GENETIC AND PHYSICAL MAPS OF THE ALCALIGENES-EUTROPHUS CH34 MEGAPLASMID PMOL28 AND ITS DERIVATIVE PMOL50 OBTAINED AFTER TEMPERATURE-INDUCEDMUTAGENESIS AND MORTALITY

We describe the construction of restriction and genetic maps of plasmi d pMOL28, which has a size of approximately 180 kb. To do so, partial BamHI-digested DNA of pMOL28 was cloned into cosmid pLAFR3, which can package up to 20-30 kb inserted DNA. Subsequently, a cosmid walking st rategy, combined with BamHI or EcoRI restriction analysis and hybridiz ation, was used to construct the restriction maps for both enzymes. On these maps, 35 BamHI fragments and 29 EcoRI fragments were placed, ac counting for a total size of approximately 180 kb. We also analyzed se veral rearranged derivatives of pMOL28 that were obtained after a proc ess of temperature-induced mortality and mutagenesis (TIMM) which is c haracteristic for Alcaligenes eutrophus CH34 and related strains. The restriction and genetic maps of pMOL50 (222 kb), an enlarged derivativ e of pMOL28 obtained after TIMM, were constructed. By comparing the pM OL28 and pMOLSO maps, at least two transposable elements were identifi ed which participated in the formation of pMOLSO from pMOL28 during TI MM. These transposable elements were IS1086, which was recently sequen ced, and a new element named IS1089, which is located on the 44-kb ins erted DNA fragment in pMOL50. Partial sequencing of IS1089 revealed si milarity of this element with IS1071 of the chlorobenzoate catabolic t ransposon Tn5271 of Alcaligenes sp. BR60. (C) 1997 Academic Press.