DE19928073B4 - Transconjugation plasmid for the mutagenesis of Pasteurellaceae - Google Patents

Abstract

Transconjugation plasmid for the introduction of unlabelled deletions into the chromosome of bacteria of the family Pasteurellaceae, consisting of:
a) mobilization region mobRP4,
b) a polylinker,
c) a replica of the Col E1 type,
d) a resistance determinant
e) a transcriptional fusion of the Bacillus subtilis sacB gene with the Actinobacillus pleuropneumoniae omlA promoter.

Description

The The present invention relates to a transconjugation plasmid for Mutagenesis of bacteria of the family Pasteurellaceae. More precisely it is a transconjugation plasmid for the introduction of unlabelled Deletions in these bacteria.

Actinobacillus (A.) pleuropneumoniae, a bacterium of the family Pasteurellaceae, is a highly infectious Pathogens in the respiratory tract of pigs, the world to significant economic losses leads (Sebunya and Saunders 1983: Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 182: 1331-1337; Fenwick and Henry 1994: Porcine pleuropneumonia. J. Am. Vet. Med. Assoc. 204: 1224-1340). At the present time are 12 serotypes, identified (based on polysaccharide antigens) (Perry et al., 1990: Structural characteristics of the antigenic capsular polysaccharides and lipopolysaccharides involved in the serological classification of Actinobacillus pleuropneumoniae strains. Serodiag. Immunother. Infect. Dis. 4: 299-308). The on the serotypes based differentiation correlates with results that both by studies on the electrophoretic mobility of outer membrane proteins and enzymes (multi-nucleus enzyme electrophoresis; Musser et al., 1987: Clonal diversity in Haemophilus pleuropneumoniae. Infect. Immune. 55: 1207-1215) as well as by toxin typing (Frey et al., 1995: Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains. Mol. Cell. Probes 9: 277-282) and analysis of relationship relationships due to restriction enzyme polymorphisms (Chevallier et al. 1998: Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae. FEMS Microbiol. Lett. 160: 209-216).

A Vaccination against A. pleuropneumoniae successfully protects against the clinical Illness. However, conventional vaccines are protective in their Effect on the serotype that is responsible for The bacterin production was used, limited and prevent beyond not a colonization by the pathogen (Nielsen 1974: Pleuropneumonia of swine caused by Haemophilus pleuropneumoniae. Studies on the protection obtained by vaccination. Nord. Vet. Med. 28: 337-338; Rosendal et al. 1981: Vaccination against pleuropneumonia in pigs caused by Haemophilus pleuropneumoniae. Can. Vet. J. 22: 34-35; Higgins et al. 1985: Evaluation of a killed vaccine against porcine pleuropneumonia due to Haemophilus pleuropneumoniae. Can. Vet. J. 26: 86-89). In contrast, animals that are a natural or survived experimental infection with an A. pleuropneumoniae serotype have, independent protected from clinical symptoms by the respective serotype (Nielsen 1984: Haemophilus pleuropneumoniae serotypes - cross protection experiments. Nord. Vet. Med. 36: 221-234; Prideaux et al. 1999: Vaccination and Protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the APXII operon. Infect. Immune. 67: 1962-1966), although one Colonization with heterologous serotypes is still possible (Cruijsen et al. 1995: Convalescent pigs are protected completely against infection with a homologous Actinobacillus pleuropneumoniae strain but incompletely against a heterologous serotype strain. Infect. Immune. 63: 2341-2343). Recent work has shown that cross-protection also by vaccination with an attenuated live vaccine to achieve (Prideaux et al 1999: Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the APXII operon. Infect. Immune. 67: 1962-1966).

US 5,801,018 discloses vaccines against Actinobacillus pleuropneumoniae. The vaccines contain at least one transferrin-binding protein and / or a cytolysin of Actinobacillus pleuropneumoniae and / or an Actinobacillus pleuropneumoniae APP4. In addition, the DNA sequences encoding these proteins, the vectors containing these sequences, and the host cells transformed with these vectors are disclosed. The vaccines can be used to treat or prevent respiratory infections in pigs.

US 5, 429, 818 describes unencapsulated mutants of normally encapsulated Actinobacillus pleuropneumoniae strains in use as a vaccine. In particular, an unencapsulated mutant of Actinobacillus (Haemophilus) pleuropneumoniae is described, which protects against pleuropneumonia in pigs.

Methods already described for Actinobacillus pleuropneumoniae mutagenesis have been limited to insertion knockout mutagenesis (Jansen et al., 1995): Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils. 63: 27-37; Fuller et al 1996: A riboflavin auxotroph of Actinobacillus pleuropneumoniae attenuated in swine Infect Immun 64: 4659-4664; Prideaux et al 1999: Vaccination and protection of pigs against pleuropneumo Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the APXII operon. Infect. Immune. 67: 1962-1966) or transposon shuttle mutagenesis techniques (Tascon et al 1993: Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative J. Bact 175: 5717-5722 Tascon-Cabrero et al 1997 Actinobacillus pleuropneumoniae does not require urease activity to produce acute swine pleuropneumonia, FEMS Microbiol., Lett., 148: 53-57). In addition, there are no selectable markers for the use of this bacterium. It is therefore desirable to develop attenuated and phenotypically distinguishable mutants without antibiotic markers as candidate vaccines. Counterselection using the Bacillus subtilis sacB gene has been successfully established in a number of Gram-negative bacteria (Reyrat et al 1998: Counterselectable markers: Untapped tools for bacterial genetics and pathogenesis, Infect. Immun. 66: 4011-4017).

A Method for generating unlabelled deletions in the genome of Helicobacter pylori is from Copass et al. (1997: Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker. Infect immune. 65: 1949-1952). This is a two-stage Transkonjugationssystem described on a plasmid with a kan-sacB cassette under the control of the H. pylori-specific flagellin promoter based.

The Selection of allelic mutant replacements in mycobacteria is reported in U.S. Patent 5,843,664. The used Method is based on a plasmid that as fusion the sacB gene and contains the mutated sequence of the gene to be inactivated.

From Jost et al. (1997: The sacB gene can not be used as a counter-selectable marker in Pasteurella multocida. Mol. Biotechnol. 8: 189-191) It has been shown that the use of the sacB gene as a marker for Counterselection in trunks Pasteurella multocida is not suitable.

task The present invention is therefore a simple and effective genetic tool for introduction unlabelled deletions into the chromosome of family bacteria Pasteurellaceae and here in particular the genus Actinobacillus to disposal to deliver.

These Task is solved by a Transkonjugationsplasmid, which from a mobilizing region mobRP4, a polylinker, one Replicon of the Col E1 type, a resistance-mediating gene and a transcriptional fusion between the Bacillus subtilis sacB gene and the omlA promoter of Actinobacillus pleuropneumoniae. For ease of description of the present invention Be the called plasmid referred to as pBMK1.

A further preferred embodiment The invention is characterized in that a segment of exogenous DNA was used in the polylinker of the plasmid. This segment exogenous DNA includes e.g. a modified Actinobacillus pleuropneumoniae acidC gene in the described polylinker.

A Another object of the present invention is to provide a simple and effective method for the insertion of unlabelled deletions into the chromosome of bacteria of the family Pasteurellaceae and here in the particular of the genus Actinobacillus.

These Object of the present invention is achieved by a method for Mutagenesis of bacteria of the family Pasteurellaceae solved, which According to the invention, the steps transconjugation of the plasmid from an Escherichia coli mobilization strain in the mutagenized bacterial strain of the family Pasteurellaceae comprises and positive selection or screening for auxotrophy of the Pasteurellaceae strain containing the transconjugated plasmid stably integrated into the chromosome, as well as a counterselection with sucrose and a screening of the sucrose-resistant strain to a strain which has an unlabelled deletion at the corresponding Owns the site of the chromosome.

One usable according to the invention Plasmid is pBMKΔ1. This vector may e.g. as a positive test system for the inventive Procedure can be used. In a further preferred embodiment of the present invention the bacteria to be mutated into the genus Actinobacillus. Most preferred is a strain of Actinobacillus pleuropneumoniae.

Further preferred embodiments are in the dependent claims characterized.

Around a universal system for introduction unlabeled mutations in Actinobacillus pleuropneumoniae For example, the vector pBMK1 was constructed. For the use of this vector Transconjugants are necessary, the stable plasmid cointegrate contained and selected based on their antibiotic resistance are. The following counterselection requires only a single crossover event to produce an unlabelled deletion mutant. This inventive system facilitates the effective introduction an unlabelled ureC deletion into the genome of Actinobacillus pleuropneumoniae. In principle allows it is also the consecutive introduction of various unlabelled mutations. This system facilitates the construction of an unimpregnated Actinobacilluspleuropneumoniae live vaccine, the, in addition to have a determinable and easily detectable phenotype, a differentiation between vaccinated and infected animals.

Around a transconjugation plasmid according to the invention to disposal To put, was a transcriptional fusion between the Actinobacillus pleuropneumoniae omlA promoter and the Bacillus subtilis sacB gene carried out. Surprisingly could be shown that a Escherichia coli Actinobacillus pleuropneumoniae shuttle vector, containing this construct, in the presence of 10% sucrose effectively from the Actinobacillus pleuropneumoniae transformants could be cured. This means that sacB is counterselective Marker in Actinobacillus pleuropneumoniae can be used while in the literature is described that this in Pasteurella (P.) multocida, also a relative the family Pasteurellaceae, is not the case (Jost et al 1997: The sacB gene can not be used as a counter-selective marker in Pasteurella multocida. Mol. Biotechnol. 8: 189-191).

Surprisingly could be shown that the Method based on a transcriptional fusion between the sacB gene and the Actinobacillus pleuropneumonia omlA promoter including a subsequent positive and negative selection, used unmarked knockout mutants in Actinobacillus pleuropneumoniae, a relative of the family Pasteurellaceae, to construct.

The The invention will be better understood by the following detailed description of present invention, in conjunction with the appended claims, the Description and the attached figures, better understood, wherein:

1 shows the physical map of the plasmid constructed within the scope of the invention. The construction of plasmids pJUK04, pJUOK14 and pJUOK27 required some intermediate cloning steps. Briefly, a BamHI-PstI fragment (containing the sacB gene) was ligated into the BamHI-PstI site of the pBluescript SK vector; then the PstI fragment from pUC4K (carrying kanamycin resistance (km ^r )) was ligated into the PstI site to give plasmid pSB5. Finally, the omlA promoter was amplified by PCR using primers olp1B and olp2, the PCR product was digested with BamHI and BglII and ligated into the unique BamHI site in both orientations, resulting in plasmid pSOP12 (opposite direction) and pSOP15 (transcriptional fusion ) resulted. Thereafter, the ureC-sacB-km ^r cassette was constructed. Briefly, the ureC gene was amplified by PCR using primers ureC2 and ureX, the PCR product was cut with BamHI and SalI and ligated into a BamHI and SalI cut pBluescript SK vector. Thereafter, the SphI-BstEII fragment was removed from the ligated ureC gene, the enzyme cleavage sites were blunt-ended, and a blunt-ended XbaI-SalI fragment containing the sacB-km ^r cassette from pSB5, pSOP12 or pSOP15 was inserted. From these pBluescript derivatives, the ureC-sacB-km ^r cassette was located on a blunt-ended ApaI-SacI fragment and ligated into the blunt-ended ClaI sites of pJFF244 resulting in plasmids pJUK04, pJUOK14 and pJUOK27. The plasmids pRUOK23 and pRUOK29 contain the same ApaI-SacI fragment as present in pJUOK 27; it was ligated in both orientations into the blunt-ended EcoRI site of pBOR8. The construction of plasmid pBMK1 also required some intermediate cloning steps. The sacB-km ^r cassette was removed from pSOP15 on a SacI-SalI fragment, blunt-ended, and cloned into the blunt-ended EcoRI site of pBOR8, resulting in the plasmid pROKB1. A BamHI fragment from pBOR (containing mobRP4) was blunt-ended and ligated into the SacI-cut and blunt-ended pBluescript-SK resulting in the vector pBluescript-RP4. Thereafter, a blunt-ended BamHI-SacI fragment from pROKB1 (containing the sacB-km ^r cassette) was ligated into a blunt-ended KpnI site of pBluescript-RP4, thus constructing the pBMK1. The open boxes marked with "C" and the arrows indicate the position and orientation of the ureC sequence The open arrow labeled sacB and km ^r indicates the location and orientation of the sacB gene and kanamycin resistance mediated determinants Arrowhead at one end of the sacB gene marks the location and orientation of the omlA promoter (omlA-P). The unique restriction enzyme sites in pBMK1 are marked with an asterisk.

2 shows the results of the Plasmid'curing 'experiment with Actinobacillus pleuropneumoniae AP76 transformants grown in PPLO broth without (a, b) and with (c, d) sucrose. The transformants were plated on PPLO agar plates without (a, c) and with (b, d) kanamycin selection. The filled symbols show the arithmetic mean of four independent experiments, the bars document the standard deviation.

3 Figure 4 shows a schematic representation of the individual crossover events that led to Actinobacillus pleuropneumoniae AP76 41 (unlabeled urease negative). The filled boxes on the intact ureC gene indicate the presence of the sequence; the open boxes indicate the section which was deleted in ureC. The broken line marks the homologous recombination events.

4 shows the PCR analysis of Actinobacillus pleuropneumoniae AP76 (1), AP76 41 (2) and the negative control (3). (A) PCR with the primers ureC2 and ureX; (B) Southern blot analysis with BstEII and SphI digested DNA using the ureC gene as a probe; (C) pulsed-field gel electrophoresis of ApaI, AscI and NotI-digested DNA; the white arrowhead marks the ApaI fragment on which the ureC gene could be mapped.

Based on the results of the plasmid'curing' experiment followed up the construction of an unlabeled, mutagenic mutant.

The ureC gene was selected as a suitable target because urease-negative mutants very fast due to their phenotype can be identified. This not only appeared beneficial to the mutagenesis procedure but also the loss of urease activity is a excellent phenotypic Markers for known attenuated Actinobacillus pleuropneumoniae live vaccines, obtained by chemical mutagenesis (Inzana et al., 1993: Safety, stability and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines. Infect. Immune. 61: 1682-1686; Byrd and Hooke 1997: Temperature-sensitive mutants of Actinobacillus pleuropneumoniae induce protection in mice. Infect. Immune. 65: 2206-2210); so is the vast one Majority of clinical Actinobacillus pleuropneumoniae isolates urease positive and the absence of urease activity affects the virulence of Actinobacillus pleuropneumoniae not (Tascon-Cabrero et al. 1997: Actinobacillus pleuropneumoniae does not require urease activity to produce acute swine pleuropneumonia. FEMS Microbiol. Lett. 148: 53-57).

The original approach to construct an unlabelled ureC mutant was based on the directed mutagenesis system described for Actinobacillus pleuropneumoniae (Mulks and Buysse 1995: A targeted mutagenesis system for Actinobacillus pleuropneumoniae, Gene 165: 61-66). The transconjugation vectors pRUOK23 and pRUOK29 carried the sacB-km ^r cassette in the ureC gene and thus reflected the general characteristics of the plasmids previously used for the introduction of unlabelled mutations into other bacteria (Blomfield et al 1991: Allelic exchange in Microbiol 5: 1447-1457; Copass et al 1997: Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker. 65: 1949-1952). This approach requires an initial double crossover recombination as well as a second transconjugation step before counterselection. Our futile effort to obtain a single urease-negative, kanamycin-resistant and sucrose-sensitive Actinobacillus pleuropneumoniae serotype 7 mutant indicates low recombination activity.

Example 1: 8. subtilis sacB Gene Mediated Plasmid'curing 'in Actinobacillus pleuropneumoniae

The clinical Actinobacillus pleuropneumoniae serotype 7 isolate AP76 was electrotransformed with the plasmids designated pJUK04, pJUOK14 and pJUOK27 ( 1 ). All three plasmids are based on the shuttle vector pJFF224, a low copy plasmid, that replicates in Actinobacillus pleuropneumoniae (Frey 1992: Consturction of a broad-spectrum shuttle vector for gene cloning and expression in Actionobacillus pleuropneumoniae and other Microbiol 143: 263-269) All plasmids carried a cassette containing the sacB gene from 8. subtilis (Gay et al 1983: Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the genes in Escherichia coli J. Bacteriol 153: 1424-1431) and the Tn903-derived kanamycin resistance determinant (km ^r ) located in Actinobacillus pleuropneumoniae ureC gene (Bosse and McInnes 1997) between the plasmids was the presence and orientation of the omlA promoter derived from Actinobacillus pleuropneumoniae (Gerlach et al., 1993: Molecular characterization of a protec tive outer membrane protein (OmlA) from Actinobacillus pleuropneumonia serotype 1. Infect. Immune. 61: 565-572; 1 ; Table 1). To check the efficiency of plasmid'curing 'in the transcriptional fusion between the sacB gene product and the omlA promoter, the Actinobacillus pleuropneumoniae transformants were grown in liquid culture with and without the addition of 10% sucrose and then on plates with and without Kanamycin plated ( 2 ). The total number of colony-forming units (KbE) remained constant at approximately 5 × 10 ⁷ CFU / ml over an incubation period of 4 hours, irrespective of the presence of 10% sucrose ( 2a . 2c ). During this incubation period, a decrease in plasmid-containing cells could be observed even in the absence of sucrose since the number of kanamycin-resistant (km ^r ) colony forming units decreased from 1 x 10 ⁷ CFU / ml to 1 x 10 ⁵ CFU / ml ( 2 B ). In the presence of sucrose, the number of plasmid-bearing bacterial cells decreased significantly from 1 × 10 ⁷ CFU / ml to less than 40 CFU / ml within 60 minutes for the transformants that contained sacB-omlA transcriptional promoter fusion. In contrast, no increased plasmid loss during this time was observed in bacteria containing sacB without transcriptional fusion with the Actinobacillus pleuropneumoniae omlA promoter ( 2d ).

These Results show that plasmids, the one transcriptional fusion between the sacB gene and the omlA promoter, as a suicide vector in Actinobacillus pleuropneumoniae can be used.

Example 2: Construction an unlabelled urease-negative Actinobacillus pleuropneumoniae knockout mutant

The ureC-km ^r -sacB cassette of pJUOK27 was isolated in both orientations in pBOR8 (Herrero et al., 1990: Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes into gram-negative bacteria. Bacteriol 172: 6557-6567) and gave pRUOK23 and pRUOK29 ( 1 ). Both plasmids were transfected into Actinobacillus pleuropneumoniae AP 76 using E. coli β2155 as a donor strain (Dehio and Meyer 1997: Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella hensalae following conjugal plasmid transfer from Escherichia coli. J. Bacteriol 179: 538-540). Although total pleuropneumoniae transconjugants were examined more than 500 km of ^r Actinobacillus, no ureasenegativen transconjugants were obtained. However, since double crossover mutants are required for subsequent counterselection (Blomfield et al 1991: Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon, Mol. Microbiol. 5: 1447-1457, Copass et al., 1997: Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker, Inf. Immun., 65: 1949-1952), this method was evaluated as inappropriate.

On the other hand, another approach has been followed that does not require two consecutive, double crossover mutational events, but works based on two consecutive single crossover mutations ( 3 ). The vector pBMK1 (Table 1; 1 ) was constructed and the ureC gene ligated with an internal 870 bp deletion (ure C) adjacent to the km ^r -sacB cassette. The resulting plasmid was designated pBMKU 1 (Table 1, 1 ) and transconjugated into Actinobacillus pleuropneumoniae AP76. Between ten and twenty transconjugants could be obtained per trial and all Km ^r transconjugants were urease positive; 20% were sucrose-sensitive and contained plasmid cointegrates. In the counter-selection with sucrose, 5% of the sucrose-resistant colonies were also urease-negative. Five of these independent experiments colonies all contained identical double crossover mutations without foreign DNA, which could be confirmed by polymerase chain reaction (PCR) and Southern blot ( 4a . 4b ). For one of these mutants designated as Actinobacillus pleuropneumoniae AP76 41, it was possible to demonstrate in pulsed-field gel electrophoresis that no major deletions or chromosomal rearrangements took place ( 4c ). In addition, nucleotide sequence analysis revealed an identical ureC sequence in pBMKU 1 and in Actinobacillus pleuropneumoniae AP76 41, demonstrating that, as expected, a double crossover mutation has occurred ( 3 ). Electro-transformation of Actinobacillus pleuropneumoniae AP7641 with plasmids pFOKU1 and pFOKU4 (Table 1) restores urease activity when the intact open reading frame of ureC forms a transcriptional fusion unit with the vector-derived T4 promoter. These results show that the plasmid pBMK1, which contains mobRP4, a polylinker containing the Tn903-derived kanamycin determinant and a transcriptional fusion of the B.subtilis sacB gene and the Actinobacillus pleuropneumoniae omlA promoter, can be used to efficiently construct unlabeled mutations in Actinobacillus pleuropneumoniae.

The bacterial strains, plasmids and primers used in the present invention are listed in Table 1. E. coli strains were amplified in Luria-Bertani medium (Sambrook et al., 1989: Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, NY) with addition of the appropriate Chemotherapeutic agents (ampicillin 100 μg / ml, kanamycin 50 μg / ml, chloramphenicol 25 μg / ml). For the cultivation of E. coli β2155 (dap), diaminopimelic acid (1 mM, Sigma Chemical Company, Deisenhofen) was added. Actinobacillus pleuropneumoniae strains were cultured in PPLO medium (Difco GmbH, Augsburg) supplemented with nicotinamide dinucleotide (NAD 10 μg / ml, Merck, Darmstadt), L-glutamine (100 μg / ml, Serva, Heidelberg), L-cysteine hydrochloride (260 μg / ml, Sigma), L-cysteine dihydrochloride (10 μg / ml, Sigma) and dextrose (1 mg / ml). Kanamycin (25 μg / ml) was added for the selection of Actinobacillus pleuropneumoniae transconjugants. For the sucrose counterselection, a salt-free stock solution (2x) was prepared for use in liquid and plate culture. Has been described in brief Bacto Beef Heart Infusion For ^® (37 g / l, Difco) for 1 h at 50 ° C, boiled for 3 minutes and filtered to remove solid components to. It was Bacto peptone ^® (7.5 g / l, Difco) was added; The solution was adjusted to pH 7.4 and sterile filtered. Prior to use, this stock solution was enriched as described above, the appropriate antibiotic added and then mixed with an equal volume of 20% sucrose or to prepare solid nutrient media with 20% sucrose and 3% Bacto agar (Difco).

The urease became like this determined: bacterial colonies were transferred to nitrocellulose platelets; these were then transferred to empty petri dishes and with indicator agarose (0.5% agarose cooled to 50 ° C and enriched with urea (20 mg / ml) and phenol red (100 μg / ml)). The urease distinctive red coloring developed after 2 to 10 minutes.

to Modification of the DNA were DNA-modifying enzymes of New England Biolabs (Bad Schwalbach) and according to the manufacturer's instructions used. The DNA or plasmid DNA was for use in PCR and prepared by standard protocols in the Southern blot (Sambrook et al., 1989). transformations Gel electrophoresis, PCR and Southern blots were performed according to appropriate Standard procedures performed (Sambrook et al., 1989).

The Electro transformation was performed with a Gene Pulser (BioRad, Munich) according to the instructions of the Manufacturer, but with the addition of Tween 80 (0.02%, Roth, Karlsruhe), tryptone and yeast extract into the electrotransformation medium (Tung and Chow 1995). DNA from E. coli β2155 was on Actinobacillus pleuropneumoniae via filter-mating technique transferred to Dehio and Meyer (1997).

After transconjugation, Km ^r Actinobacillus pleuropneumoniae colonies were assayed for the presence of crossover events in the PCR with the primer pairs ureC2 and ureX (Reaction 1) and the primers ureC2 and kan2 (Reaction 2, Table 1). Colonies with the correct PCR profile were confirmed by Southern blot analysis using the kanamycin determinant and the ureC gene as probes.

For the counterselection The supplemented PPLO medium was 1/10 volume overnight a confirmed, single crossover mutant inoculated and for Shaking for 2 h incubated. The bacteria were pelleted to a sucrose-containing fertile soil plated and over Incubated night. Sucrose resistant colonies were transferred to nitrocellulose platelets and urease activity tested.

Ureasenegative mutants were analyzed by PCR and Southern blotting as described above. Additional confirmation was obtained by pulsed field gel electrophoresis (Chevailler et al 1998: Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae, FEMS Microbiol., Lett: 160: 209-216) using restriction endonucleases ApaI, AscI and NotI, as well as by nucleotide sequence analysis of ureC Gene.

Claims (13)

Transconjugation plasmid for the introduction of unlabelled deletions in the chromosome of bacteria of the family Pasteurellaceae, consisting out: a) mobilization region mobRP4, b) a polylinker, c) a replica of the Col E1 type, d) a resistance determinant e) a transcriptional fusion of the Bacillus subtilis sacB gene with the Actinobacillus pleuropneumoniae omlA promoter. Plasmid according to claim 1, characterized in that the Resistance determinant of Tn903-derived kanamycin resistance determinant is. Plasmid according to one of the preceding claims, characterized in that a Segment of exogenous DNA is inserted into the polylinker. Plasmid according to claim 3, characterized in that the inserted into the polylinker exogenous DNA segment is mutated. Plasmid according to claim 3, characterized in that the Segment of exogenous DNA is a modified Actinobacillus pleuropneumoniae acidC gene contains which is inserted in the polylinker. Method for mutagenesis of a bacterium of the family Pasteurellaceae, comprising the steps a) a transconjugation the plasmid according to claim 3 from an Escherichia coli mobilization strain into one to be mutagenized Strain of a bacterium of the family Pasteurellaceae, b) one positive selection or screening for auxotrophy of Pasteurellaceae Strain that stably integrates the transconjugated plasmid into the chromosome wearing, c) a counterselection with sucrose and d) a screening of sucrose-resistant strains on a strain containing an unlabelled deletion at the point of interest Body of the chromosome carries. Method according to claim 6, characterized in that the Actinobacillus pleuropneumoniae to be mutagenized Trunk is. Bacterium of the family Pasteurellaceae, characterized that this A bacterium containing the plasmid according to claim 3 is transformed. Bacterium according to claim 8, characterized in that the Bacterium is an auxotrophy caused by the transformation contains. Bacterium according to claim 8, characterized in that the Bacterium is a strain of the genus Actinobacillus. Host cells stably transformed with a plasmid according to claim 1. Host cells stably transformed with a plasmid according to claim Third Kit for introduction unlabelled deletions into the chromosome of a bacterium of the family Pasteurellaceae containing the plasmid according to claim 3.