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A bacterial model for studying effects of human mutations in vivo: Escherichia coli strains mimicking a common polymorphism in the human MTHFR gene

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A bacterial model for studying effects of human mutations in vivo: Escherichia coli strains mimicking a common polymorphism in the human MTHFR gene
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  Mutation Research 578 (2005) 175–186 A bacterial model for studying effects of human mutationsin vivo:  Escherichia coli  strains mimicking a commonpolymorphism in the human  MTHFR  gene Joanna Jak ´obkiewicz-Banecka a , Anna Kloska b , Magdalena Stepnowska b , 1 ,Bogdan Banecki c , Alicja W˛egrzyn a , Grzegorz W˛egrzyn b , ∗ a  Laboratory of Molecular Biology 2  , I nstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Kładki 24,80-822 Gda´ nsk, Poland  b  Department of Molecular Biology, University of Gda´ nsk, Kładki 24, 80-822 Gda´ nsk, Poland  c  Intercollegiate Faculty of Biotechnology, University of Gda´ nsk and Medical University of Gda´ nsk, Kładki 24, 80-822 Gda´ nsk, Poland  Received 1 September 2004; received in revised form 17 March 2005; accepted 13 May 2005Available online 14 June 2005 Abstract Asimplebacterialmodelforstudyingeffectsofhumanmutationsinvivo,whenhomologousgenesexistinbacterialandhumancells,ispresented.Wehaveconstructed  Escherichiacoli strainsbearingdifferentallelesofthe metF  gene,anortologueofhuman  MTHFR gene,codingfor5,10-methylenetetrahydrofolatereductase.Thesestrainsbearanullmutationinthechromosomal metF  gene and different  metF   alleles on plasmid(s), and thus there are merozygotes mimicking wild-type homozygotes, heterozygotesand recessive mutant homozygotes. The A177V mutantion in  metF   corresponds to one of the most common  MTHFR  polymor-phism,A222V,whichhasbeenshowntobeassociatedwithincreasedlevelsofhomocysteineinplasmathat,inturn,causesmanyserious medical problems. Results of relatively simple and quick experiments with these strains are compatible with previouslypublished reports on effects of the A222V substitution in the product of   MTHFR  gene. In addition, these results suggest eitherimpairment of formation of heterodimers and/or heterotetramers by wild-type and A177V  metF   variants or dominance of thewild-type polypepides in such structures. Moreover, positive effects of folic acid and vitamins B 2  and B 12  on physiology of themutant cells, suggested on the basis of clinical studies, is confirmed. Therefore, we conclude that the bacterial model describedin this report may be a useful tool in studies on human mutations.© 2005 Elsevier B.V. All rights reserved. Keywords:  Hyperhomocysteinemia;Human  MTHFR gene;  MTHFR polymorphism;  EscherichiacolimetF  gene;5,10-Methylenetetrahydrofolatereductase ∗ Corresponding author. Tel.: +48 58 346 3014; fax: +48 58 301 0072.  E-mail address:  wegrzyn@biotech.univ.gda.pl (G. W˛egrzyn). 1 Present address: Department of Biology and Genetics, Medical University of Gda´nsk, D˛ebinki 1, 80-211 Gda´nsk, Poland. 2 Affiliated with the University of Gda´nsk.0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2005.05.002  176  J. Jak´ obkiewicz-Banecka et al. / Mutation Research 578 (2005) 175–186  1. Introduction Itisobviousthatstudiesonasimplemodelaretech-nically easier and can give results significantly quickerthan analogous studies on a more complicated sys-tem. Therefore, it is not surprising that developmentof molecular genetics was based on prokaryotic mod-els for many years. Although molecular experimentsarecurrentlycommoninhumanbiologyandmedicine,stillstudiesoneffectsofparticularhumanmutationsoncellular functions are usually more complicated, timeconsuming and technically difficult relative to anal-ogous studies performed with bacteria. On the otherhand, some biological systems are evolutionary con-served and particular processes proceed very similarly,if not identically, in both bacterial and human cells.In fact, our knowledge about functions of many humangenesandproteinsoriginatedfromresultsofstudiesonbacteria that were subsequently repeated using morecomplicated eukaryotic systems. Examples of evolu-tionaryconservedproteinsareheatshockproteins[1,2]or small GTP-binding proteins [3,4]. A procedure for studying bacterial ortologue of particular eukaryoticproteinandthencheckingwhetherthelatterhassimilarproperties is well known. Moreover, it is easy to imag-inebenefitsofinvestigatingeffectsofcertainmutationsin the bacterial gene on activity of its product, whichcan subsequently be extrapolated to its human homo-logue. We assumed that bacterial mutants could alsobetestedininvivoassays,givingveryimportantinfor-mation about effects of mutations on cell physiology.However, one limitation had to be overcome, namelydue to a haploid nature of bacterial genomes, studieson effects of human heterozygotes might be problem-atic. Human genetic disorders are not always of reces-sive nature, thus phenotypes and features of heterozy-gotes may be very important from a medial point of view.Human  MTHFR  gene codes for 5,10-methyl-enetetrahydrofolate reductase (EC 1.5.1.20), whosedysfunction or decreased activity leads to increasedlevels of homocysteine (scheme of homocysteinemetabolism is shown in Fig. 1), as this enzyme cataly- sesconversionof5,10-methylenetetrahydrofolateto5-methyltetrahydrofolate,whichservesasamethyldonorintheremethylationofhomocysteinetomethionine[5].Hyperhomocysteinemia is a serious metabolic defect,and elevated levels of homocysteine are considered arisk factor for many diseases, including colorectal can-cer, cardiovascular disease, renal failure, inflammatory Fig. 1. Scheme of homocysteine metabolism (see [6,10] and references therein, for details). Abbreviations: DHF, dihydrofolate; THF, tetrahy-drofolate.   J. Jak´ obkiewicz-Banecka et al. / Mutation Research 578 (2005) 175–186   177 boweldiseaseandstroke(forreviewssee[6,7]).Recent studies revealed that moderately increased homocys-teine level causes the risk of osteoporotic fracture andit may be a predictive factor for hip fracture in olderpersons [8,9].The common polymorphism of the  MTHFR  gene,A222V, is caused by C to T transition at nucleotideposition 677 [7]. The A222V protein is less active and reveals increased thermolability relative to the wild-type form [7]. Homozygotes with two 677C → T alle-les have a high concentration of plasma homocysteine,while heterozygotes may reveal moderate hyperhomo-cysteinemia. Therefore, all three  MTHFR  genotypes(677Cand677Thomozygotes,andheterozygotes)con-fer different levels of risk for at least several seriousdiseases [7,10].  Escherichia coli metF   gene, whose dysfunc-tion leads to methionine auxotrophy, codes fora protein that is homologous to human 5,10-methylenetetrahydrofolate reductase [11]. The level of  similarity between these two proteins is so high thatparticular amino acid residues in MetF can be ascribedto their counterparts is MTHFR; A222 in MTHFRcorresponds to A177 in MetF [12]. On the basis of  structural and biochemical properties of the mutatedform of MetF (A177V), a specific mechanism lead-ing to decreased activity and increased thermolabilityof the A222V variant of MTHFR was suggested [12].However, no in vivo studies on the effects on A177Vsubstitution in MetF on cell phenotype were reported.Therefore, we aimed to construct an  E. coli  experi-mental system that could be used in studies on mecha-nisms and effects of decreased activity of human 5,10-methylenetetrahydrofolate reductase in both homozy-gousandheterozygousstatesofthe677C → T  MTHFR allele (the 530C → T allele of   metF  ). 2. Materials and methods 2.1. Bacterial strains, plasmids and bacteriophages E. coli  strains used in this work are listed in Table 1,and plasmids are characterized in Table 2. Bacterio-phageP1 vir  wasusedintransductionexperiments[13]. 2.2. Culture media All strains were cultured in Luria-Bertani (LB)nutrient medium [14] or MM minimal medium [15] supplemented with 1% glucose or 1% glycerol,10  g/ml thiamine and, when indicated, 50  g/mlamino acids ( l -methionine,  l -arginine). Minimal agarplates were prepared by supplementation of minimalmedium with 1.5% (final concentration) bacteriolog-ical agar. Media were supplemented with appropriateantibiotics (when required) as follows: ampicillin at50  g/ml, kanamycin at 50  g/ml and chlorampheni-col at 34  g/ml. Vitamin stock solutions were pre-pared as follows: 0.05mg/ml riboflavine (Vitamin B 2 )in water, 1mg/ml cobalamin (Vitamin B 12 ) in waterand0.5mg/mlfolicacidin100mMNaHCO 3 .Vitaminsolutions were sterilized by filtration (Millex 0.22  m,Millipore). 2.3. Disruption of the chromosomal metF gene in E. coli The chromosomal copy of the  metF   gene in  E.coli MG1655strainwasdisruptedusingPCR-mediatedsite-specific mutagenesis and in vivo recombination,accordingtothemethoddescribedpreviously[16].Theampicillin-resistance gene,  bla , was amplified by PCR Table 1  Escherichia coli  strainsStrain Gentotype ReferenceMG1655 Wild-type [33]W3110 Wild-type [34], ATCC 27325HME30 W3110   lacU169 ,  gal::bla ,  λ cI857   ( cro-bioA ) From Donald L. CourtDY330 W3110   lacU169 ,  gal490 ,  λ cI857   ( cro-bioA ) [16]DY330  MetF2 As DY330 but   metF::bla  This work, by PCR-mediated mutagenesis  MetF2 As MG1655 but   metF::bla  This work, by P1 transductionAB1909  lacY1  (or  lac24 ),  tsx-6, supE44 ,  galK5 ,  xyl-5 (or  xyl-7  ),  metF27  ,  argH1  (or  argE3 )[19]  178  J. Jak´ obkiewicz-Banecka et al. / Mutation Research 578 (2005) 175–186  Table 2PlasmidsPlasmid Characteristics ReferencepUC19  ori -pMB1-like,  bla ,  lacZ   [35]pUC19-metF As  pUC19  but bearing  metF   under control of   p lac  This work pACYC177  ori-p15A, kan ,  cat   [36]pMET1 As pACYC177 but bearing  metF   under control of   p lac  This work pCattTrE18  ori -pMB1,  cat  ,  tetR ,  p tet - lacZ  ’ [37]pMET2 As pCattTrE18 but bearing  metF   under control of   p tet  This work pBAD30  ori -p15A,  bla ,  araC  ,  p ara  [38]pMET5a As pBAD30 but bearing  metF   under control of   p ara  This work pMET5 As pMET5a but bearing  kan  instead of   bla  This work pUC19-metF177 As pUC19-metF but bearing 530C → T substitution in  metF   ( metF177   allele) This work pMET4 As pACYC177 but  bla ::  p lac - metF177   This work pMET9 As pACYC177 but  kan ::  p lac - metF177   This work pAD325  cat  ,  lacI  Q [39] using  E. coli  HME30 chromosome as a template. PCRprimers (synthesized by Interactiva Biotechnologie)were:delamp(5  -CATCCTGAAGTTTTTTCATCTTCC CTG ATT TTT CCT CAC CAT CAT TGG TCAGAG TTG GTA GCT CTT GAT C-3  ) and delamprev(5  -TAT TTT CCC GCC CTC ATT TCG AGG CAGCAT CTT GTG CTC TGT TTA AAA TTT GCA TTCAAATATGTATCCGCTC-3  ).Eachoftheseprimersconsisted of two parts: a 5  end, homologous to flank-ing regions of   metF  , and a 3  end, homologous to oneend (either 5  or 3  ) of the  bla  gene. Linear DNA (PCRproduct) was introduced into  E. coli  DY330 compe-tent cells using electroporation (1.8kV, 25  F, 200  ).Recombinant colonies were selected on LB plates forampicillin-resistance.Then,thechromosomefragmentencompassing  metF  :: bla  was transferred into  E.coli MG1655 strain by P1 transduction [13] to generate  E.coli   MetF2 strain, bearing the  bla  gene integratedinto  metF   locus. The transductants were selected onplates supplemented with ampicillin and checked forcolony formation on minimal medium plates with andwithoutmethionine,toconfirmdysfunctionofthechro-mosomal copy of   metF   (the mutant cells were notable to form colonies on plates without methionine).All genetic constructs were verified by DNA sequenc-ing [14], using the ABI Prism 310 DNA sequencer (Applied Biosystems). 2.4. Subcloning of the metF gene Molecular cloning procedures were as describedpreviously[14],andallconstructsdescribedinthissec-tion were verified by DNA sequencing as noted above.ForconstructionofthepUC19-metFplasmid,the metF  coding region was amplified by PCR using  E. coli MG1655chromosomeasatemplateandprimerspPstN(5  -AAC TGC AGA TTG ATG AGG TAA GGT ATGAGCTTTT-3  ,whichintroducesa Pst  Irestrictionsite)and pSmaC (5  -TCC CCC GGG TTA TAA ACC AGGTCG AAC CCC-3  , carrying a  Sma I restriction site)(Interactiva Biotechnologie). In the PCR, the anneal-ing temperature was 48 ◦ C for 10 cycles and 68 ◦ Cfor 25 cycles. The 924bp PCR product, digested with Pst  I and  Sma I, was inserted into the  Pst  I- Sma I sites of dephosphorylated pUC19 vector to generate plasmidpUC19-metF.For construction of plasmid pMET1, the pUC19-metF plasmid was digested with  Pvu II, and 1212bpgel-purifiedDNAfragmentwasinsertedintothe  Hin cIIsite of the dephosphorylated vector pACYC177.For construction of plasmid pMET2, the pUC19-metF plasmid was digested with  Pst  I and  Sma I, andthe 911bp gel-purified DNA fragment was insertedinto  Hin dIII(filled-inusingKlenowfragment)and Pst  Idigested, and dephosphorylated vector pCattTrE18.For construction of plasmid pMET5, the 911bp Pst  I– Sma I fragment (see preceding paragraph) wascloned into the  Sda I–  Hin dIII (filled-in using Klenowfragment) sites of dephosphorylated pBAD30 vector,giving plasmid pMET5a. This construct was digestedwith  Sca I to generate 4192bp fragment and, whichwasthenligatedwiththe1430bp  Eco 47IIIfragmentof pACYC177, containing a kanamycin resistance gene,to produce pMET5.   J. Jak´ obkiewicz-Banecka et al. / Mutation Research 578 (2005) 175–186   179 2.5. Site-directed mutagenesis of metF  The530C → Tsubstitutioninthe metF  gene,result-ing in the A177V substitution in MetF protein, wasgenerated using the site-directed mutagenesis methodwith four oligonucleotides [17]. External primers usedin PCR reaction (with pUC19-metF as a template)werepPstNandpSmaC(seeabove),andthe530C → Tsubstitution was introduced using primers c177t (5  -GGA TGC CGG GAT CAA TCG CGC-3  ) and c177t-rev (5  -GCG GTT GAC TCC GGC ATC CAC T-3  ) (synthesized by Interactiva Biotechnologie). ThePCR product, digested with  Pst  I– Sma I, was insertedinto the  Pst  I– Sma I sites of dephosphorylated pUC19vector to generate plasmid pUC19-metF177. DNAsequence of this construct was verified by automatedsequencing using the ABI Prism 310 DNA sequencer(Applied Biosystems). The pUC19-metF177 plasmidwas digested with  Pvu II, and 1212bp gel-purifiedDNA fragment was inserted into the  Hin cII site of dephosphorylated vector pACYC177 to generate plas-mid pMET4 or into the  Sma I site of pACYC177 togenerate plasmid pMET9. 2.6. Estimation of plasmid copy number  Plasmid copy number in bacterial cells was esti-mated as described previously [18]. Briefly, plasmidDNA was isolated from known number of bacterialcells, and following linearization with a restrictionenzyme the material was separated during agarose gelelectrophoresis.AmountofplasmidDNAineachsam-ple was estimated densitometrically by comparisonwithaknownamountofplasmidDNAseparatedonthesame gel. Then, average number of plasmid monomersper cell was calculated. 2.7. Quantitative RT-PCR Quantitative RT-PCR was based on the methodsdescribed previously [14]. Total RNA was isolated from bacterial cells using TOTAL RNA kit (A&ABiotechnology). DNase (RNase free) (Roche) wasaddedandsampleswereincubatedfor30minat37 ◦ C.Following addition of 0.1 volume of 25mM EDTA(pH 8.0) the samples were transferred to 65 ◦ C for10min. Three micrograms of total RNA was mixedwith a solution of a specific primer (RT-kan, 5  -CTCGTC CAA CAT CAA TAC A, or RT-met, 5  -AATCTT TCA CTC CTT CAC G) and nuclease-free water(Promega) to obtain primer concentration of 20pmol(final reaction volume was 11  l). Following incuba-tionat37 ◦ Cfor5minandchillinginice-bath,20unitsof the RNase inhibitor (Fermentas) dNTPs (final con-centrationof1mMeach)andtheconcentratedreactionbuffer (Fermentas) were added. Then, samples wereincubated for 5min at 37 ◦ C, and following addition of 200 units of M-MuLV Reverse Transcriptase (Fermen-tas) the incubation was prolonged for 60min at 42 ◦ C.The reverse transcriptase was inactivated by incuba-tion at 70 ◦ C for 10min, and PCR was performed withtwo alternative pairs of primers: kan-F (5  -GAT AATGTC GGG CAA TCA GGT G) and kan-R (5  -AATCACTCGCATCAACCAAACC)ormet-F(5  -CAGAAG TCC AGG GGC AGA TTA AC) and met-R (5  -GCA TCA TCA TCC AGA CCG TCG), according tothe following scheme: initial incubation for 5min at95 ◦ C, then 30 cycles of denaturation for 40s at 95 ◦ C,annealing for 40s at 63 ◦ C and extension for 50s at72 ◦ C. Final extenstion was carried out for 7min at72 ◦ C. Products of the reaction were sepatared duringagarose gel electrophoresis and quantified densitomet-rically. 2.8. Measurement of bacterial growth in cultures Bacteria were cultured overnight at 30 ◦ C inMM minimal medium supplemented with methion-ine (50  g/ml). The cultures were diluted 1:50 in theminimal medium with methionine and incubated withshaking at 25, 30, 37 or 43 ◦ C to an optical densityat 575nm (OD 575 ) of 0.2. The cells were pelleted bycentrifugation(2000 × g for5min),washedtwicewiththe minimal medium lacking methionine, and resus-pended in the same medium. Following 30min culti-vation with shaking at adequate temperature, bacteriawereinducedwithIPTG(atfinalconcentrations:0.001,0.01, 0.05, 0.1, 0.2 or 0.5mM), autoclaved chlortetra-cycline (at final concentrations: 0.5, 1, 5, 10, 15 or30  g/ml) and/or  l -arabinose (at final concentrations:0.02or0.2%).Opticaldensityofcultureswasmeasuredat indicated times using SmartSpec3000 spectropho-tometr (BioRad). Generation times of bacterial cellswere calculated when indicated. In these experiments,repeated several times, standard deviation was alwaysbelow 10%.
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