First Chromosomal Restriction Map ofActinobacillus pleuropneumoniaeand Localization of Putative Virulence-Associated Genes

Abstract

Combined physical and genetic maps of the genomes ofActinobacillus 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, andSalI. 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, andrhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.

Actinobacillus pleuropneumoniaeserotype 7 siderophore receptorFhuAis not required for virulence

“…The fhuA gene is localized on fragments APA3, ASC1 and NOT1, respectively (Fig. 2D), and therefore not co-localized with any virulence-associated genes previously mapped [12].…”

Identification ofBrachyspira hyodysenteriae-specific DNA fragments using representational difference analysis

“…Gel electrophoresis, Southern blot, transformations and PCR were done following standard protocols[17]. For pulsed‐field gel electrophoresis (PFGE) bacteria were washed off the plates using PET IV solution (1 M NaCl, 10 mM Tris–HCl [pH 8], 10 mM Na 2 EDTA), and DNA‐imbedded agarose plugs were prepared as described previously[18]. Agarose plugs were cleaved with the endonucleases Eag I (isoischizomer to Ecl XI) and Sal I (both New England Biolabs, Bad Schwalbach, Germany), and were separated in a 1% agarose gel in 0.5× TBE (45 mM Tris–borate [pH 8], 1 mM Na 2 EDTA) using a CHEF‐DR ® III pulsed‐field electrophoresis system (Bio‐Rad, München, Germany) with the settings: 6 V cm −1 , 12°C, block I from 7 to 12 s for 11 h and block II from 20 to 65 s for 13 h. For an improved separation of fragments with sizes between 300 and 1000 kb ramped switch times from 20 to 80 s for 24 h were used.…”

Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis

“…Gel electrophoresis, Southern blot, transformations and PCR were done following standard protocols [17]. For pulsed-¢eld gel electrophoresis (PFGE) bacteria were washed o¡ the plates using PET IV solution (1 M NaCl, 10 mM Tris^HCl [pH 8], 10 mM Na 2 EDTA), and DNAimbedded agarose plugs were prepared as described previously [18]. Agarose plugs were cleaved with the endonucleases EagI (isoischizomer to EclXI) and SalI (both New England Biolabs, Bad Schwalbach, Germany), and were separated in a 1% agarose gel in 0.5U TBE (45 mM Tris^borate [pH 8], 1 mM Na 2 EDTA) using a CHEF-DR 0 III pulsed-¢eld electrophoresis system (Bio-Rad, Mu « nchen, Germany) with the settings: 6 V cm 31 , 12 ‡C, block I from 7 to 12 s for 11 h and block II from 20 to 65 s for 13 h. For an improved separation of fragments with sizes between 300 and 1000 kb ramped switch times from 20 to 80 s for 24 h were used.…”