Novel cassette-based shuttle vector system for gram-positive bacteria

Abstract

Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

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-att_S (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), P_blaZ (constitutive β-lactamase promoter module), and P_cad-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 P_blaZ. 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 P_cad-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.