Construction of a physical and preliminary genetic map of Aerornonas hydrophila IMP636

Department of Microbiology Queensland, Australia 4072 University 2 Author for correspondence: John M. Pemberton. Tel: +61 7 33654640. Fax: +61 7 33654620.e-mail: pembertoa biosci.uq.edu.au ABSTRACT SUMMARY: A physical and preliminary genetic map of the Aeromonas hydrophila JMP636 chromosome has been constructed. The topology of the genome was predicted to be circular as chromosomal DNA did not migrate from the origin during PFGE unless linearized by 51 nuclease. Cleavage of the chromosome with Pacl and PmeI produced 23 and 14 fragments, respectively, and enabled calculation of the genome size at 4.5 Mb. Digestion of the chromosome with I-Ceul produced 10 fragments, indicating that 10 rrl(23S) genes were likely t o be present. Hybridizations between DNA fragments generated with Pad, PmeI and I-Ceul were used t o initially determine the relationship between these segments. To accurately map genes previously characterized from JMP636, the suicide vector pJP5603 was modified to introduce restriction sites for Pad and PmeI, producing pJP9540. Following cloning of genes into this vector and recombinational insertion into the JMP636 chromosome, Pad and Pmel cleavage determined the location of genes within macrorestriction fragmentswith the additional bands produced forming hybridization probes. From the data generated, it was possible t o form a physical map comprising all the fragments produced by Pacl and Pmel, and assign the contig of I-Ceul fragments on this map. The preliminary genetic map defines the location of six loci for degradative enzymes previously characterized from JMP636, while the locations of the 10 sets of ribosomal genes were assigned with less accuracy from hybridization data. Keywords: Aeromonas hydrophila, extracellular enzymes, rrn operons, physical map, genetic map