Kudrin Supplemental Material - Antimicrobial Agents and...

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    SupplementalMaterialSub-inhibitory concentrations of bacteriostatic antibiotics induce relA-dependentandrelA-independenttoleranceto-lactamsPavel Kudrin1,, Vallo Varik1,2,3, Sofia Raquel Alves Oliveira1, JelenaBeljantseva1, Teresa Del Peso Santos2,3, Ievgen Dzhygyr2,3, Dominik Rejman4,FelipeCava2,3,TanelTenson1,VasiliHauryliuk1,2,3,*University of Tartu, Institute of Technology, Tartu, Estonia1; Department ofMolecularBiology,UmeUniversity,Ume, Sweden2; Laboratory forMolecularInfectionMedicineSweden (MIMS),UmeUniversity,Ume,Sweden3; Instituteof Organic Chemistry and Biochemistry, Czech Academy of Sciences v.v.i.,Flemingovonm.2,16610Prague6,CzechRepublic4Runninghead:relA-dependentandrelA-independent-lactamtolerance*addresscorrespondencetoVasiliHauryliuk,vasili.hauryliuk@umu.seP.K.andV.V.contributedequallytothiswork

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    SupplementaryMethodsPreparationofthiostreptonstocksolutionsSeveral crystals of thiostrepton (Tocris Bioscience, validated by massspectrometry) dissolved in 300 l of 100% DMSO (Sigma Aldrich) and theconcentration(600mM)ismeasuredbyabsorbanceat280nmwith=0.027M-1cm-1. DMSO stock is then used to prepare the working stock in 1xHepes:polymixbuffer(25mMHepes-KOHpH7.5,15mMMgCl2,0.5mMCaCl,95mMKCl,5mMNH4Cl,8mMputrescine,1mMspermidine,5mMK3PO4pH7.3and 1 mM DTT (1)) supplemented 0.1% Pluronic F-127 (Sigma Aldrich) asfollows.ThiostreptonstockinDMSOwasmixedwith20%(v/w)PluronicF-127inDMSO and 1xHepes:polymix bufferwas gradually added to final volume toachievethefinalconcentrationof12.5M.ThefinalDMSOconcentrationshouldbekeptbelow3%andallthepreparationsshouldbedoneatroomtemperaturetoavoidprecipitation.Workingstockwasbrieflycentrifuged(21,000rcf,5min)atroomtemperatureinordertoremoveanytracesofprecipitatedthiostrepton,supernatant transferred into a new tube and the final concentration ofthiostreptonre-measuredusingabsorbanceat280nmusing1xHepes:polymixsupplementedwith3%DMSOand0.1%PluronicF-127asablankreference.PreparationofppGppppGpp was produced enzymatically using either RelSeq enzyme fromStreptococcus equisimilis (2) or RelQ enzyme from Enterococcus faecalis (3)essentiallyasdescribedinMecholdetal.(4).Reactionmixturecontaining5mMGDP,10mMATP,15mMMgCl2,30mMTris-HCl(pH8.0),100mMNaClinMilliQH2Owaspreincubatedfor5minat37Cfollowedbytheadditionof50MRelSeqor5MRelQ.SinceRelSeq isstrongly inhibitedby freeMg2+ ions, therefore thetotalnucleotideconcentration(GDP+ATP)shouldbeequaltothatofMgCl2;inthecaseofusingRelQfreeMg2+isnotaconcernsinceduetoRelQsinsensitivity.After 2 hours of incubation at 37 C with constant shaking, nucleotides wereextracted by the addition of acidic phenol (pH 4.5) (Amresco); directly afterphenol addition to the reaction (1:1), the mixture was subjected to vigorousvortexingfollowedbycentrifugation(16krcf,4C,30min)inordertoseparatethephases.To completely remove the tracesofphenol this stepwas repeated.UpperphasewascollectedandappliedontoMonoQTM5/50GL(GEHealthcare)columnonequilibratedinbufferA(2.5mMTris-HCl(pH8.0),0.5mMEDTAand0.5 mM LiCl in MilliQ H2O). Nucleotides were separated by gradient elutionincreasing concentration of LiCl up to 2M.Wehave collected the ppGpppeakthatelutsat250mMLiCl,andthenucelotidewasprecipitatedbytheadditionof1MLiCl(f.c.)and3volumesofcold96%EtOHfollowedincubationeitherat-20 C (overnight) or at -80 C (2 hours). The precipitate was vortexed,transferred to centrifugation tubes and pelleted at 16k rcf, 4 C for 30 min.Supernatantwasdiscarded,thepelletwashedwith-20C70%EtOHinordertoremove traces of LiCl and the pellet (ppGpp) dissolved in MilliQ H2O. TheconcentrationofppGppwasdeterminedwithextinctioncoefficientEa=13,600M-1cm-1at252nm.

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    PreparationofRelAandEF-GC-terminally6His-taggedRelA(5)wasoverexpressedinE.coliBL21(DE3)frompET28a plasmid; a colony from overnight grown on LB/Kanamycin agar platewasinoculatedinto3mlofLBmediaandgrownshakingoverdayat37Cinthepresenceof25g/mlKntoOD600=0.5.Afterthattheculturewastransferredinto200mloffreshpre-warmed2xYTmedia(with25g/mlKn)andgrownshakingat37CtoOD600=0.5followedbytheinductionofRelAoverexpressionbyadding1mMIPTG(Sigma).Straightlyafterinductionthetemperaturewasdecreasedto30Candbacteriawas left togrowfor1.5hours.Afterwards thebacterialcellmass was collected by centrifugation. For the lysis step bacteria wereresuspended inabuffer, consistingof25mMTris (pH7.5),2mMMgCl2and1mM2-mercaptoethanol (ME)with the addition of 100 M PMSF and 1 U/mlDNase I (Thermo Scientific). Cells were lysed with Stansted SPCH-10homogenizer and the lysate centrifugedwith following application for Ni-NTAaffinitychromatographyinGEHealthcareHisTrapHPcolumnonktaPrimePlus(GEHealthcare).Therunningbufferconsistedof25mMTris(pH7.5-8),1MKCl,350mMNaCl, 2mMMgCl2, 5mM imidazole, 1mM MEwhile in the elutionbuffer the concentration of imidazole was increased to 300 mM. RelA-correspondent peakwas collected and the protein concentrated using AmiconUltra-15 (Millipore) centrifugal filter devices with a 50 kDa cut-off. The finalpreparations were aliquoted by 15 l, shock-frozen with liquid nitrogen andstoredat-80 C inbuffer containing25mMHEPES-KOH,pH7.5,0.7MKCl, 2mMDTT,15mMMgCl2and10%glycerol.N-terminally6His-taggedEF-Gproteinwasoverexpressed inE.coliBL21(DE3)from pQE30 plasmid provided by J. Remme (6). The overnight-grown culturewas transferred into 200ml of fresh 2x YTmedia (with 100 g/mlAmp) andgrown shaking at 37 C to OD600=0.5 followed by the induction of EF-Goverexpressionbyadding1mMIPTG(Sigma).After the induction thebacteriawere left to grow shaking for 2more hours at 37 Cwith the cellmass beingcollectedby centrifugation in the end.Cellswere lysedwith StanstedSPCH-10homogenizerinbufferA(50mMTris-HClpH7.5,10mMMgCl2,200mMPMSFandDNaseI)andclarifiedbycentrifugationat40krpminTi50rotorfor40min(Beckman&Coulter).ClarifiedlysatewasloadedonHisTrapHPNi-NTAaffinitychromatography column (GE Healthcare) and the protein was eluted by agradient of buffer B (A supplemented with 300 mM imidazole). EF-G wasconcentratedandstored-80Cinbuffercontaining25mMHEPES-KOH,pH7.5,0.7MKCl,2mMDTT,15mMMgCl2and10%glycerol.fMet-tRNAfMetpurificationonBDsepharoseion-exchangecolumnPreparation was done essentially as described in (7). The starting material,deacylatedE.colitRNAifMetwaspurchasedfromChemblock.Theaminoacylationand formylation reactionmixture containing 100 Mdeacylated tRNAifMet, 300M 3H-methionine (Perkin Elmer), 1 M E.coli MetRS, 1 M E.coli formylase(FMT),1mMTHF,2mMATP,3mMPEP,0.05g/lphosphokinase,0.01g/lyeastpyrophosphatase (100 U/mg, Sigma-Aldrich), 0.002 g/l myokinase, 3 mM -mercaptoethanol,50mMHEPES,50mMKCland10mMMgCl2wasincubatedat

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    37Cfor30minandthereactionwasstoppedbyadding10mLofcoldbufferA(20mMNaOAcpH5.1,1mMEDTA,5mMBME).100mlBDSepharosecolumnwas equilibrated with 5 CV of buffer A at room temperature. The reactionmixture diluted in 10 mL of buffer A was loaded at speed of 1 ml/min,equilibratedwith bufferA until the baseline, and 3H-fMet-tRNAifMetwas elutedwitha two-stepgradientof thebufferB (B=A+1MNaCl):1.5CV0-50%(B)followed by 2.5 CV 50-100% (B). The fractions containing fMet-tRNAifMet peak(95%B) (identified by scintillation counting) was collected and precipitatedwith 3 volumes of EtOH (95%) and 300 mM KOAc at -20C overnight. Theprecipitatewascentrifugedat20000rpminTi45rotor(Backman&Coulter)for40min,thepelletdissolvedin4mlinKOAc5mM(pH5.1)andre-precipitatedagainwithKOAcandEtOHasstatedabovefor30minat-80C.Precipitatewascentrifugedat21,000rcffor15mininthebench-topcentrifuge.Thepelletwaswashedwith500l EtOH70%,dried at room tepmeratrue anddissolved in5mMKOAc(pH5.1)toyielda100Mstocksolution.Concentrationandcharginglevels of fMet-tRNAifMetwere determined spectrophotometrically (1 AU260 = 40ng/l RNA, Mw tRNA = 25 kDa) and by scintillation counting using 3H-methionineusedforchargingreactionasastandard.PreparationofribosomalinitiationcomplexesWild typeMRE600E.coli andA1067U (providedby J.Remme)70S ribosomeswere prepared as per (7). Model mRNA(MF) 5'-GGCAAGGAGGUAAAAAUGUUCAAA-3' was purchased from Sigma-Aldrich. Toformtheinitiationcomplexes,8M70Sribosomes,3.1MIF1,4.4MIF2,3.1M IF3, 12 M mRNA, 12 M [3H]fMet-tRNAfMet, 1 mM GTP were mixed inHepes:polymixbuffer(finalreactionvolume500l;2mMDTTand5mMMg2+).Themixturewasincubatedat37Cfor30minutes,chilledonicefor5minutes,Mg2+concentrationadjustedto15mMandthereactionmixturewasloadedon400 l sucrose cushion (1.1 M sucrose, 1x Hepes:polymix, 15 mM Mg2+) andcentrifuged at 45,000 rpm in TLS-55 rotor (Beckman & Coulter) for 2 hours,supernatantdiscardedandthepelletdissolvedin200lofpolymixbuffer(5mMMg2+), aliquoted, snap-frozen with liquid nitrogen and stored at -80 C.Ribosomal concentrationwas determined by diluting 2 l of IC 2000 times inwaterandmeasuringtheabsorbanceat260nm:1AU260=23nM70S.[3H]fMet-tRNAfMet concentrationwas determined by scintillation counting. IC occupancywascalculatedasratiobetweenfMet-tRNAifMetand70Sconcentrations.IsolationofpeptidoglycanandUPLCanalysisPeptidoglycan of E. coli was isolated as described previously (8), with minormodifications.E.colistrainsweregrowninMOPSmedia(9),supplementedwith0.4%glucoseand25g/mlof20aminoacids.600mlofmediawasinoculatedfrom1mlofovernightcultureandgrownat37CwithvigorousaerationuntilOD600 = 0.5, then the corresponding antibiotics were added for furtherincubation. Cells were harvested by 10 min at 5000 g at room temperature,washed with 10 ml PBS, harvested again and snap frozen in liquid nitrogen.Pelletswerere-suspendedin3mlofPBS,addedtoanequalvolumeof10%SDSinaboilingwaterbathandvigorouslystirredfor3h,thenstirredovernightat37

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    C.Theinsolublefraction(peptidoglycan)waspelletedat60000rpm,15min,20CusingaTL100BeckmanrotorinanOptimaMAX-TLultracentrifuge(BeckmanCoulter),andthenwashed3timeswithMQ-water.Samplesweredigestedfor1hat60CwithpronaseE(100g/ml)in10mMTris-HClpH7.5andNaCl0.06%(w/v), to remove Brauns lipoprotein. The reaction was heat-inactivated byaddingSDStoafinalconcentrationof1%(w/v)andboilingfor10mininawaterbath. SDS wa