First chromosomal restriction map of Actinobacillus pleuropneumoniae and localization of putative virulence-associated genes

Abstract

Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The single SalI site within the chromosome of strain A. pleuropneumoniae AP76 was defined as position 1 of the map; for the map of A. pleuropneumoniae ATCC 27088, the corresponding SalI site was chosen. Putative virulence-associated genes (apx, omlA, sodA, tbpBA, ureC, and a repeat element) and housekeeping genes (glyA, metJ, recA, and rhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.