Functional Pathogenomics of Mucosal Immunity    R.E.W Hancock Laboratory    The Brinkman Laboratory
Pseudomonas aeruginosa PAO1mini-Tn5 lux transposon mutant library

Mutant Construction and Transposon Insertion Mapping

The mini-Tn5-lux transposon was delivered to P. aeruginosa PAO1 by conjugation with the donor strain E. coli S17-1 lambda-pir (pUTmini-Tn5-luxCDABE-Tc) (Winson et al., 1998). Biparental mating mixtures of the donor and recipient (1:2) were incubated on LB agar for 6-8 hours at 37°C. Cells were resuspended in PBS and plated onto large 22 X 22 cm LB agar plates containing 50 µg/ml tetracycline (Tc) to select for P. aeruginosa transconjugants. This Tc concentration kills the E. coli donor. Transconjugants were robotically picked to 96-well plates containing 100 µl of LB broth + 50 µg/ml Tc, grown overnight and frozen after the addition of DMSO (7% final concentration) for long-term storage at -80°C.
A high-throughput inverse PCR protocol was developed for the mapping of transposon sites in which all steps were performed in a 96-well format with the use of a DNA Engine Tetrad gradient cycler (MJ Research, Waltham, MA). Cells were harvested from 24 hour cultures (1.2 ml) and genomic DNA was isolated using the DNeasy Kit (Qiagen, Valencia, CA) according to manufacturer’s recommendations. Genomic DNA was examined for purity and quantity on 1% agarose-Tris-acetate-EDTA (TAE) gels. For amplication of DNA flanking the left side of the transposon, SstII digestion, ligation and inverse PCR were performed (SstII-IPCR) while for amplification of the right side of the transposon, NarI digestion, ligation and inverse PCR (NarI-IPCR) were performed. Alternatively, SphI was used instead of SstII for amplification of the left side of the transposon. These restriction enzymes were chosen because their cut sites are present in the transposon and they were likely to cut near the site of insertion due to the high number of cut sites in the PAO1 genome. Between 250-500 ng genomic DNA was digested overnight with 2 U of NarI (NEB, Mississauga, Canada) or SstII (Invitrogen, Carlsbad, CA) (20 µl reaction volume) and heat inactivated at 65°C (SstII or 85°C (NarI). T4 DNA Ligase (0.5U) (Invitrogen, Carlsbad, CA) and its corresponding buffer was added to the digestion reaction to bring the volume to 25 µl, and incubated overnight with temperatures cycling between 10°C and 30°C. The ligation product was used as template for an inverse PCR reaction. [see supplementary information for PCR conditions and primer sequences]. The PCR reaction products were run on 1% agarose-TAE gels and all samples yielding a single major band were selected for clean up with magnetic beads (Agencourt, Beverly, MA) and sequencing. PCR products were sequenced with nested primers at the University of Victoria Sequencing Centre (Victoria, Canada) or occasionally in our lab using Big Dye Terminator chemistry (Applied Biosystems, Foster City, CA) on a Basestation 51 Fragment Analyzer (MJ Research, Waltham, MA).
Transposon insertion sites in the PAO1 genome were determined using an automated process whereby a PERL script parsed the top scoring BLASTN alignment to each query sequence. The coordinates for each insertion were then used to reference an annotation table of ORFs and intergenic regions. A second PERL script was used to identify transcriptional versus non-transcriptional fusions given the orientation of the gene where the insertion had occurred, the restriction enzyme used in the initial digestion and the orientation of the transposon relative to the origin of replication in the PAO1 genome.

Winson, M.K., S. Swift, P.J. Hill, C.M. Sims, G. Griesmayr, B.W. Bycroft, P. Williams, and Stewart, G.S. 1998. Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs. FEMS Microbiol Lett 163: 193-202.

McPhee, J.B., S. Lewenza, and Hancock, R.E.W. 2003. Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50: 205-217.

Supplementary information