Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis Form an "Operational Group B. amyloliquefaciens" within the B. subtilis Species Complex

Abstract

The plant growth promoting model bacterium FZB42^T was proposed as the type strain of Bacillus amyloliquefaciens subsp. plantarum (Borriss et al., 2011), but has been recently recognized as being synonymous to Bacillus velezensis due to phylogenomic analysis (Dunlap C. et al., 2016). However, until now, majority of publications consider plant-associated close relatives of FZB42 still as "B. amyloliquefaciens." Here, we reinvestigated the taxonomic status of FZB42 and related strains in its context to the free-living soil bacterium DSM7^T, the type strain of B. amyloliquefaciens. We identified 66 bacterial genomes from the NCBI data bank with high similarity to DSM7^T. Dendrograms based on complete rpoB nucleotide sequences and on core genome sequences, respectively, clustered into a clade consisting of three tightly linked branches: (1) B. amyloliquefaciens, (2) Bacillus siamensis, and (3) a conspecific group containing the type strains of B. velezensis, Bacillus methylotrophicus, and B. amyloliquefaciens subsp. plantarum. The three monophyletic clades shared a common mutation rate of 0.01 substitutions per nucleotide position, but were distantly related to Bacillus subtilis (0.1 substitutions per nucleotide position). The tight relatedness of the three clusters was corroborated by TETRA, dDDH, ANI, and AAI analysis of the core genomes, but dDDH and ANI values were found slightly below species level thresholds when B. amyloliquefaciens DSM7^T genome sequence was used as query sequence. Due to these results, we propose that the B. amyloliquefaciens clade should be considered as a taxonomic unit above of species level, designated here as "operational group B. amyloliquefaciens" consisting of the soil borne B. amyloliquefaciens, and plant associated B. siamensis and B. velezensis, whose members are closely related and allow identifying changes on the genomic level due to developing the plant-associated life-style.

Keywords: Bacillus amyloliquefaciens; Bacillus subtilis group; Bacillus taxonomy; average amino acid identity (AAI); average nucleotide identity (ANI); digital DNA–DNA hybridization; phylogenomics.

Figure 1

Phylogeny of the Bacillus subtilis species complex based on the core genomes of representative type strains. The core genome of Bacillus cereus ATCC14579 was used as outgroup. The roman letters at the branching points designate the four clades identified in this analysis. The numbers at the branching points designate the number of CDS calculated for the core genome of a given subset of genomes. Bootstrap values of 200 (100%) are indicated below the CDS numbers (see Materials and Methods). Percentage of identity according to type strains B. subtilis subsp. subtilis 168^T, B. amyloliquefaciens DSM7^T, B. licheniformis DSM13^T, and B. pumilus SAFR032, respectively. Note that within clade II (“amyloliquefaciens”) the group with B. amyloliquefaciens subsp. plantarum FZB42^T, B. velezensis KCTC 13012^T, and B. methylotrophicus KACC 13105^T is conspecific. The same is true for the group within clade IV (“pumilus”) consisting of B. altitudinis 41KF2b^T, B. stratosphericus LAMA 585^T, and B. aerophilus C772^T. The scale bar corresponds to 0.1 substitutions per site.

Figure 2

NJ phylogenetic tree, extracted from 66 complete rpoB nucleotide sequences with high similarity to B. amyloliquefaciens DSM7^T (>98% identity). B. subtilis subsp. Subtilis 168^T was used as outgroup. The consensus tree was reconstructed from 1000 trees according to the extended majority rule (SEQBOOT program). Bootstrap values >90%, based on 1000 repetitions, are indicated at branch points. Strain and accession numbers are indicated. Type strains for B. amyloliquefaciens (DSM7^T), B. siamensis (KCTC13613^T) and B. vanillea (XY18^T), and the conspecific group containing FZB42^T as the type strain for B. amyloliquefaciens subsp. Plantarum, B. velezensis KCTC13012^T, and B. methylotrophicus KACC13105^T are in bold. Bar, 0.01 substitutions per nucleotide position. For further characterization of strains and genomes see Table 1.

Figure 3

NJ phylogenomic tree, constructed from the 66 core genomes with the highest similarity to DSM7^T (Table 1). The B. subtilis genome was used as outgroup. The number of core genome CDSs is indicated at the nodes. They were calculated for the respective subsets of genomes. Bootstrap values obtained from 200 repetitions are also indicated at the nodes. Type strains (T) are indicated by bold letters. Bar, 0.02 substitutions per nucleotide position.

Figure 4

Dendrogram of the type strains of clade I (operational group “B.subtilis”) and II (operational group “B. amyloliquefaciens”) based on their median ANIb values (upper part of the Table) and Tetra-nucleotide correlation signatures (lower part of the Table). The median nucleotide percent identity values between the orthologous genes of the core of the selected genomes after pairwise BLASTN comparison are indicated. Standard deviation values are given in parentheses.