Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative

Tascon, R.I.; Rodríguez-Ferri, Elías Fernando; Gutiérrez-Martín, César B.; Rodríguez-Barbosa, José-Ignacio; Berche, Patrick; Vázquez‐Boland, José A.

doi:10.1128/jb.175.17.5717-5722.1993

Cited by 61 publications

(33 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1). The BglII sites on the 5.2-kb plasmid were filled in by reaction with Klenow enzyme and ligated to the 1.2-kb SalI fragment of pUC4K (also blunt ended) containing the Tn903 npt gene (34), which confers kanamycin resistance (Kan r ) to A. pleuropneumoniae (52). The resulting suicide vector was designated pAp12KB.…”

Section: Methodsmentioning

confidence: 99%

Association ofActinobacillus pleuropneumoniaeCapsular Polysaccharide with Virulence in Pigs

et al. 2003

View full text Add to dashboard Cite

The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan r ) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan r gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074⌬cps1N and strain 4074⌬cps1B, respectively. Strain 4074⌬cps1N produced no detectable CP, but strain 4074⌬cps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074⌬cps1N to produce 4074⌬cps1N(pABcps101), 4074⌬cps1N(pJMLcps53), and 4074⌬cps1N(pABcps55), respectively. Strain 4074⌬cps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074⌬cps1N (pJMLcps53) and 4074⌬cps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074⌬cps1N(pABcps101) > strain 4074⌬cps1N > strain 4074⌬cps1B. Strain 4074⌬cps1N(pJMLcps53) was less virulent than strain 4074⌬cps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

show abstract

Section: Methodsmentioning

confidence: 99%

Association ofActinobacillus pleuropneumoniaeCapsular Polysaccharide with Virulence in Pigs

et al. 2003

View full text Add to dashboard Cite

show abstract

“…The suicide plasmid pLOF/Km containing a miniTn10 transposon was conjugally transferred from E. coli S17-1(-pir) into CM5 Nal r as previously described (39). Transconjugants were selected on BHIV-NAL-KAN, and loss of the suicide plasmid was confirmed by patching colonies onto BHIV-NAL-KAN-AMP.…”

Section: Methodsmentioning

confidence: 99%

Novel Genes Affecting Urease Activity inActinobacillus pleuropneumoniae

2001

View full text Add to dashboard Cite

Characterization of a series of urease-negative transposon mutations of Actinobacillus pleuropneumoniae revealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster. A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes. As well, a partial ORF, apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation. The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators. Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system. The cbiM and cbiQ genes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and the cbiO gene encodes a cobalt transport ATP-binding protein homologue. The product of the cbiK gene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein. Escherichia coli clones containing this putative transport operon together with the urease genes of A. pleuropneumoniae were urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl 2 for full urease activity. This result supports the hypothesis that nickel is a substrate for this permease system. The sixth ORF, utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function in A. pleuropneumoniae remains to be determined.The genes required for urease activity in various bacterial species typically include those encoding the structural subunits and those encoding the accessory proteins involved in insertion of two nickel ions within the catalytic site (24). In bacteria with urea-inducible gene clusters, the regulatory gene, ureR, is also present (24). Because of the requirement for nickel as a cofactor, genes such as Helicobacter pylori nixA and Alcaligenes eutrophus hoxN that encode nickel uptake systems have also been shown to affect urease activity (23,41).We recently identified the urease gene cluster of the gramnegative swine pathogen Actinobacillus pleuropneumoniae (4). The organization of the A. pleuropneumoniae gene cluster was found to be similar to that of other bacterial ureases, with the first three genes encoding the structural subunits (UreABC), and the accessory proteins (UreEFGD) encoded by four contiguous genes downstream. There was no evidence of a regulatory gene (ureR) upstream of the cluster.In order to further characterize the urease activity of A. pleuropneumoniae, a bank of transposon mutants of strain CM5 Nal r was generated and screened for urease-negative isolates. A large number of insertions in the urease-negative mutants mapped upstream of the urease gene cluster. Therefore, the 5-kbp reg...

show abstract

“…Random transposon mutagenesis is a valuable technique for the discovery of new virulence factors in bacteria. For A. pleuropneumoniae, the transposon Tn10 has been the most widely used (8,20,38,39,44,46). We (44) and others (20), have modified the Tn10 delivery vehicle described by Tascon et al (46) to allow high-throughput screening for virulence factors using signature-tagged mutagenesis.…”

mentioning

confidence: 99%

New Plasmid Tools for Genetic Analysis of Actinobacillus pleuropneumoniae and Other Pasteurellaceae

Bossé

Durham

Rycroft

et al. 2009

Appl Environ Microbiol

View full text Add to dashboard Cite

We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae. A tandem reporter plasmid, pMC-Tandem, carrying promoterless xylE and gfpmut3 genes downstream of a multiple-cloning site (MCS), can be used for identification of transcriptional regulators and conditions which favor gene expression from different cloned promoters. The ability to detect transcriptional regulators using the tandem reporter system was validated in A. pleuropneumoniae using the cloned rpoE ( E ) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of E , among a bank of random transposon mutants, as well as to detect induction of E following exposure of A. pleuropneumoniae to ethanol or heat shock. pMCsodCP, carrying the cloned sodC promoter of A. pleuropneumoniae, was functional in A. pleuropneumoniae, Haemophilus influenzae, Haemophilus parasuis, Mannheimia haemolytica, and Pasteurella multocida. Two general expression vectors, pMK-Express and pMCExpress, which differ in their antibiotic resistance markers (kanamycin and chloramphenicol, respectively), were constructed for the Pasteurellaceae. Both plasmids have the A. pleuropneumoniae sodC promoter upstream of the gfpmut3 gene and an extended MCS. Replacement of gfpmut3 with a gene of interest allows complementation and heterologous gene expression, as evidenced by expression of the Haemophilus ducreyi nadV gene in A. pleuropneumoniae, rendering the latter NAD independent.Actinobacillus pleuropneumoniae is the etiological agent of pleuropneumonia, an economically significant disease responsible for substantial morbidity and mortality in the worldwide pig industry (47). Understanding the molecular basis of pathogenicity is important in the design and implementation of vaccine and treatment strategies. Established virulence factors include surface polysaccharides (5, 24), Apx toxins (19, 29), iron uptake systems (24), components of anaerobic metabolism (3,4,10,11,23), and outer membrane proteins (12). In general, these have been discovered through hypothesis-driven research (9). As with other bacterial pathogens, further advances will be facilitated by the availability of microarrays and genetic tools such as transposons and reporter gene plasmids.Whole-genome sequences are available for A. pleuropneumoniae serovars 3 (accession no. NC_010278), 5b (accession no. NC_009053), and 7 (accession no. NC_010942), with others in progress. Microarrays, developed from the whole-genome sequences, have been used for genotyping (22) as well as to identify genes important for iron uptake (15), anaerobicity (10, 11), interaction with host cells (2), and the role of the global regulators H-NS and RpoE in biofilm formation (J. T. Bossé, S. Sinha, C. A. O'Dwyer, J. H. E. N. Nash, A. N. Rycroft, J. S.Kroll, and P. R. Langford, submitted for publication). It is envisaged that ...

show abstract

Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative

Cited by 61 publications

References 36 publications

Association ofActinobacillus pleuropneumoniaeCapsular Polysaccharide with Virulence in Pigs

Association ofActinobacillus pleuropneumoniaeCapsular Polysaccharide with Virulence in Pigs

Novel Genes Affecting Urease Activity inActinobacillus pleuropneumoniae

New Plasmid Tools for Genetic Analysis of Actinobacillus pleuropneumoniae and Other Pasteurellaceae

Contact Info

Product

Resources

About