Abstract
The genus Bacillus constitutes a plethora of species that have medical, environmental, and industrial applications. While genus Bacillus has been the focus of several studies where genomic data have been used to resolve many taxonomic issues, there still exist several ambiguities. Through the use of in-silico genome-based methods, we tried to resolve the taxonomic anomalies of a large set of Bacillus genomes (n = 178). We also proposed species names for uncharacterized strains and reported genome sequence of a novel isolate Bacillus sp. RL. In the hierarchical clustering on genome-to-genome distances, we observed 11 distinct monophyletic clusters and investigated the functional pathways annotated as the property of these clusters and core-gene content of the entire dataset. Thus, we were able to assert the possible outlier strains (n = 17) for this genus. Analyses of secondary metabolite potential of each strain helped us unravel still unexplored diversity for various biosynthetic genes.
| Original language | English |
|---|---|
| Pages (from-to) | 3191-3200 |
| Number of pages | 10 |
| Journal | Genomics |
| Volume | 112 |
| Issue number | 5 |
| Early online date | 5 Jun 2020 |
| DOIs | |
| Publication status | Published - 13 Jun 2020 |
Bibliographical note
Funding Information:The study was supported by grants received from the Indian Council of Agricultural Research funded National Bureau of Agriculturally Important Microorganisms (NBAIM), India [Grant No. NBAIM/AMAAS/2017-20/GF/1a ] and Department of Biotechnology (DBT), India [ BT/PR22797/BCE/8/1413/2016 ].
Funding Information:
This work was supported by funds from ICAR and DBT-funded projects. HK and MS thank ICMR and CSIR for providing research fellowships, respectively. HV thanks Ramjas College, University of Delhi, for providing infrastructural support. RL thanks The National Academy of Sciences, India, for support under the NASI-Senior Scientist Platinum Jubilee Fellowship Scheme.
Funding Information:
The hierarchical clustering of estimated pairwise distance values between 178 genomes were used to construct a dendrogram. The Pearson's correlation analysis based on GGDs clustered strains in 11 distinct clusters designated A to K (Fig. 1). To the best of our knowledge, this is the first description of phylogenetically placing different Bacilli into unique clusters and also highlighting their ambiguous taxonomic assignments. The dDDH values of strain RL with other genomes were well below the cut-off for species designation, i.e., 70% reflecting its status as a novel species under genus Bacillus (Fig. S1). The above-mentioned results were concordant with genome-to-genome distance values. The majority of the strains had pairwise dDDH values in close agreement with 70% for species delineation; however, ambiguities were observed in the phylogenetic placement of certain strains. In cluster D, the pairwise dDDH values between strains Bacillus gibsonii FJAT-10019 and Bacillus murimartini LMG21005 was 90.4%, Bacillus subtilis susbsp. subtilis 168 and Bacillus murimartini LMG21005 was 96.6%, Bacillus gibsonii FJAT-10019 and Bacillus subtilis susbsp. subtilis 168 was 90.7%, which, although represent different species according to the current species affiliation, should be reclassified as strains of Bacillus subtilis. We here proposed the species name for the paradoxical strains based on the first validly described and published species among the comparing species [1, 40?44]. This relatedness was also validated through measures of average nucleotide identity (ANI) and values ?99% were recorded for each pair of genomes compared (Table S2). Also, the dDDH estimates for the following pairs of genomes, Bacillus encimensis SGD-V-25 and Bacillus badius DSM 30822 was 81.6%, Bacillus indicus LMG 22858 and Bacillus cibi DSM 16189 was 80.3%, Bacillus malacitensis NRRL B-41618 and Bacillus axarquiensis NRRL B-41617 was 93.0%. These values far diverged from the threshold for species demarcation supported by ANI values greater than 98% for each pairwise comparison. While proposing their reclassification, we found that such strains have already been updated with their new names in the public databases as for instance, Bacillus encimensis SGD-V-25 had been classified as Bacillus badius; Bacillus cibi DSM 16189 as Bacillus indicus and Bacillus malacitensis NRRL B-41618 and Bacillus axarquiensis NRRL B-41617 as Bacillus halotolerans. Fallacious trends were observed among strains of Bacillus megaterium group (cluster F). Both the dDDH and ANI values between Bacillus megaterium MSP20.1 and other strains of Bacillus megaterium (strains QM B1551, DSM 319 and WSH-002) included in this study were well below the threshold value of 70% for dDDH and 96% for ANI (ANI boundary value for species demarcation is >96%) [45,46], demanding its reclassification as different species (Table S2). Cluster G consisted of 30 strains, of which the pair Bacillus anthracis Ames and Bacillus thuringiensis serovar konkukian 97?27 showed dDDH value of 81.1% whereas the pair of strains Bacillus bombysepticus Wang and Bacillus cereus ATCC 14579 showed dDDH value of 73.9%. These results were further supported by ANI values between the strains. The former pair had ANI value of 98%, and the latter one had 97% ANI. The congruent results of both dDDH and ANI pointed out the need for reclassification of these species. As stated earlier, we here proposed the new species name based on the first described species among the pair. Among Bacillus anthracis Ames and Bacillus thuringiensis serovar konkukian 97?27, strain Ames was first characterized in 1981 [47?49]. Thus, based on the above mentioned dDDH and ANI values and similarities in genomic attributes, we proposed that Bacillus thuringiensis serovar konkukian 97?27 and Bacillus anthracis Ames are conspecific and Bacillus thuringiensis serovar konkukian 97?27 should be reclassified under Bacillus anthracis. Likewise, Bacillus cereus ATCC 14579 was described first by Frankland and Frankland in 1887 [50?52]; thus, Bacillus bombysepticus Wang should be classified as a strain of Bacillus cereus species. Similarly, for pairs of genomes, Bacillus weihenstephanensis WSBC 10204 and Bacillus mycoides ATCC 6462, the dDDH value was 77.8%, and ANI value was 98%. Hence, according to our analysis, both these strains should be classified under the same species, and our proposition was congruent to their current species affiliation in public databases as Bacillus mycoides. This was also reported by Soufiane et al. [53], who observed similarity in these species based on their growth patterns and homology between signature sequences. The dDDH value between Bacillus okuhidensis DSM 13666 and Bacillus halodurans C-125 was 92.1%, and ANI value was 99% reflecting that these strains had higher phylogenetic relatedness and thus we proposed that Bacillus okuhidensis DSM 13666 should be reclassified under Bacillus halodurans based on predated valid characterization of Bacillus halodurans DSM 13666 [54,55]. Another sub-group within this cluster, Bacillus lehensis G1 and Bacillus oshimensis DSM 18940 had 89.6% dDDH, Bacillus lehensis G1 and Bacillus plakortidis DSM 19153 had 89.9% dDDH, Bacillus oshimensis DSM 18940 and Bacillus plakortidis DSM 19153 had dDDH value of 98.5%. These higher dDDH measures were supported by ANI values, which were greater than or equal to 99% for each pair of comparing genomes. Thus, our proposition was that all these strains should be reclassified under Bacillus oshimensis as this species was described first [41,56?58]. Cluster H consisted of five erroneously classified species reflected by their higher dDDH values (in parentheses) between the pairs of taxonomically different species as Bacillus obstructivus VT-16-70 and Bacillus oleronius DSM 9356 (90.4%), Bacillus farraginis DSM 16013 and Bacillus obstructivus VT-16-70 (86.1%), and Bacillus farraginis DSM 16013 and Bacillus oleronius DSM 9356 (84.8%). They all should belong to Bacillus farraginis as this species was first validly characterized among the comparing species [59?61]. The ANI values for each pair of genomes were ?98%. In cluster J, the dDDH estimates between the genome pairs, Bacillus aerophilus C772 and Bacillus altitudinis YNP4-TSU, Bacillus aerophilus C772 and Bacillus cellulasensis NIO-1130, Bacillus aerophilus C772 and Bacillus stratosphericus LK18, Bacillus stratosphericus LK18 and Bacillus cellulasensis NIO-1130 were 85.7%, 82.7%, 82.9%, and 86.3%, respectively. Also, the dDDH values of the pairs Bacillus altitudinis YNP4-TSU and Bacillus stratosphericus LK18, Bacillus altitudinis YNP4-TSU and Bacillus cellulasensis NIO-1130 were 83.2% and 82.9%, respectively. ANI values were 98% for each of the pairwise comparisons. The above mentioned results indicated that different members, Bacillus aerophilus C772, Bacillus altitudinis YNP4-TSU, Bacillus cellulasensis NIO-1130 and Bacillus stratosphericus LK18 should be reclassified as belonging to single species Bacillus altitudinis due to predated valid characterization of this species [62?66]. Also, not belonging to this particular sub-group, pair of species Bacillus clausii KSM-K16 and Bacillus rhizosphaerae SC-N012 showed relatively higher dDDH value of 93.5% and ANI value of 99% demanding reclassification of Bacillus rhizosphaerae SC-N012 as Bacillus clausii because it was first validly described among the pair [67,68]. For uncharacterised species, we had proposed the species name based on its phylogenetically nearest neighbour; Bacillus sp. 2A57CT2 (cluster B) should be classified as Bacillus oceanisediminis 2A57CT2 on the basis of its 91.00% dDDH and 99% ANI with Bacillus oceanisediminis 2691. Similarly, cluster E had an uncharacterised species Bacillus sp. BT1BCT2 for which the species Bacillus licheniformis was proposed on the basis of 97.9% dDDH and 100% ANI with Bacillus licheniformis.The study was supported by grants received from the Indian Council of Agricultural Research funded National Bureau of Agriculturally Important Microorganisms (NBAIM), India [Grant No. NBAIM/AMAAS/2017-20/GF/1a] and Department of Biotechnology (DBT), India [BT/PR22797/BCE/8/1413/2016].
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