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The complete nucleotide sequence of a 7.7kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (sigma(E)) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of sigma(E), among a bank of random transposon mutants, as well as to detect induction of sigma(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 pMC-Express, 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.

A novel fibronectin-binding protein from Pasteurella multocida (PM1665) that binds to the fibronectin type III(9-10) modules via two helix-hairpin-helix motifs has recently been described [1]. This protein shares homology with competence-related DNA-binding and uptake proteins (ComEA and ComE) from Gram-positive and Gram-negative bacteria. Here, we show that recombinant PM1665 (now designated ComE1) also binds to DNA through the same helix-hairpin-helix motifs required for fibronectin-binding. This binding to DNA is non sequence-specific and is confined to double-stranded DNA. We have cloned and expressed ComE1 proteins from five members of the Pasteurellaceae in order to further investigate the function(s) of these proteins. When expressed as recombinant GST-fusion proteins, all of the homologues bound both to fibronectin and to double-stranded DNA. Inactivation of the gene encoding the ComE1 homologue in Actinobacillus pleuropneumoniae indicates major roles for these proteins in at least two processes: natural transformation, and binding of bacteria to fibronectin.

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease in pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organisation was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as "complete", indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Grace's Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of the PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu-like phage. [ABSTRACT FROM AUTHOR]

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen Actinobacillus pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆ hfq , which showed slightly reduced growth throughout the 24 h time course, and the complemented Ap8∆ hfq C mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella , and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae , but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes. [ABSTRACT FROM AUTHOR]

Evidence of plasmids carrying the tetracycline resistance gene, tet (B), was found in the previously reported whole genome sequences of 14 United Kingdom, and 4 Brazilian, isolates of Actinobacillus pleuropneumoniae. Isolation and sequencing of selected plasmids, combined with comparative sequence analysis, indicated that the four Brazilian isolates all harbor plasmids that are nearly identical to pB1001, a plasmid previously found in Pasteurella multocida isolates from Spain. Of the United Kingdom isolates, 13/14 harbor plasmids that are (almost) identical to pTetHS016 from Haemophilus parasuis. The remaining United Kingdom isolate, MIDG3362, harbors a 12666 bp plasmid that shares extensive regions of similarity with pOV from P. multocida (which carries blaROB-1 , sul2 , and strAB genes), as well as with pTetHS016. The newly identified multi-resistance plasmid, pM3362MDR, appears to have arisen through illegitimate recombination of pTetHS016 into the stop codon of the truncated strB gene in a pOV-like plasmid. All of the tet (B)-carrying plasmids studied were capable of replicating in Escherichia coli , and predicted origins of replication were identified. A putative origin of transfer (oriT) sequence with similar secondary structure and a nic -site almost identical to that of RP4 was also identified in these plasmids, however, attempts to mobilize them from an RP4-encoding E. coli donor strain were not successful, indicating that specific conjugation machinery may be required. [ABSTRACT FROM AUTHOR]

Objectives: To characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae, and analyse the functional nature of the encoded FloR.Methods: ICEApl2 was identified in the genome of A. pleuropneumoniae MIDG3553. Functional analysis was done using conjugal transfer experiments. MIDG3553 was tested for susceptibility to the antimicrobials for which resistance genes are present in ICEApl2. Lack of florfenicol/chloramphenicol resistance conferred by the encoded FloR protein was investigated by cloning and site-directed mutagenesis experiments in Escherichia coli.Results: ICEApl2 is 92660 bp and contains 89 genes. Comparative sequence analysis indicated that ICEApl2 is a member of the SXT/R391 ICE family. Conjugation experiments showed that, although ICEApl2 is capable of excision from the chromosome, it is not self-transmissible. ICEApl2 encodes the antimicrobial resistance genes floR, strAB, sul2 and dfrA1, and MIDG3553 is resistant to streptomycin, sulfisoxazole and trimethoprim, but not florfenicol or chloramphenicol. Cloning and site-directed mutagenesis of the floR gene revealed the importance of the nature of the hydrophobic amino acid residues at positions 160 and 228 in FloR for determining resistance to florfenicol and chloramphenicol.Conclusions: Our results indicate that the nature of hydrophobic residues at positions 160 and 228 of FloR contribute dynamically to specific efflux of florfenicol and chloramphenicol, although some differences in resistance levels may depend on the bacterial host species. This is also, to our knowledge, the first description of an SXT/R391 ICE in A. pleuropneumoniae or any member of the Pasteurellaceae. [ABSTRACT FROM AUTHOR]

Bacterial respiratory diseases are responsible for considerable mortality, morbidity and economic losses in the swine industry. Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is one of the most important disease agents, but its identification and surveillance can be impaired by the existence of many other related bacteria in normal swine microbiota. In this work, we have evaluated a BOX-A1R-based repetitive extragenic palindromic- PCR ( BOX- PCR) sequence characterised amplified region ( SCAR) marker for the specific identification of A. pleuropneumoniae and its use in a multiplex PCR to detect additionally Haemophilus parasuis and Pasteurella multocida, two other major respiratory pathogens of pigs that are members of the family Pasteurellaceae. PCRs based on the BOX- SCAR fragment developed were rapid, sensitive and differentiated A. pleuropneumoniae from all swine-related members of the Pasteurellaceae family tested. Single and multiplex BOX- SCAR fragment-based PCRs can be used to identify A. pleuropneumoniae from other bacterial swine pathogens and will be useful in surveillance and epidemiological studies. [ABSTRACT FROM AUTHOR]

We have identified a highly transformable strain of Actinobacillus pleuropneumoniae whose competence is regulated by the competence-activator Sxy as in other Pasteurellaceae. Other strains were poorly transformable or nontransformable. The genomes of two poorly transformable strains contain intact sets of competence genes. Moreover, we show that the low competence of one of these strains is not due to an inability to induce sxy expression or to a defect in Sxy function, suggesting that some other component of the competence system is defective. Although the A. pleuropneumoniae sxy gene has only 24% identity to its Haemophilus influenzae homologue, both genes fully complemented an H. influenzae sxy knockout, demonstrating that Sxy function is conserved throughout the Pasteurellaceae. [ABSTRACT FROM AUTHOR]

A small (3.9 kb) plasmid (p518), conferring resistance to florfenicol (MIC >8 μg/mL) and chloramphenicol (MIC >8 μg/mL) was isolated from an Actinobacillus pleuropneumoniae clinical isolate from Southeastern Brazil. To date, this is the smallest florfenicol resistance plasmid isolated from a member of the Pasteurellaceae. The complete nucleotide of this plasmid revealed a unique gene arrangement compared to previously reported florfenicol resistance plasmids found in other members of the Pasteurellaceae . In addition to the floR gene and a lysR gene, common to various florfenicol resistance plasmids, p518 also encodes strA and a partial strB sequence. An origin of replication ( oriV ) similar to that in the broad host range plasmid, pLS88, was identified in p518, and transformation into Escherichia coli MFD pir confirmed the ability to replicate in other species. Mobilisation genes appear to have been lost, with only a partial mobC sequence remaining, and attempts to transfer p518 from a conjugal donor strain ( E. coli MFD pir ) were not successful, suggesting this plasmid is not mobilisable. Similarly, attempts to transfer p518 into a competent A. pleuropneumoniae strain, MIDG2331, by natural transformation were also not successful. These results suggest that p518 may be only transferred by vertical descent. [ABSTRACT FROM AUTHOR]