DIFFERENTIAL GENOME ANALYSIS OF BACTERIA BY GENOMIC SUBTRACTIVE HYBRIDIZATION AND PULSED-FIELD GEL-ELECTROPHORESIS

A comprehensive analysis of the differences between the genomes of two closely related bacterial strains should give insight into the molecu lar basis of their individual phenotypic and genotypic characteristics . Here we present an integrative approach including two different stra tegies for the thorough investigation of genomic divergence. We have c ombined two techniques including genomic subtractive hybridization and comparative genome mapping by pulsed field gel electrophoresis (PFGE) techniques. The subtractive method for which a protocol is given here in results in the production of a library of specific DNA sequence tag s present only in one strain, while the construction of macrorestricti on maps of the bacterial chromosomes yields data about the overall gen ome organization and the arrangement and distance of gene loci. Compar ison of the physical and genetic maps and determination of the map pos itions of the strain-specific DNA sequences reveals gross chromosomal modifications, insertions or deletions of additional genetic material, and transpositional events. The further investigation of the strain-s pecific regions yields information about the nature and origin of the acquired DNA and their influence on the evolution of the individual ba cterial genome. The two methods were applied to differential genome an alysis of clonal divergence in Pseudomonas aeruginosa choosing two clo ne C isolates from diverse habitats.