Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
Tables
- TABLE 1.
Bacterial strains and plasmids
Strain or plasmid Relevant characteristic(s) Source or reference Strains E. coli DH5α Host for DNA cloning Lab strain collection TOP10 Host for DNA cloning Invitrogen Life Technologies S. aureus NCTC8325 Propagating strain for typing phage 47 Lab strain collection RN11 Harbors pI258; lysogenic for φ11, φ12, and φ13 Lab strain collection RN27 RN450 lysogenic for 80α, φ13 16 RN450 NCTC8325 cured of φ11, φ12, and φ13 16 RN1329 8325-4; lysogenic for φ11; Spr [Tn554(aad9)] Lab strain collection RN2424 8325 (pT181) Lab strain collection RN2442 8325-4 (pE194) Lab strain collection RN4220 Restriction-defective derivative of RN450 11 RN4253 8325 (pRN8061) Lab strain collection RN4416 RN27 (pSA0331) Lab strain collection RN5830 8325-4 (pC194) Lab strain collection RN6851 RN6502 (pRN6680) Lab strain collection RN6734 agr+; 8325-4 derivative Lab strain collection RN7206 RN6734Δagr::tet(M) Lab strain collection RN7885 JM109 (pRL189) Lab strain collection RN8972 RN4220 (pRN6998) 25 RN9011 RN4220 (pRN7023) 25 Plasmids pUC19 Apr; ColE1 compatibility group origin of replication 867; 2686 bp 32 pT181 pT181wt; Tcr; Inc3; naturally occurring; ±22 copies/cell; 4,440 bp 1, 17 pRN8061 pT181cop-623; Tcr; Inc3; ±400 copies/cell; 4,440 bp 1, 17 pSA0331 pT181cop-634repC4; Tsr; Tcr; Inc3; ±145 copies/cell; 4,440 bp 1, 17 pI258 Naturally occurring β-lactamase plasmid; 28 kb 21 pE194 Emr (ermC); Inc11; naturally occurring; 3725 bp 5 pAT21 Apr Kmr; pBR322Ω1.5-kb pJH1 ClaI (aphA-3) 28 pC194 Cmr (cat194); Inc8; naturally occurring; 2,910 bp 6 pRN6680 Apr Tcr; pBluescriptΩ2.9-kb pMVN6 Smal-HindII [tetA(M)] 15 pRN6998 pE194tsΩpUC18-MCSΩSaPI1sek-int-attS; 8,319 bp 25 pRN7023 pC194tsΩpUC19-MCSΩSaPI1int 25 pRL189 luxAB Gift of Peter Wolk pBD4 LITMUS28Ωgfpmut2-ermC; 4,784 bp Gift of D. J. Bartels and E. P. Greenberg - TABLE 2.
Oligonucleotide primers used for plasmid construction
Primer Sequence (5′-3′)a F or Rb DNA template Cassette 672 GAA TAT GCA TGC GAT AAT GGC GCCTTT GCG GAA AGA GTT AGT AAG TTA ACA G F pT181, pRN8061, pSA0331 pT181 replicon 671 TGA ACG GGC CCAATA AAA GCA ATC AAT GAA CCA AG R pT181, pRN8061, pSA0331 pT181 replicon P386 TAC TTG GGG CCCTCG ATG ATT ACC AGA AGT TCT C R pI258 pI258 replicon P387 TAG CAT GGC GCCGTT TTA TCT TCA TCA CTT GTA TTA ATC F pI258 pI258 replicon 673 GAT CAT CTC GAG CGG CCG CAT AGT TAA GCC AGC CCC GAC F pUC19 amp ColE1 ori 623 GAT TAG CAT GCA GCG GCC GCC AGC TCA CTC AAA GGC GG R pUC19 amp ColE1 ori 675 CTC TGC GGG CCC ACC TAG GAA TTG AAT GAG ACA TGC TAC F pE194 ermC 674 TCT CTT CTC GAG CGC CGC GGA AAA CTG GTT TAA GCC GAC R pE194 ermC 599 GTT TAA GGG CCC ACC TAG GCA AAT ATG CTC TTA CGT GC F pRN6680 tet(M) 600 CTA TGA CTC GAG GCC GCG GAA ATA TTG AAG GCT AGT CAG R pR6680 tet(M) 617 GTT TAA GGG CCC ACC TAG GTA TTA TCA AGA TAA GAA AG F pC194 cat194 618 CTA TGA CTC GAG GCC GCG GCC TTC TTC AAC TAA CGG GG R pC194 cat194 619 GTT TAA GGG CCC ACC TAG GGG TTT CAA AAT CGG CTC CG F pAT21 aphA-3 620 CTA TGA CTC GAG GCC GCG GCG CTC GGG ACC CCT ATC TAG C R pAT21 aphA-3 676 GTT TAA GGG CCC ACC TAG GAT CGA ATC CCT TCG TGA GCG F Tn554 aad9 677 TCT CTT CTC GAG GCC GCG GTA ATA AAC TAT CGA AGG AAC R Tn554 aad9 583 TAC GTT GCA TGCAGC TTA CTA TGC CAT TAT T F pI258 PblaZ 584 ATT CGT CTG CAGAAT AAA CCC TCC GAT ATT AC R pI258 PblaZ 594 TGC GAT GCA TGCGCA CTT ATT CAA GTG TAT TT F pI258 Pcad-cadC 595 AAT AAT GTC GAC CTG CAGGTT CAG ACA TTG ACC TTC AC R pI258 Pcad-cadC 616 TCG ATA GAA TTC GTT AAC TAA TTA ATA TCG GAG GGT TTA TTT TG F pI258 blaZ 628 AAC AGT GGC GCCTGT CAC TTT GCT TGA TAT ATG AG R pI258 blaZ 589 TCG ATA GAA TTC GTT AAC TAA TTA ATT TAA GAA GGA GAT ATA CAT ATG AG F pDB4 gfpmut2 662 AGC ATA GGC GCG CCT TAT TTG TAT AGT TCA TCC ATG CCA R pDB4 gfpmut2 601 AAG ATT GAA TTC GTT AAC TAA TTA ATC ACC AAA AAG GAA TAG AGT ATG AAG F pRL189 luxAB 612 AAG TAT GGC GCG CCA AAA AGG CAA TCT AAT ATA GAA ATT GCC R pRL189 luxAB 658 TTA TCT GAA TTC AGG CGC GCCTAT TCT AAA TGC ATA ATA AAT ACT G F pI258 TT 628 AAC AGT GGC GCCTGT CAC TTT GCT TGA TAT ATG AG R pI258 TT 655 TTA ATC CGC GGT GTC ATT ATT TC F φ11 φ11 EcoRI-K 656 ATC TTC TCG AGT AAA AGG CTT CGG AAG TAG R φ11 φ11 EcoRI-K P805 TTG ATG GGC CCG AAA GAT GTT GGT ATT GAT AAA GAA G F pRN6991 SapI1-attS P806 TGG AAC CTA GGA GCT GGA AAT ATT CGT TAT TTA TG R pRN6991 SapI1-attS
