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A combined physical and genetic map of thePseudomonas putida KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization. Circular genome size was estimated at 6.0 Mb by adding the sizes of 19Swa I, 9Pme I, 6Pac I, and 6 I-Ceu I fragments. A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome withPme I,Swa I, I-Ceu I, andPac I as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I andEscherichia coli , asP. putida DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of theP. putida genome eluted from agarose gels. As an alternative, 10 random mini-Tn5 -Km mutants ofP. putida KT2440 were used as a source of DNA, and the band carrying the mini-Tn5 in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn5 as a probe. We established a circular genome map with an average resolution of 160 kb. Among the 63 genes located on the genetic map were key markers such asoriC , 6rrn loci (rnnA to -F ),recA ,ftsZ ,rpoS ,rpoD ,rpoN , andgyrB ; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds. The genetic map ofP. putida KT2440 was compared to those ofPseudomonas aeruginosa PAO1 andPseudomonas fluorescens SBW25. The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.

Combined Physical and Genetic Map of the Pseudomonas putida KT2440 Chromosome