Reclassification of Paenibacillus riograndensis as a Genomovar of Paenibacillus sonchi: Genome-Based Metrics Improve Bacterial Taxonomic Classification

Abstract

Species from the genus Paenibacillus are widely studied due to their biotechnological relevance. Dozens of novel species descriptions of this genus were published in the last couple of years, but few utilized genomic data as classification criteria. Here, we demonstrate the importance of using genome-based metrics and phylogenetic analyses to identify and classify Paenibacillus strains. For this purpose, Paenibacillus riograndensis SBR5^T, Paenibacillus sonchi X19-5^T, and their close relatives were compared through phenotypic, genotypic, and genomic approaches. With respect to P. sonchi X19-5^T, P. riograndensis SBR5^T, Paenibacillus sp. CAR114, and Paenibacillus sp. CAS34 presented ANI (average nucleotide identity) values ranging from 95.61 to 96.32%, gANI (whole-genome average nucleotide identity) values ranging from 96.78 to 97.31%, and dDDH (digital DNA-DNA hybridization) values ranging from 68.2 to 73.2%. Phylogenetic analyses of 16S rRNA, gyrB, recA, recN, and rpoB genes and concatenated proteins supported the monophyletic origin of these Paenibacillus strains. Therefore, we propose to assign Paenibacillus sp. CAR114 and Paenibacillus sp. CAS34 to P. sonchi species, and reclassify P. riograndensis SBR5^T as a later heterotypic synonym of P. sonchi (type strain X19-5^T), with the creation of three novel genomovars, P. sonchi genomovar Sonchi (type strain X19-5^T), P. sonchi genomovar Riograndensis (type strain SBR5^T), P. sonchi genomovar Oryzarum (type strain CAS34^T = DSM 102041^T; = BR10511^T).

Keywords: Paenibacillus; Paenibacillus riograndensis; Paenibacillus sonchi; average nucleotide identity; dDDH; phylogeny; taxonomy.

FIGURE 1

16S rRNA gene phylogeny of Paenibacillus species. The 16S rRNA gene rooted tree was constructed using the maximum-likelihood method. aLRT values greater than 70% are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. P. polymyxa is the outgroup. Bacteria of P. riograndensis/P. sonchi clade are in bold. This tree is the same of Supplementary Figure S2, although only taxa of interest were kept.

FIGURE 2

Synteny analysis of P. riograndensis SBR5^T and the complete genome sequences of related species (A), and mapping of draft genome sequences of related species to the P. riograndensis SBR5^T genome (B). Blue line denotes syntenic scaffold alignments. The horizontal bar at the bottom indicates coverage of the matches to the reference scaffold, SBR5^T, with maximal coverage indicated with black, fading to light gray with less coverage. Red indicates uncovered regions.

FIGURE 3

Comparison of predicted proteins of Paenibacillus spp. against the P. riograndensis SBR5 translated genome sequence. The innermost black line circle represents the P. riograndensis SBR5 genome sequence. Each one of the outer rings represents a Paenibacillus strain, as depicted in the legend box. Colored regions of each ring symbolize tblastn hits with at least 30% of identity.

FIGURE 4

Relationship between ANI and 16S rRNA gene identity levels among Paenibacillus species. Each red circle represents a comparison between two Paenibacillus species. Traced line shows the 16S rRNA gene identity threshold for species demarcation. Gray background represents the ANI range for species demarcation. All six red circles over the gray background are pairwise comparisons of P. riograndensis, P. sonchi, Paenibacillus sp. CAR114, and Paenibacillus CAS34.

FIGURE 5

Phylogenies of genes other than 16S rRNA gene of Paenibacillus species. The trees were built using the maximum-likelihood method. Details are as shown in Figure 1, unless specified otherwise.

FIGURE 6

AMPHORA multiprotein phylogeny of Paenibacillus species. The multiprotein rooted tree was constructed using the maximum-likelihood method. Details are as shown in Figure 1, unless specified otherwise.

FIGURE 7

Core-proteome phylogeny of Paenibacillus species. The core-proteome rooted tree was constructed using the Neighbor Joining method. Details are as shown in Figure 1, unless specified otherwise. Bootstrap values greater than 70% are shown next to the branches.