PHYSICAL MAPPING OF 32 GENETIC-MARKERS ON THE PSEUDOMONAS-AERUGINOSA PAO1 CHROMOSOME

The Pseudomonas aeruginosa chromosome was fractionated with the enzyme s 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 mappe d. 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 definit ion 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 probe s, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and sin gle-stranded DNA prepared from phagemid libraries that had been random ly sequenced. We used a PCR approach to clone fragments of the putativ e 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 aco A, catR, hemD pheS, pro5, oprD, pyo and rpsB gene homologues. The desc ribed genomic methods permit the rapid mapping of the P. aeruginosa ge nome without linkage analysis.