Comparative Genomics of Actinobacillus pleuropneumoniae Serotype 8 Reveals the Importance of Prophages in the Genetic Variability of the Species

Abstract

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia. Currently, there are 18 different serotypes; the serotype 8 is the most widely distributed in the United States, Canada, United Kingdom, and southeastern Brazil. In this study, genomes of seven A. pleuropneumoniae serotype 8 clinical isolates were compared to the other genomes of twelve serotypes. The analyses of serotype 8 genomes resulted in a set of 2352 protein-coding sequences. Of these sequences, 76.6% are present in all serotypes, 18.5% are shared with some serotypes, and 4.9% were differential. This differential portion was characterized as a series of hypothetical and regulatory protein sequences: mobile element sequence. Synteny analysis demonstrated possible events of gene recombination and acquisition by horizontal gene transfer (HGT) in this species. A total of 30 sequences related to prophages were identified in the genomes. These sequences represented 0.3 to 3.5% of the genome of the strains analyzed, and 16 of them contained complete prophages. Similarity analysis between complete prophage sequences evidenced a possible HGT with species belonging to the family Pasteurellaceae. Thus, mobile genetic elements, such as prophages, are important components of the differential portion of the A. pleuropneumoniae genome and demonstrate a central role in the evolution of the species. This study represents the first study done to understand the genome of A. pleuropneumoniae serotype 8.

Figure 1

Analysis of similarity between predicted amino acid sequences of A. pleuropneumoniae serotype 8 and the other serotypes. The protein-coding sequences were clustered according to similarity in percentages to serotype 8. The serotypes were also characterized in relation to virulence.

Figure 2

Use of codons and their respective amino acids of A. pleuropneumoniae. (a) The trend in the use of codons is represented in the circular map. Methionine, tryptophan, and stop codons were omitted. Synonymous codons for an amino acid used with equal frequency have RSCU = 1, indicated by the red circular line. (b) Percentage of the use of amino acids represented in the circular map. A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine.

Figure 3

Synteny analysis of A. pleuropneumoniae genomes. The genomes represented correspond to serotype 3 (JL03 strain), serotype 5 (L20 strain), serotype 7 (AP76), and serotype 8 (MIDG2331, MV460, MV518, MV597, MV780, MV1022, and MV5651 strains). Horizontal bars represent the size of the genome (kb). The region identified in 1 represents the acquisition and loss of genomic information, and region 2 represents a recombination event.

Figure 4

Phylogenetic relationships between sequences related to complete prophages found in A. pleuropneumoniae. The phylogenetic tree using the Neighbor-Joining method with 2000 bootstraps was generated by the MEGA 6 program after alignment by MAFFT and GBLOCKS. The scale is represented below, with 0.1 nucleotide substitutions per site.

Figure 5

Heat map analysis from identity matrix generated by global alignment of 16 complete prophage genomes. The alignment was done using Clustal Omega, and the identity matrix generated was used to create the heat map by R software.