DIRECTED INTRODUCTION OF DNA CLEAVAGE SITES TO PRODUCE A HIGH-RESOLUTION GENETIC AND PHYSICAL MAP OF THE ACINETOBACTER SP STRAIN ADP1 (BD413UE) CHROMOSOME

The natural transformability of the soil bacterium Acinetobacter sp. A DP1 (BD413UE), formerly classified as A. calcoaceticus, has facilitate d previous physiological and biochemical investigations, In the presen t studies, the natural transformation system was exploited to generate a physical and genetic map of this strain's 3780+/-191 kbp circular c hromosome. Previously isolated Acinetobacter genes were modified in vi tro to incorporate a recognition sequence for the restriction endonucl ease NotI. Following transformation of the wild-type strain by the mod ified DNA, homologous recombination placed each engineered NotI cleava ge site at the chromosomal location of the corresponding gene. This al lowed precise gene localization and orientation of more than 40 genes relative to a physical map which was constructed with transverse alter nating field electrophoresis (TAFE) and Southern hybridization methods , The positions of NotI, Ascl and I-Ceul recognition sites were determ ined, and the latter enzyme identified the presence of seven ribosomal RNA operons. Multiple chromosomal copies of insertion sequence IS1236 were indicated by hybridization. Several of these copies were concent rated in one region of the chromosome in which a spontaneous deletion of approximately 100 kbp occurred. Moreover, contrary to previous repo rts, CoIE1-based plasmids appeared to replicate autonomously in Acinet obacter sp. ADP1.