraction [8], the present study, which described E. coliK12 BW25113membrane protein complexes, displays NarG andNarH, two components of Nar complex that were not detectedin the previous report, on the gels. We were interested in the

d testing for survival capability. A–E, Histogram of MIC assay25113 for SM (A), GEN (B), CAZ(C), TET (D), NA (E). F–J, Testingontrol E. coli K12 BW25113 for SM (F), GEN (G), CAZ(H), TET

Fig. 4 (continued).

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complex due to its role in bacterial energy metabolism [24].Furthermore, we revealed that the low abundance of the twoproteins became a characteristic feature in five antibiotic-resistant E. coli strains. Thus, Nar complex may play a crucialrole in antibiotic resistance and shows antibiotic-related-dependent characteristics.

Nar complex is a membrane-bound and multi-subunitoxidoreductase comprising a large catalytic subunit (NarG), anelectron-transfer subunit (NarH) and a membrane-anchoringsubunit (NarI) as well as a nitrate reductase chaperone(NarJ), which is not a part of the active nitrate reductase[24]. It catalyzes the following reaction: ferrocytochrome +nitrate Y ferricytochrome + nitrite in E. coli. An electron trans-port scheme from formate to nitrate and including possiblequinone participation has been reported. NarGHI is cytoplas-mically oriented [27]. The function, activity, location andbiological characteristics of the enzyme complex have beenwidely investigated in E. coli [24,28,29], but information regardingto association with antibiotic resistance is not available. In thepresent study, we have demonstrated that the deletion of narGor narH results in higher survival capabilities in exposure to SM,GEN and CAZ compared with the control. MIC assay furthervalidates the critical role of the two proteins in SM and GENexposure. We have no proof why the loss of narG or narH leadsto lower survival capabilities when the mutants exposed to NAand TET, but the present study demonstrates that low

abundance of Nar complex may be a critical strategy for E. colito resist aminoglycoside antibiotics andCAZ. Allison et al. showthat specific metabolic stimuli enable the killing of E. colipersisters with aminoglycosides by promoting generation of aproton-motive force which facilitates aminoglycoside uptake[11]. Nar complex contributes to the generation of proton-motive force. Our results show the low abundance of Narcomplex in SM-R and GEN-R. The low abundance of Narcomplex may decrease the generation of proton-motive forceand thus limits the uptake of aminoglycoside, which results inthe elevation of bacterial resistance to aminoglycoside antibi-otics. Thus, the present study indicates that decrease of theproton-motive force generation is not only a resistant mecha-nism in the persisters [11], but also an important strategy inaminoglycosides- and CAZ-resistant E. coli. Our results furtherhighlight the importance of the metabolic environment toantibiotic treatment.

Interestingly, the mutants grew faster in the medium withaminoglycosides and CAZ and lower in the medium with NAandCTC,whichmay suggest that the loss of the genes results inthe limitation of NA and CTC uptake. The results indicate thatdifferential regulation mechanisms to different classes ofantibiotics are related toNar complex. Allison et al. also indicatethat the specific metabolic stimuli enable the killing of E. colipersisters with aminoglycosides by the generation of aproton-motive force that is determinedonly in aminoglycosides

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but not other antibiotics [11]. Our results demonstrate that lowabundance of Nar complex promotes bacterial resistance toboth aminoglycosides and CAZ, but further investigation isrequired to answer why the loss of the complex elevates thebacterial susceptibility to NA and TET.

5. Conclusion

N-PAGE based proteomic approach was used to investigateE. coliK12 BW25113membrane protein complexes and resultedin the detection of NarG and NarH, two main components ofNar complex. The decreased abundance of the two proteinswas characterized in SM-R, GEN-R, CAZ-R, TET-R and NA-R.Functional tests using narG- and narH-deleted mutants dem-onstrated distinctly elevated resistance to aminoglycosidesand CAZ. It is documented that low abundance of Nar complexis required for E. coli resistance to aminoglycosides and CAZ,which is the first report on E. coli Nar metabolism which altersbacterial resistance to antibiotics. This finding can serve topropose novel strategies for treating infections caused byaminoglycoside- and CAZ-resistant E. coli through activationof Nar activity.

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.jprot.2013.05.019.

Acknowledgments

This work was sponsored by grants from the NSFC projects(30972279, 40976080), Guangdong Provincial Science andTechnology projects (2012A031100004) and Doctoral Fund ofMinistry of Education of China (100171110029).

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