Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km r ) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km r -sacB cassette. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Upon iron restriction, Actinobacillus pleuropneumoniae has been shown to express the transferrin-binding proteins TbpB and TbpA, both of which have been implied to be important virulence factors. In order to identify additional iron-regulated proteins, we cloned and analyzed the region upstream of the transferrinbinding protein genes in an A. pleuropneumoniae serotype 7 strain. We located immediately upstream of the tbpB gene two open reading frames which were 43% homologous to the neisserial ExbBD protein genes. By raising specific antibodies, we showed that ExbB is expressed under iron-limiting growth conditions only, and RT-PCR analysis revealed that the exbBD genes and the tbpB gene are transcribed on a single polycistronic mRNA. By constructing an isogenic and nonpolar exbBD mutant, we showed that the exbBD genes are required by A. pleuropneumoniae for utilization of transferrin-bound iron. Using PCR and Western blotting, we showed that the genetic organization found in A. pleuropneumoniae serotype 7 is similar in all 12 A. pleuropneumoniae serotype reference strains.

Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km(r)-sacB cassette.

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.

Summary During a search for conserved target sequences on the genome of Coxiella burnetii for a diagnostic PCR a NotI/EcoRV DNA fragment was cloned and sequenced from the isolate “Nine Mile”, Phase I (sequence data are registered under accession number X70045 in data bases EMBL, GENE‐BANK and DDBJ). On this fragment we have found a sequence of 1741 base pairs which showed an exceptionally high homology (60 % on the nucleic acid level and 49.6 % on the amino acid level) to the sporulation gene “spoIIIE” of the grampositive bacterium Bacillus subtilis. This is first evidence of the molecular basis for formation of spores in Coxiella burnetii which has been postulated in the literature by electron microscopic investigations.