Novel cassette-based shuttle vector system for gram-positive bacteria.
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.
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, NYU Medical Center, New York, USA. emmanuelle.charpentier@univie.ac.at.
Emmanuelle
E
Charpentier
Ana I
AI
Anton
Peter
P
Barry
Berenice
B
Alfonso
Yuan
Y
Fang
Richard P
RP
Novick
D019677:Artificial Gene Fusion; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors; D006094:Gram-Positive Bacteria / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012093:Replicon; D015183:Restriction Mapping; D013204:Staphylococcus Phages / Q000235:genetics; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity; D014774:Virulence / Q000235:genetics
D004269:DNA, Bacterial; D005819:Genetic Markers