pGSTp: An IVET-Compatible Promoter Probe Vector Conferring Resistance to Trimethoprim

Promoter probe plasmids that can integrate into the chromosome via homologous recombination are an important tool in studying gene regulation. These vectors have proved useful in identifying bacterial genes that are specifically expressed during infection of cultured cells or animal models. A common system that is used is in vivo expression technology (IVET) (9). The original IVET-based vectors involve cloning random chromosomal DNA fragments upstream of a promoterless purA gene (9). Bacterial genes, which are active during infection, are identified by the ability of their cognate promoters to effect the complementation of a purA auxotroph. One disadvantage of these systems is the requirement for a bacterial strain containing a nonrevertable auxotrophic mutation. This potential drawback can be circumvented by using a promoterless antibiotic resistance gene and selecting for bacterial co-integrate clones that resist antibiotic challenge during infection (10). A promoterless chloramphenicol acetyltransferase (cat) gene has been found to be useful in this respect, especially for studying pathogens that can adopt an intracellular lifestyle, as chloramphenicol can penetrate mammalian cells (10,16). Plasmids such as pIVET8 and pGY2 (10,16), which contain the bla gene (and also the aadA gene in the case of pGY2), are only useful in Gram-negative bacteria that are sensitive to β-lactams and streptomycin. However, many pathogenic bacteria, such as members of the genus Burkholderia, exhibit high levels of resistance to both antibiotics. Here, we describe the construction and use of a promoter probe suicide vector that contains a trimethoprim resistance marker, which allows selection in many bacteria that are naturally resistant to most βlactams and aminoglycosides. The promoter probe vector pKK2328 (Amersham Pharmacia Biotech, Little Chalfont, UK) contains a 660-bp promoterless cat gene preceded by stop codons in all three reading frames (1). Using oligonucleotide primers designed to incorporate single BamHI, KpnI, SmaI, BglII and XbaI sites upstream of cat and a restriction site for XhoI downstream of cat, the complete polypeptide coding sequence, together with its cognate ribosome binding site but excluding the stop codons, was amplified by PCR (13). The primers used were: catf, 5′-GCGGGATCCGGTACCCGGGAGATCTTCTAGAAGGAGCTAAGGAAGCTAA-3′ and catr, 5′-GCGCTCGAGCACTTATTCAGGCGTAGC-3′ (cat Shine-Dalgarno and stop codon sequences are underlined). The 880-bp PCR product was digested with BamHI and XhoI and cloned between the compatible BglII and SalI sites of the mobilizable suicide vector pUTmini-Tn5Cm (3) to create pGS18. This step resulted in substitution of the promoterless cat cassette for the mini-Tn5Cm transposon and adjacent tnp gene. Benchmarks