Draft Genome Sequences of Actinobacillus pleuropneumoniae Serotypes 2 and 6

Abstract

Actinobacillus pleuropneumoniae is a bacterial pathogen that causes highly contagious respiratory infection in pigs and has a serious impact on the production economy and animal welfare. As clear differences in virulence between serotypes have been observed, the genetic basis should be investigated at the genomic level. Here, we present the draft genome sequences of the A. pleuropneumoniae serotypes 2 (strain 4226) and 6 (strain Femo).Previous studies of different serotypes of Actinobacillus pleuropneumoniae showed that there are significant variations among them at the DNA sequence level, supposed to cause differences in pathogenicity and immunogenicity (2,8). However, it is difficult to carry out more general studies of the immunity mechanisms of different serotypes, typing-based diagnosis, and multivalent genetically engineered vaccines due to the lack of complete genome sequences of the different serotypes (11).Draft genome sequences of serotypes 2 and 6 were assembled by combining Roche 454-FLX reads with Illumina Genome Analyzer IIx paired-end reads. The final assembly (CLCGenomicsWorkbench version 3.6, CLCbio) of the serotype 2 genome has a length of 2,314,315 bp (38 contigs), while the assembly of serotype 6 has a length of 2,375,501 bp (36 contigs). The average GC contents are 41.17% and 40.95% in serotypes 2 and 6, respectively, similar to those reported for other serotypes (1, 12). Using EasyGene (6, 9), 2,100 putative open reading frames were predicted for serotype 2 and 2,168 for serotype 6. Approximately 86% of the nucleotides were predicted to be involved in coding sequences, which is similar to results reported for other finished A. pleuropneumoniae genomes (1, 12). There are 86 and 136 genes found to be specific for serotypes 2 and 6, respectively, that do not have any homologue in other reported serotypes. rRNA genes were identified by RNAmmer (5). Serotype 2 harbors six rRNA operons (16S-23S-5S rRNA) and three additional 5S rRNA genes, while six rRNA operons and one additional 5S rRNA gene are present in serotype 6. Using the tRNAscan-SE server (7), 61 and 59 tRNA operons were predicted for serotypes 2 and 6, respectively.Since porcine pleuropneumonia caused by A. pleuropneumoniae leads to large economic losses for the swine industry (3), it is of considerable interest to investigate the virulence factors of the different serotypes. We compiled a list of 105 known and putative virulence genes of A. pleuropneumoniae from published literature and performed a diversity study of these genes at the nucleotide level, comparing serotypes 2 and 6. Sixty-two conserved virulence genes (Ͼ99% identity or fewer than three mismatches) were found between these two serotypes, while 28 virulence genes showed a larger degree of dissimilarity (Ͻ95% identity or more than 20 mismatches), including candidates like the apxIVA gene encoding RTX toxin and the cysI gene encoding NADPH-sulfite reductase hemoprotein. Capsular polysaccharides (CPS) produced by A. pleuropneumoniae are considered to be important virulence f...

Experimental Identification of Actinobacillus pleuropneumoniae Strains L20 and JL03 Heptosyltransferases, Evidence for a New Heptosyltransferase Signature Sequence

“…Similarly five homologues of these heptosyltransferases are found in A. pleuropneumoniae genome strains JL03 (serotype 3) [27] and AP76 (serotype 7) [28]. The presence of these heptosyltransferases is in apparent agreement with the five heptose residues present in the L20 strain core LPS (3 L,D-Hep and 2 D,D-Hep) [23] (Figure 1).…”

“…These heptosyltransferases were predicted on the basis of the nature of the substrate heptose, either L,D-Hep or D,D-Hep, and the linkage to the corresponding substrate core residue. In order to attribute putative functions we performed a bioinformatic analysis based on the alignment (Clustal W) of heptosyltransferases whose function is experimentally proved with those of A. pleuropneumoniae strains L20 (serotype 5b) [26], JL03 (serotype 3) [27], and AP76 (serotype 7) [28]. After this alignment, we performed a phylogenetic analysis using the same heptosyltransferases whose function is experimentally proved and the ones obtained with A. pleuropneumoniae strains.…”

“…The analysis was performed by running the phylogenetic inference software Protpars from the PHYLIP (Phylogeny Inference Package) package version 3.5c (Department of Genetics University of Washington, Seattle) [47], using heptosyltransferases whose function is experimentally proved from enteric bacteria, Aeromonas or Pasteurella multocida with those of A. pleuropneumoniae strains L20 (serotype 5b) [26], JL03 (serotype 3) [27], and AP76 (serotype 7) [28]. The cladogram is indicative by the separation and the large of the lanes from the values obtained running the program (the scale bar indicates an evolutionary distance of 0.1 aminoacid substitutions per position).…”

Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1–18, and development of two multiplex PCRs for comprehensive capsule typing

“…Although sequences for the CPS loci of serovars 14, 15, and 16 have previously been deposited in Genbank (accession numbers AB810251 , AB701753 , KX907602 ), we have extended these sequences (accession numbers MG868948 , MG868949 , MG868950 ) to encompass the complete CPS loci in order to allow a more thorough comparison with the other serovars. Additionally, we have generated draft genomes of two serovar K2:O7 isolates in order to compare their CPS loci (accession numbers MG868951 and MG868952 ) to those in the two published serovar 2 genomes (accession numbers ADXN00000000 and ADOE00000000 ) ( Xu et al, 2010 ; Zhan et al, 2010 ). Although the genes in the K2:O7 CPS loci encode the same proteins as in the serovar 2 CPS loci, there are differences at the nucleotide level that explain amplification of both serovar 2 and 8 amplicons for K2:O7 isolates using our previously designed mPCR ( Bossé et al, 2014 ).…”

The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

“…With the availability of whole genome sequences for most serovars of A. pleuropneumoniae [36] – [39] , it is now possible, using HS143 and/or MIDG2331, to systematically mutate specific highly conserved core genes in order to determine their contribution to the biology and pathogenesis of this bacterium, with a view to improving diagnostics, therapies and vaccine strategies.…”