Molecular Cloning and Expression in Escherichia Coli

download Molecular Cloning and Expression in Escherichia Coli

of 5

Transcript of Molecular Cloning and Expression in Escherichia Coli

  • 8/9/2019 Molecular Cloning and Expression in Escherichia Coli

    1/5

    Appl Microbi ol Biotechnol (1995) 43: 65- 69 Springer-Verlag 1995

    M. Z. A. Bhuiyan K. Ueda Y. InouyeM. S ug iyamaMolecular cloning and expression in Escherichia coliof bleomycin-resistance genefrom a methicillin-resistant Staphylococcus aureusand its association with IS431 mec

    Received: 18 April 1994/Rec eived last revision: 1 July 1994/Accepted: 26 July 1994

    Abstract A gene that confers b leomycin resis tance wasc loned f rom the ch rom osom al DN A of me th ic i ll i n- re -sistant Staphylococcus aureus (MRSA) B-26 into theplasmid pUC18. I t is of chro mos oma l orig in ra therthan p l a smid and ex i s t s i n t he ch romosome mak inga c luster wi th the kanamycin -resis tanc e gene. We foundthat the nucleot ide sequence of the Neomycin-resis t -ance gene f rom the ch romosome o f MRS A B-26 i sident ical to that from a s taphylococcal p lasmid,pU Bl l 0 . The pa r t ia l sequence o f IS 4 3 1 mec was alsofound ups t ream f rom the DNA f ragmen t con ta in ingthe b leomycin- and kanam ycin-res is tance genes.

    IntroductionBleomycin , an aminoglycopep t ide , is one of the impor-tant ant ineoplast ic ant ib io t ics i so la ted from S t r ep t o my-ces vert ici l lus (Umezaw a 1975) . The mode o f ac tion o fbleomy cin has been s tudied extensively and i t wasproved that the drug causes cel l death as a resul t ofmul t ip le s t rand sc iss ions by d i rect in teract ion wi th thetum ou r cell DN A. D etai led s tudies have e lucidated themechan i sm o f Neomyc in - induced doub le -s t randbreaks in bacteria l and v i ra l DNA (Haidle and Lloyd1979).A t ransp oson Tn5, orig inal ly ident i fied as a com pon-en t o f an R fac to r f rom Klebs ie l la (Berg et al. 1975),

    carr ies a Neom ycin-resis tanc e determ inant (Geni l loudet al . 1984; M azo dier et al . 1985). Sug iyam a et al. hav ecloned two Neom ycin-resis tanc e genes, designatedb l mA a n d blmB, f rom Neomyc in -p roduc ing S. verticil-lu s and demons t ra t ed tha t t hey encode b l eomyc in -b ind ing p ro t e in and Neomyc in ace ty l t rans fe raserespectively (Sugiy ama et al . 1994). In a ddit ion, a stra inha rbour ing the s t aphy lococca l p l a smid pUBl l0(Jala nko et al . 1981) was resistant to bleom ycin (Semonet al. 1987). Alth oug h the ble omyc in-res istance genesencod ed in pUB110 an d Tn5 h ave been sequenced, thebiochemical mechan ism of res is tance has n ot yet beenelucida ted (Ma zodie r et al. 1985; Sem on et al . 1987).Alm ost al l strains of methicil l in-re sistant Staphyl -ococcus aureus (MRSA), iso la ted in Hirosh ima U niver-si ty Hos pital fr om O cto ber 1990 to April 1992, wereresis tant to Neomycin , which was not used as an ant i -bacteria l agent . From our observat ion that theNeom ycin-resis tanc e propert ies d id not d isappear,even when the M RSA cell s were t reated wi th e th id iumbromide as a p lasmid-curing agent (Bouanchaud e t a l .1969), these strains were thought to be resistant to thedrug as a resul t of the presence of the gene in thech rom osome ra the r t han in t he p la smid .In the present s tudy we c loned a gene that confersNeomyc in re s i s t ance f rom the ch romosomal DNA ofa MRSA st ra in i so la ted from Hiroshima Universi tyHospi ta l , Japan, and the possib i l ity of the b leomycin-resis tance determinant as a t ransposable e lement inMRSA strains is discussed.

    M. Z. A. Bhuiyan K. UedaDepartment of Paediatrics,Hiroshima University School of Medicine,Kasumi 1-2-3, Minami-ku, Hiroshima 734, JapanY. Inouye . M. Sugiyama (~)Institute of Pharmaceutical Sciences,Hiroshima University School of Medicine, Kasumi 1-2-3,Minami-ku, Hiroshima 734, Japan.Fax: 81-82-257-5284

    Materials and methodsBacterial strains and plasmidsMethicillin-resistantStaphylococcus aureus (MRSA) B-26 was usedto clone a gene encoding a chromosomal Neomycin-resistancedeterminant. Escherichia coli TG1 and E. coli DH5c~ were used ashosts for cloning and sequencing experiments. Plasmids pUC18,pU Cl l8 and pU Cl l9 were used as vectors. E. coli TB1 was used as

  • 8/9/2019 Molecular Cloning and Expression in Escherichia Coli

    2/5

    66a host for expression vector pMALc-2 (M aina et al. 1988) obtainedfrom New England BioLabs Inc. (Beverly, Mass., USA). E. coliharbouring the pUC-based plasmids and pM ALc-2 were cultivatedin LB br oth contain ing 100 gg/ml am picillin at 37C overnight.MRS A B-26 was cultivated in TSB b roth (Difco laboratories, De-troit, Mich., USA) containing 100 gg/ml bleom ycin A2 sulphate at37C overnight.

    Reagents usedRestriction enzymes were purchased from New England Biolabs Inc.and T oyobo Co. Ltd. (Osaka, Japan). A ligation kit containing T4DNA ligase was obtained from Gibco BRL (Gaithersburg, Md.,USA). A PCR-amp lification kit containing Taq polymerase anddNT P was purchased from Tak ara Shuzo Co. Ltd. (Kyoto, Japan).

    Prepara t ion of chromosomal DNAThe chromosomal DNA from MRSA B-26 was isolated accordingto the met hod of Davis et al. (1980) after cell lysis using lysozym e andachromopeptidase.

    Shotgun cloning of the bleomycin-resistance geneThe ch romosom al DN A was partially digested with several differentrestriction enzymes, either singly or in combination, and ligated topUC18 digested with the same enzyme. The chimeric plasmid wasintroduced into E. coli cells by electroporation using the Transpor-ator (BTX Electro Cell Manipulator, San Diego, Calif., USA), ac-cording to the manufacturer's instructions. LB agar containing am-picillin (100 ~tg/ml) and Neom ycin A2 sulphate (100 gg/ml) was usedto screen bleomycin-resistant transformants.

    Oligonucleotides and polymerase chain reactionTo amplify the structural gene for bleomycin resistance from MRS AB-26 by the polymerase chain reaction (PCR), two primers havingE coR I an d B am H I sites at the 5'- and 3'-adjacent regions respect-ive ly , des ignated pr imer I(5 ' -ATGCGAATTCATGTTACAG-TCTATCCCGGC-3 ') and primer 2 (5 ' -ATAAGGATCCTTAG-CTTTTTATTTGTTGAA-3'), were designed on the basis of thenucleotide sequence of the Neomy cin-resistanc e gene from pUB110(Semon et al. 1987) and synthesized using a Cyclo ne Plus D NAsynthesizer (MilliGen Bioresearch, Bedford, Mass., USA) by Bio-Synthesis Inc. (Lewisville, Tex., USA). A 1.6-kb B am H I-B g l I ! frag-ment containing the bleomycin-resistance gene from M RSA B-26was used as a template for the PCR. PCR amplification was per-formed according to the metho d described previously (Innis et al.1990).

    Subcloning of the PC R-amplified geneThe structural gene for bleomycin resistance, amplified by the PCRmethod, was inserted into an expression vector pMALc-2 and intro-duced into E. coli TB1. The structural gene is expected to beexpressed under the control of the ta c promoter in pMALc-2(Maina et al. 1988).

    DNA sequencingSome DNA fragments were subcloned into pUC118 and pUC119.The plasmids were isolated by the alkaline extraction procedure ofBirnboim and Doly (1979), and purified by the Magic MiniprepsDNA purification system (Promega Corp., Madison, Wis., USA),according to the manufacturer's instructions. DNA was sequencedby the dide oxy-DN A chain-termination method (Sanger et al. 1977)using the A.L.F. DNA sequencer (Pharmacia LK B Biotechnology,Uppsala, Sweden). The sequence data were analysed for open-read-ing frame and amino acid sequence homologies with the GE NET YXprogramm e (Software Development, Tokyo, Japan).

    Results

    Cloning and expression of the Neomycin-resistancegene in E. coliPreliminary experiments confirmed that no plasmidDNA was present in MRSA B-26. To clone a gene forNeomycin resistance from MRSA B-26, we partiallydigested the chromosomal DNA with several differentrestriction enzymes, either singly or in combination,ligated the fragments to pUC18 cleaved with the sameenzyme, and introduced the products into E. coli TG1.Only when the chromosomal DNA was digested withHi ndI I I , were three clones resistant to bleomycinobtained. The plasmids isolated from these clones allcontained the same 5.1 x 103-base (5.1-kb) insert ofStaphylococcus DNA in the same direction. A partialrestriction map of the plasmid, designated pl 8KBA1, isshown in Fig. 1. E. coli harbouring the plasmid wasresistant even to 1000 gg/ml bleomycin.

    Clustering with the kanamycin-resistance geneE. coli harbouring p18KBA1 was also resistant to100 gg/ml kanamycin. To localize both the Neomycin-and kanamycin-resistance genes the deletion deriva-tives ofpl8KBA1 were constructed; the plasmid, desig-nated pl8KBA2, was obtained by double digestion ofpl8KBA1 with Hi ndI I I and B a m H I and subsequentligation (Fig. 1). E. coli TG1, harbouring the 2.6-kbB a m H I - H i n d I I I insert, contained in the p 18KBA2 plas-mid, was also resistant to both antibiotics.The pl8KBA3 plasmid, which contained the 1.6-kbB g l I I - B a m H I segment from the cloned 5.1-kb Hi ndI I I -Hi ndI I I DNA fragment, was also constructed. E. coliharbouring pl8KBA3 was resistant to bleomycin, butnot to kanamycin. E. coli harbouring plasmidpl8KBA4, in which the 2.6-kb B a m H I - H i n d I I I seg-ment was deleted from the 5.1-kb Hi ndI I I -Hi ndI I IDNA fragment, no longer expressed both bleomycinand kanamycin resistance. Thus the Neomycin-resist-ance gene was cloned together with the kanamycin-resistance gene in a clustered fashion.Ubuk ata et al. (1989) have also describeda kanamycin-resistance gene, located on the chromo-some of a MRSA strain. In the present study we dem-onstrated that the bleomycin-resistance gene is clus-tered with the kanamycin-resistance gene and located

  • 8/9/2019 Molecular Cloning and Expression in Escherichia Coli

    3/5

    Fig. 1 Rest r ic t ion-mapsimilari ty between p 18KBA 1 (a)and pU B l l 0 (b ). The thickblack bar indicates insert ionfragment from methici l l in-resistant S. aureus B-26 strain,and the line indicates theplasmid pUC18. The thick whiteba r indicates the deletedfragment. The dotted lineindicates the extent of thesequence de termined, pUBl l0(Jalanko et al. 1981) wasl inearized at the E c o R I site. Inpl8KBA1, an ext ra B g l I I site ispresent , which is within theinser t ion sequence IS431mec, asshown in Fig . 2

    pl 8KBA1

    BamH Ipl 8KBA2 ]

    BamH Ipl 8KBA3 ]

    Hind IIIEcoR IamH IHind III

    Hind III~

    gl II Bgl II Pvu ][ 8amH II I l l

    Hind IIIBgl II

    BamHI

    Bgl II Pvu II BamH II I

    67

    Hind III

    BamHII

    BamHII

    a1 ;[ 1 ;]

    l k b

    pUB 11 0b

    EcoRlIkan r b le rBgl II Pvull BamHI Xbal EcoRI

    I I i I II I1 Kb

    downs t ream f rom the gene fo r kanam yc in re s is t ance a sa pU B ll 0 p lasmid (Ja lanko e t al . 1981; Semo n et al .1987), and there is a ma jor restric t ion-e nzym e site sim-ilari ty in both (Fig. 1). We a mplified the struc tural g ene(399 base pairs, bp; data not shown) for bleomycinres is t ance f rom MRS A B-26 by the P C R method , u s ingtwo ol igonucleot ide primers , which were synthesizedon the basis of the nucleot ide sequence of theb leomyc in - res i st ance gene f rom pU Bl l0 (S emon e t al .1987). The nucleot ide seque nce of the b leomycin-resis t -ance gene from M RS A B-26 was ident ical to that frompUBl l0 (da t a no t shown) . In add i t i on , we con f i rmedthat the s t ructural gene for b leomycin resis tance fromMR S A was exp ressed under t he con t ro l o f t he tacprom ote r on exp ress ion vec to r pMA Lc-2 (M aina e t a l.1988) in E. coll.To e luc ida t e the pheno meno n o f how b leomyc in -and kanam ycin-res is tance genes have been evolu-t i ona r il y i n t eg ra t ed in to t he ch romos omal DN A f romMRS A, we a lso sequenced about 300 bp from both theright and left ends of the c loned 5 .1-kb HindIII-HindIIIDN A fragmen t. We com pared the nuc l eo tide sequencesof both the regions wi th tho se o f several insert ionsequences from Staphylococcus aureus to d iscoverwhether there i s any insert ion-sequence (IS)- l ike e le-men t nea r t he Neomyc in - and kanamyc in - res i s t ance

    gene. We thus ought to f ind out why the nucleot idesequence of the b leomycin-resis tance gene of MRSAB-26 is identical to that of pU Bl l0 , a lso having a s im-i lar a l ignment wi th the kanam ycin-resis tance gene; th isled us to investigate how these two genes from pUB 110t rans fe r red to t he ch romoso mal DN A of MRS A B-26.As shown in F ig . 2 , a region wi th complete homologyto IS431rnec (Barberis-Maino et al . 1990) was founddowns t ream f rom the l e f t HindIII si te of the c loned5 .1 -kb DNA f ragmen t . The sequence ACTTTG-CAACAGAACCG i s i den t i ca l t o an inve r t ed repea t ,designated IR-r in IS431mec, which is a methicil l in-resis tance-associa ted insert ion-sequence-l ike e lement .But another inverted repeat , designated IR-1 , inIS431mec was not found in our c loned gene: IR-1 i sshow n to be located 759 bases upst r eam from the IR-rmot i f (Barberis-Maino e t a l. 1990). Up st rea m from theright HindlII si te of the frag ment, how ever, no signifi-can t hom ology wi th IS431mec was found.

    D i s c u s s i o nInsert ion sequences can reside on both p lasmids andch romosom es and p rov ide reg ions o f DN A sequence

  • 8/9/2019 Molecular Cloning and Expression in Escherichia Coli

    4/5

    68Fig. 2 Compari son of thepartial nucleotide sequences ofthe segment containing theHindIII-BgIII site (a) in thecloned 5.1-kb DNA fragment ofStaphylococcus DNA withthose of IS431mec (b). Thenucleot ide sequence ofIS431mec is numberedaccording to the systemdescribed by Barberis-Maino etal. (1990). * Identicalnucleotides in the comparedsequences, - - the IR-rsequence

    H in dllIa AAGCTTT TAA

    b AAGCTTT TAA 720

    ACTTAAACCTGACTGTCATTGTACATCGAAATATCTGAATAACCTCATTGAGCAAGATCA

    ACTTAAACCTGACTGTCATTGTACATCGAAATATCTGAATAACCTCATTGAGCAAGATCA 780

    CCGTCATATTAAAGTAAGAAAGACAAGGTATCAAAGTATCAATACAGCAAAGAATACTTT************************************************************

    CCGTCATATT AAAGTAAGAAA GACAAGGTATCA AAGTATCAATA CAGCAAAGAATA CTTT 840BglnA~KAGGTATTGAATGTATTTACGCTCTATATAAA/~Gd~CCG~2AGGTCTCCTTCAGATCT

    AAAAGGTATTG AATGTATTTAC GCTCTATATAAA AAGAACCGCAG GTCT-CTTCAGA TCT 099

    ACGGATTTTCGCCATGCCACGAAATTAGCATCATGCTAGCAAGTTAAGCGAACACTGACA

    ACGGATTTTCGCCATGCCACGAAATTAGCATCATGCTAGCAAGTTAAGCGAACACTGACA 959

    TGATAAATTAGTGGTTAGCTATATTTTTTTACTTTGCAACAGAACCG

    TGATAAATTAGTGGTTAGCTATATTTTTTTACTTTGCAACAGAACCG 1006

    for recom bina t ion (Broda 1979; Coh en 1976; I ida e t a l.1983). In addit ion, plasmids also can act as vectors fort r ansposab l e DNA e l ement s o r t r ansposons (Kl eckne r1981).The f requent p re sence of I S 4 3 1 o n c h r o m o s o m a l an dpl a smi d DNA of s t aphyl ococc i i n va r i ous copy num-bers and loca t ions p oints to the mo bi le chara c ter of thi se lement . I S 4 3 1 i s al so present in the mercury-re s i s tancede t e rmi nan t , m e r o f S . a u r e u s (Barber i s -Maino e t a l .1987) . The methic i l lin-res i stance de term inant m e c in S.a u r e u s i s thought to be t ransposable (Trees and Ian-dolo 1987) and has a chro mo som al loca t ion (Ku hl e t al.1978). Th ere is indirect evidenc e tha t b oth m e t an d m e cde t e rmi nan t s t r anspose (Tree s and Iandol o 1987;Shali ta et al . 1980; Witte et al. 1986; Ly on an d Sk urr ay1987) . However , di rec t evidence for the mobi l i ty of1 S 4 3 1 i s not ava i lable yet . Judg ing from the obser va t ionof above researchers and our f indings in S . a u r e u s B-26,we hypo thes ize tha t this bleo mycin-res i s tance gene wasor i g ina l ly p re sen t i n p l a smi d pU B l l 0 c l us te red wit ha kana mycin -res i s tance gene , which was la te r integ-ra t ed i n to t he chr omo some of MR SA, wi t h t he medi -a t i on of I S 4 3 1 m e c , l ike the mercury- and te t racycl ine-res i s tance genes (Skinner e t a l. 1988; Barbe r i s -Ma inoet al . 1987). Studies are in progress in ou r lab or ato ry tode t e rmi ne t he s t ruc ture and func t i on of a p ro t e i nencoded by t he b l eomyc i n- re s i s tance gene in MR SA.Fur ther d e ta i led studies a re a l so needed of ho w the

    b l eomyc i n- and kanamyc i n- re s i s t ance genes f romp U B l l 0 t r an s f er r ed to t h e c h r o m o s o m e i n M R S A .AcknowledgementsWe are grateful to Professor T. Yokoyama,Hiroshima University Hospital for gifts of MRSA strains. We tha nkMr. H. Yaju, Pharmacia Biotech K.K., Tokyo, for his skilful analysisof nucleotide sequencing. We thank Ni ppon Kayaku Co. Ltd. for thegift of bleomycin A2 sulphate. We wish to thank the Research Centrefor Molecular Medicine, Hiroshima Universi ty School of Medicine,for the use of their facilities.

    ReferencesBarberis-Maino L, Berger-Bachi B, Weber H, Beck WD, Kayser FH(1987) IS431, a staphylococcal inser tion sequence-like elementrelated to IS26 from Proteus vulgaris. Gene 59:107-113Barberis-Maino L, Ryffel C, Kayser FH, Berger-Bachi B (1990)Complete nucleotide sequence of IS431mec in Staphylococcusaureus. Nucleic Acids Res 18:5548Berg DE, Davies J, Allet B, Rochaix J-D (1975) Transposition offactor R genes to bacteriophage 2. Proc. Natl Acad Sci USA72:3628 3632.Birnboim HC, Doly J (1979) A rapid alkaline extraction procedurefor screening recombinant plasmid. Nucleic Acids Res7:1513 1523Bouanchaud HH, Scavizzi MR, Chabbert YA (1969) Elimina tion byethidium bromide of antibiotic resistance in enterobacteria andstaphylococci. J Gen Microbiol 54:417-425Broda P (1979) Plasmids. Freeman, San Francisco, pp 23-51Cohen SN (1976) Transposab le genetic elements and plasmid evolu-

    tion. Nature 263:731-738

  • 8/9/2019 Molecular Cloning and Expression in Escherichia Coli

    5/5

    69Davis RW , Botstein D, R oth JR (1980) Adva nced bacte rial genetics.A manual for genetic engineering. Cold Spring Harbo r L abora t-ory, Cold Spring Harbor, New YorkGenilloud O, Garrido M C, Mo rreno F (1984) The transposon Tn5carries a bleom ycin-resistance determinant. G ene 32:225-233Haidle CW, Lloyd RS (1979) In: Hahn FE (ed) Bleomycin in anti-biotics, vol V, part II. Springer, Berlin Heidelberg New York, pp124-151Iida S, Meyer J , Arber W (1983) Prok aryoti c IS dements. In: ShapiroJA (ed) Mo bile genetic elements. Academic Press, New Y ork, pp159-221Innis MA, G elfand DH, Sninsky JJ , White TJ (1990) PCR protocols:a guide to m ethods and applications. Academic Press, San Diego,CalifJalanko A, Palva I, Soederlund H (1981) Restriction maps of plas-mids pUB110 an d pBD9. Gene 14:325 328Kleckner N (1981) Transposable elements in Prokary otes. Rev Gen -et 15:341 404Kuhl SA, Patee PA, Baldwin JN (1978) Chromosom al map lo cationof the methicillin resistance determinant in Staphylococcus aure-

    us . J Bacteriol 135:460-465.Lyon BR, Sku rray R (1987) Antimicrobial resistance of S ta p h y lo co c -cus aureus: genetic basis. M icrobiol Rev 51:88-134

    Maina CV, Riggs PD, Grandea III AG, Slatko BE, Moran LS,Tagliamonte JA, McReynolds LA, Gu an CD (1988) An Es-cherichia coli vector to express and purify foreign proteins byfusion to and separation from ma ltose-binding protein. Gene74:365-373Mazodier P, Cossart P, Giraud E, Gasser F (1985) Completion ofthe nucleotide sequence of the central region of Tn5 confirms thepresence of three resistance gene. Nuc leic Acids Res 13:195-205

    Sanger F, Nivcklen S, Coulson AR (1977) DNA sequencing withchain-terminating inhibitors . Proc Natl Acad Sci USA74:5463-5467Semon D, Movva NR, Smith TF, Alama ME, Davies J (1987)Plasmid-determined bleomycin resistance in Staphylococcusaureus. Plasmid 17:46-53Shalita Z, M urphy E, Novick RP (1980) Penicillinase plasmids ofStaphylococcus aureus: structural and evolutionary relationships.Plasm id 3:291.-311Skinner S, Inglis B, Matthews PR, Stewart PR (1988) Mercury andtetracyclin e resistance genes and fla nking repeats associa ted withmethicillin resistance on the chromosom e of Staphylococcusaureus. Mol Microbiol 2:289-297Sugiyama M, Thompson CJ, Kumagai T, Suzuki K, Deblaere R,Villarro el R, Davies J (1994) Char acterisa tion by mol ecular clon-ing of two genes from Streptomyces vert ic i l lus encoding resistanceto bleomycin. Gene 151:11-16Trees DL, Ian dolo JJ (1987) Isolation of a transposon (Tn4291) th atcodes for resistance to m ethicillin in Staphylococcus aureus. AbstrAnnu Meet Am Soc Microbiol 87:56Ubukata K, Nonoguchi R, Matsuhashi M, Song MD, KonnoM (1989) Restriction maps of the regions coding for methicillinand tobramycin resistances on chromosomal DNA in methicil-lin-resistant s taphylococci. Antimicrob Agents Chemother33:1624 1626Umezawa H (1975) Bleomycin. In: Corcoran JW and Ha hn FE (eds)Antibiotics, vol III. Springer, Berlin Heidelberg New York, pp21-33Witt e W, Gree n L, Mi sra TK , Silver S (1986) Resistance to mercuryand to cadm ium in chromosom ally resistant Staphylococcusaureus. Antimicrob Agents Chemother 29:663-669