Comparative Genomics of the First and Complete Genome of “Actinobacillus porcitonsillarum” Supports the Novel Species Hypothesis

Abstract

“Actinobacillus porcitonsillarum” is considered a nonpathogenic member of thePasteurellaceaefamily, which phenotypically resembles the pathogenActinobacillus pleuropneumoniae. Previous studies suggested that “A. porcitonsillarum” may represent a new species closely related toActinobacillus minor, yet no full genome has been sequenced so far. We implemented the Oxford Nanopore and Illumina sequencing technologies to obtain the highly accurate and complete genome sequence of the “A. porcitonsillarum” strain 9953L55. After validating ourde novoassembly strategy by comparing theA. pleuropneumoniaeS4074Tgenome sequence obtained by Oxford Nanopore Technology combined with Illumina reads with a PacBio-sequenced S4074Tgenome from the NCBI database, we performed comparative analyses of the 9953L55 genome with theA. minortype strain NM305T,A. minorstrain 202, andA. pleuropneumoniaeS4074T. The 2,263,191 bp circular genome of 9953L55 consisted of 2168 and 2033 predicted genes and proteins, respectively. The lipopolysaccharide cluster resembled the genetic organization ofA. pleuropneumoniaeserotypes 1, 9, and 11, possibly explaining the positive reactions observed previously in serotyping tests. In contrast to NM305T, we confirmed the presence of a completeapxIICABDoperon in 9953L55 and 202 accounting for their hemolytic phenotype and Christie-Atkins-Munch-Petersen (CAMP) reaction positivity. Orthologous gene cluster analysis provided insight into the differential ability of strains of theA. minor/“porcitonsillarum” complex andA. pleuropneumoniaeto ferment lactose, raffinose, trehalose, and mannitol. The four strains showed distinct and shared transposable elements, CRISPR/Cas systems, and integrated prophages. Genome comparisons based on average nucleotide identity andin silicoDNA-DNA hybridization confirmed the close relationship among strains belonging to theA. minor/“porcitonsillarum” complex compared to otherActinobacillusspp., but also suggested that 9953L55 and 202 belong to the same novel species closely related toA. minor, namely, “A. porcitonsillarum.” Recognition of the taxon as a separate species would improve diagnostics and control strategies of pig pleuropneumonia.