Physical mapping of 32 genetic markers on the Pseudomonas aeruginosa PAO1 chromosome

1 Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver BC, Canada V6T 1Z3 2 Microbiologie Moléculaire et Génie des Protéines, Département de Microbiologie, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4 3 Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 4 Department of Medical Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada T2N 4N1 5 Author for correspondence: Roger C. Levesque. Tel: +1 418 656 3070. Fax: +1 418 656 7176. e-mail: rclevesq@rsvs.ulaval.ca ABSTRACT The Pseudomonas aeruginosa chromosome was fractionated with the enzymes Spel and Dpnl, and genomic fragments were separated by PFGE and used for mapping a collection of 40 genes. This permitted the localization of 8 genes previously mapped and of 32 genes which had not been mapped. We showed that a careful search of databases and identification of sequences that were homologous to known genes could be used to design and synthesize DNA probes for the mapping of P. aeruginosa homologues by Southern hybridization with genomic fragments, resulting in definition of the locations of the aro-2, dapB, envA, mexA, groEL, oprH, oprM, oprP, ponA, rpoB and rpoH genetic markers. In addition, a combination of distinct DNA sources were utilized as radioactively labelled probes, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and single-stranded DNA prepared from phagemid libraries that had been randomly sequenced. We used a PCR approach to clone fragments of the putative yhhF, sucC, sucD, cypH, pbpB, murE, pbpC, soxR, ftsA, ftsZ and envA genes. Random sequencing of P. aeruginosa DNA from phagemid libraries and database searching permitted the cloning of sequences from the acoA, catR, hemD, pheS, proS, oprD, pyo and rpsB gene homologues. The described genomic methods permit the rapid mapping of the P. aeruginosa genome without linkage analysis. Keywords: Pseudomonas aeruginosa chromosome, physical mapping, genetic markers