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Genetics and Molecular Biology

Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria

Emmanuelle Charpentier, Ana I. Anton, Peter Barry, Berenice Alfonso, Yuan Fang, Richard P. Novick
Emmanuelle Charpentier
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, New York, New York
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  • For correspondence: emmanuelle.charpentier@univie.ac.at novick@saturn.med.nyu.edu
Ana I. Anton
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, New York, New York
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Peter Barry
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, New York, New York
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Berenice Alfonso
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, New York, New York
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Yuan Fang
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, New York, New York
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Richard P. Novick
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Department of Microbiology, NYU Medical Center, New York, New York
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  • For correspondence: emmanuelle.charpentier@univie.ac.at novick@saturn.med.nyu.edu
DOI: 10.1128/AEM.70.10.6076-6085.2004
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  • FIG. 1.
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    FIG. 1.

    Cassette restriction maps of shuttle vector pCN series. The gene designations include pT181 cop-wt repC, pT181 cop-623 repC, pT181 cop-634 repC4, and pI258 replicon for replication in Staphylococcus. Each pT181 cassette contains the single-stranded origin of replication, double-stranded origin of replication, the copy control system, and the repC gene encoding the replication protein RepC of the pT181 rolling-circle replicon. The pI258 replicon includes the plasmid's replication origin, rep protein gene, and, presumably, the copy control system, though this has not been defined to date. Antibiotic resistance modules consist of aad9 (aminoglycoside adenyltransferase-encoding gene of Tn554 for spectinomycin resistance), aphA-3 (for aminoglycoside 3′-phosphotransferase, the kanamycin resistance gene of pAT21), cat194 (chloramphenicol acetyltransferase-encoding gene of pC194 for chloramphenicol resistance), ermC (ribosomal methylase-encoding gene of pE194 for erythromycin resistance), and tet(M) (gene of pRN6680 for tetracycline resistance). Additional modules include φ11 EcoRI-K (enabling high-frequency transduction), SaPI1-attS (enabling site-specific integration into the SaPI1 chromosomal attachment site in the presence of SaPI1 integrase in trans), amp ColE1 ori (bla gene conferring ampicillin resistance and ColE1ori for replication in E. coli), blaZ (gene from pI258 encoding the β-lactamase reporter), gfpmut2 (fluorescence-activated cell sorting-optimized mutant version gfpmut2 of the green fluorescent gene of A. victoria encoding the GFP reporter), luxAB (gene encoding the luciferase determinant from V. fischeri), TT (blaZ transcriptional terminator), PblaZ (constitutive β-lactamase promoter module), and Pcad-cadC (cadmium-inducible promoter module). For transcriptional fusions, a three-way translational stop codon was inserted upstream of each promoterless reporter gene. The transcriptional terminator of blaZ was inserted downstream of each reporter gene to prevent any read-through of the repC gene from the promoter to be tested. The MCS from pUC19 is symbolized by a black box. Each cassette is flanked by restriction sites introduced into the PCR product used for its cloning, as indicated. Adventitious sites corresponding to those of the pUC19 MCS are indicated. Those occurring in any of the three backbone cassettes are crossed out in the representation of the MCS.

  • FIG. 2.
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    FIG. 2.

    Series of pCN shuttle vectors. The first generation of pCN shuttle vectors is here represented in linear form, opened at the NarI site between the replicon and MCS cassettes. Each of these was assembled using the flanking restriction sites shown in Fig. 1. In cases where two sites are shown, the outer one was used. Note that the replicon cassettes were cloned using their ApaI and SphI sites and that the MCS was subsequently inserted between the SphI and NarI sites at the left end of the cassette. Any cassette can be removed and replaced using these same restriction enzymes. For details of each module, please refer to Fig. 1.

  • FIG. 3.
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    FIG. 3.

    Analysis of the pT181-based staphylococcal modules. Whole-cell sheared minilysates of S. aureus RN4220 strains containing the indicated plasmids were separated on 1% agarose in Tris-borate buffer for 18 h at 2.5 V/cm, stained with ethidium bromide, and photographed. Samples corresponding to equivalent numbers of cells were used to permit comparison. (A) Strains were grown at 32°C. Lane 1, RN4220; lane 2, RN2424; lane 3, RN4253; lane 4, RN4416; lane 5, RN9582, lane 6, RN9586; lane 7, RN9593. (B) Experiments were conducted at 43°C. Lane 1, RN4220; lane 2, RN9582; lane 3, RN9593. The heavy upper band corresponds to sheared chromosomal DNA. The supercoiled plasmid bands are indicated with arrows. Intermediate plasmid bands correspond to topoisomers and multimers.

  • FIG. 4.
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    FIG. 4.

    Analysis of the constitutive promoter cassette PblaZ. Data are for RN4220 derivatives containing the indicated plasmid. Expression of the gfpmut2 reporter gene was monitored during growth at 37°C. The vertical axis represents relative fluorescence versus OD620 of the culture.

  • FIG. 5.
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    FIG. 5.

    Analysis of the cadmium-inducible promoter cassette Pcad-cadC. Data are for RN4220 derivatives containing the indicated plasmid. Expression of the blaZ reporter gene was monitored during growth at 37°C. Low-density cultures were incubated at 37°C for 90 min in the absence or presence of different concentrations of Cd2+. Samples equalized to the same number of cells were analyzed. (A) The vertical axis represents β-lactamase activity (initial reaction rate in milliunits of OD490/min). (B) Total RNA was prepared and analyzed by Northern blot hybridization with a blaZ-specific probe. For loading controls, an rRNA 16S-specific probe was used.

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  • TABLE 1.

    Bacterial strains and plasmids

    Strain or plasmidRelevant characteristic(s)Source or reference
    Strains
        E. coli
            DH5αHost for DNA cloningLab strain collection
            TOP10Host for DNA cloningInvitrogen Life Technologies
        S. aureus
            NCTC8325Propagating strain for typing phage 47Lab strain collection
            RN11Harbors pI258; lysogenic for φ11, φ12, and φ13Lab strain collection
            RN27RN450 lysogenic for 80α, φ13 16
            RN450NCTC8325 cured of φ11, φ12, and φ13 16
            RN13298325-4; lysogenic for φ11; Spr [Tn554(aad9)]Lab strain collection
            RN24248325 (pT181)Lab strain collection
            RN24428325-4 (pE194)Lab strain collection
            RN4220Restriction-defective derivative of RN450 11
            RN42538325 (pRN8061)Lab strain collection
            RN4416RN27 (pSA0331)Lab strain collection
            RN58308325-4 (pC194)Lab strain collection
            RN6851RN6502 (pRN6680)Lab strain collection
            RN6734 agr+; 8325-4 derivativeLab strain collection
            RN7206RN6734Δagr::tet(M)Lab strain collection
            RN7885JM109 (pRL189)Lab strain collection
            RN8972RN4220 (pRN6998) 25
            RN9011RN4220 (pRN7023) 25
    Plasmids
        pUC19Apr; ColE1 compatibility group origin of replication 867; 2686 bp 32
        pT181pT181wt; Tcr; Inc3; naturally occurring; ±22 copies/cell; 4,440 bp 1, 17
        pRN8061pT181cop-623; Tcr; Inc3; ±400 copies/cell; 4,440 bp 1, 17
        pSA0331pT181cop-634repC4; Tsr; Tcr; Inc3; ±145 copies/cell; 4,440 bp 1, 17
        pI258Naturally occurring β-lactamase plasmid; 28 kb 21
        pE194Emr (ermC); Inc11; naturally occurring; 3725 bp 5
        pAT21Apr Kmr; pBR322Ω1.5-kb pJH1 ClaI (aphA-3) 28
        pC194Cmr (cat194); Inc8; naturally occurring; 2,910 bp 6
        pRN6680Apr Tcr; pBluescriptΩ2.9-kb pMVN6 Smal-HindII [tetA(M)] 15
        pRN6998pE194tsΩpUC18-MCSΩSaPI1sek-int-attS; 8,319 bp 25
        pRN7023pC194tsΩpUC19-MCSΩSaPI1int 25
        pRL189 luxAB Gift of Peter Wolk
        pBD4LITMUS28Ωgfpmut2-ermC; 4,784 bpGift of D. J. Bartels and E. P. Greenberg
  • TABLE 2.

    Oligonucleotide primers used for plasmid construction

    PrimerSequence (5′-3′)aF or RbDNA templateCassette
    672GAA TAT GCA TGC GAT AAT GGC GCCTTT GCG GAA AGA GTT AGT AAG TTA ACA GFpT181, pRN8061, pSA0331pT181 replicon
    671TGA ACG GGC CCAATA AAA GCA ATC AAT GAA CCA AGRpT181, pRN8061, pSA0331pT181 replicon
    P386TAC TTG GGG CCCTCG ATG ATT ACC AGA AGT TCT CRpI258pI258 replicon
    P387TAG CAT GGC GCCGTT TTA TCT TCA TCA CTT GTA TTA ATCFpI258pI258 replicon
    673GAT CAT CTC GAG CGG CCG CAT AGT TAA GCC AGC CCC GACFpUC19 amp ColE1 ori
    623GAT TAG CAT GCA GCG GCC GCC AGC TCA CTC AAA GGC GGRpUC19 amp ColE1 ori
    675CTC TGC GGG CCC ACC TAG GAA TTG AAT GAG ACA TGC TACFpE194 ermC
    674TCT CTT CTC GAG CGC CGC GGA AAA CTG GTT TAA GCC GACRpE194 ermC
    599GTT TAA GGG CCC ACC TAG GCA AAT ATG CTC TTA CGT GCFpRN6680 tet(M)
    600CTA TGA CTC GAG GCC GCG GAA ATA TTG AAG GCT AGT CAGRpR6680 tet(M)
    617GTT TAA GGG CCC ACC TAG GTA TTA TCA AGA TAA GAA AGFpC194 cat194
    618CTA TGA CTC GAG GCC GCG GCC TTC TTC AAC TAA CGG GGRpC194 cat194
    619GTT TAA GGG CCC ACC TAG GGG TTT CAA AAT CGG CTC CGFpAT21 aphA-3
    620CTA TGA CTC GAG GCC GCG GCG CTC GGG ACC CCT ATC TAG CRpAT21 aphA-3
    676GTT TAA GGG CCC ACC TAG GAT CGA ATC CCT TCG TGA GCGFTn554 aad9
    677TCT CTT CTC GAG GCC GCG GTA ATA AAC TAT CGA AGG AACRTn554 aad9
    583TAC GTT GCA TGCAGC TTA CTA TGC CAT TAT TFpI258PblaZ
    584ATT CGT CTG CAGAAT AAA CCC TCC GAT ATT ACRpI258PblaZ
    594TGC GAT GCA TGCGCA CTT ATT CAA GTG TAT TTFpI258Pcad-cadC
    595AAT AAT GTC GAC CTG CAGGTT CAG ACA TTG ACC TTC ACRpI258Pcad-cadC
    616TCG ATA GAA TTC GTT AAC TAA TTA ATA TCG GAG GGT TTA TTT TGFpI258 blaZ
    628AAC AGT GGC GCCTGT CAC TTT GCT TGA TAT ATG AGRpI258 blaZ
    589TCG ATA GAA TTC GTT AAC TAA TTA ATT TAA GAA GGA GAT ATA CAT ATG AGFpDB4 gfpmut2
    662AGC ATA GGC GCG CCT TAT TTG TAT AGT TCA TCC ATG CCARpDB4 gfpmut2
    601AAG ATT GAA TTC GTT AAC TAA TTA ATC ACC AAA AAG GAA TAG AGT ATG AAGFpRL189 luxAB
    612AAG TAT GGC GCG CCA AAA AGG CAA TCT AAT ATA GAA ATT GCCRpRL189 luxAB
    658TTA TCT GAA TTC AGG CGC GCCTAT TCT AAA TGC ATA ATA AAT ACT GFpI258TT
    628AAC AGT GGC GCCTGT CAC TTT GCT TGA TAT ATG AGRpI258TT
    655TTA ATC CGC GGT GTC ATT ATT TCFφ11φ11 EcoRI-K
    656ATC TTC TCG AGT AAA AGG CTT CGG AAG TAGRφ11φ11 EcoRI-K
    P805TTG ATG GGC CCG AAA GAT GTT GGT ATT GAT AAA GAA GFpRN6991SapI1-attS
    P806TGG AAC CTA GGA GCT GGA AAT ATT CGT TAT TTA TGRpRN6991SapI1-attS
    • ↵ a Restriction sites are underlined. Bases complementary to the template are italicized. Bases in boldface represent stop codons.

    • ↵ b F, forward; R, reverse.

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Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria
Emmanuelle Charpentier, Ana I. Anton, Peter Barry, Berenice Alfonso, Yuan Fang, Richard P. Novick
Applied and Environmental Microbiology Oct 2004, 70 (10) 6076-6085; DOI: 10.1128/AEM.70.10.6076-6085.2004

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Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria
Emmanuelle Charpentier, Ana I. Anton, Peter Barry, Berenice Alfonso, Yuan Fang, Richard P. Novick
Applied and Environmental Microbiology Oct 2004, 70 (10) 6076-6085; DOI: 10.1128/AEM.70.10.6076-6085.2004
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KEYWORDS

Genetic Vectors
Gram-Positive Bacteria
plasmids

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