Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004

doi:10.21203/rs.3.rs-1603963/v1

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Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004

https://doi.org/10.21203/rs.3.rs-1603963/v1

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In the present study, we attempted to clarify the relationship between Anoxybacillus karvacharensis K1^T and Anoxybacillus kestanbolensis NCIMB13971^T by using whole-genome phylogenetic analysis. The genome sequence of A.kestanbolensis NCIMB13971^T was not available in any database, so it was sequenced in this study. The 16S rRNA gene sequence obtained from the genome of A.kestanbolensis NCIMB13971^T had 99.93% similarity with A.karvacharensis K1^T. Also, A.karvacharensis K1^T and A.kestanbolensis NCIMB 13971^T formed a robust branch different from other type strains of Anoxybacillus in the phylogenetic trees based on whole-genome sequences and 16S rRNA gene sequences.

The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA–DNA hybridization (DDH) values between A.karvacharensis K1^T and A.kestanbolensis NCIMB13971^T were greater than the threshold values for species demarcation. The present results indicate that both strains were considered to belong to the same species and A. karvacharensis K1^T is later heterotypic synonym of A.kestanbolensis NCIMB13971^T.

Anoxybacillus karvacharensis

Anoxybacillus kestanbolensis

genome-based reclassification

The genus Anoxybacillus, a member of the phylum Firmucutes, was first described by Pikuta et al. (2000) describing the type species of this genus Anoxybacillus pushchinoensis. According to the list of LPSN (https://lpsn.dsmz.de/genus/bacillus), the genus Anoxybacillus includes 24 species with validly published names. The taxonomy of Anoxybacillus members was predominantly based on 16S rRNA gene sequence analysis and DNA-DNA hybridization (DDH). However, it is widely known that the resolving power of 16S rRNA gene analysis is generally limited and DDH is difficult to reproduce and sometimes varies depending on the particular method of the laboratory used. Phylogeny using whole-genome sequence based metrics such as average nucleotide identity (ANI), digital DDH and average amino acid identity (AAI) has become an important tool for the delineation of prokaryotic taxa (Orata et al. 2018) and is being used for the reclassifcation of several bacterial taxa (Liu et al., 2019; Rao et al., 2022).

Anoxybacillus. kestanbolensis NCIMB 13971^T was isolated from hot spring in Turkey by Dulger et al. in 2004 and described as validly named species based on a polyphasic taxonomic approach. Anoxybacillus karvacharensis K1^T was isolated from the Karvachar geothermal spring in Nagorno-Karabakh by Panosyan et al. in 2021 and proposed as validly named species based mainly on genomic average nucleotide identity (ANI) value and pairwise digital DNA–DNA hybridization values between the A. karvacharensis K1^T and A. flavithermus, A. flavithermus subsp. yunnanensis, A. tengchongensis, A. mongoliensis, A. pushchinoensis, A. thermarum, A. ayderensis, A. kamchatkensis, A. eryuanensis, A. gonensis, A. rupiensis. Phylogenetic tree based on 16S rRNA gene sequences in the original article showed that A. karvacharensis K1^T, A. flavithermus DSM 2641^T and A. kestanbolensis NCIMB 13971^T clustered together. However, in the original article Panosyan et al. determined only A. flavithermus DSM 2641^T as the nearest neighbour based on 16S rRNA gene sequences similarity and phylogenetic tree, they did not identify A. kestanbolensis NCIMB 13971^T as the nearest neighbour. The genome sequence of A. kestanbolensis NCIMB 13971^T was not available in any database, so Panosyan et al. did not determine (ANI) value and pairwise digital DNA–DNA hybridization values between the A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T. Hence, in the present work A. kestanbolensis NCIMB 13971^T was sequenced and we attempted to clarify, employing genomics-based methods, the relationship between the type strains of A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T. The data presented in this study provides evidence that A. karvacharensis K1^T is later heterotypic synonym of A. kestanbolensis NCIMB 13971^T.

A. kestanbolensis NCIMB 13971^T was purchased from the National Collection of Industrial Food and Marine Bacteria, and it was grown on Nutrient Agar medium incubated at 50°C for 24 h. The genome sequencing of A. kestanbolensis NCIMB 13971^T was performed in this study, while genome sequence of A. karvacharensis K1^T (MQAD00000000) was downloaded from GenBank database. For whole genome sequencing, genomic DNA was isolated from culture of A. kestanbolensis NCIMB 13971^T by using the QIAamp DNA Mini Kit according to the manufacturer's instructions (Qiagen, Hilden-Germany). Whole-genome sequencing of A. kestanbolensis NCIMB 13971^T was performed on an Illumina HiSeq 2500 platform using 2 × 250 bp paired-end reads by MicrobesNG (University of Birmingham, United Kingdom). The reads were assembled using the full SPAdes assembly strategy on the patric web server (https://patricbrc.org/) (Wattam et al. 2017).The genome annotation was performed by using the Rapid Annotations Using Subsystems Technology (RAST) server (Aziz et al. 2008). The obtained draft genome sequence was deposited in the National Centre for Biotechnology Information (NCBI) database under accession number JALLIW000000000. The 16S rRNA gene sequence was extracted from the draft genome sequence of A. kestanbolensis NCIMB 13971^T using RNAmmer (version 1.2) (Lagesen et al. 2007). The 16S rRNA gene sequence similarity values between A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T were calculated using the pairwise alignment feature implemented on the EZBioCloud server (https://www.ezbiocloud.net/tools/pairA).

The 16S rRNA gene sequences of closely related type strains were downloaded from EzBioCloud server (https://www.ezbiocloud.net/) (Yoon et al. 2017a) and edited by using the BioEdit program (Hall, 1999). Multiple sequence alignment of 16S rRNA gene sequences was performed using the clustal_w (Thompson et al. 1994). Phylogenetic trees based on 16S rRNA gene sequences were reconstructed by three algorithms with the neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) methods, using MEGA version 7.0 (Kumar et al. 2016). Evolutionary distance matric was calculated according to Kimura’s two-parameter model (Kimura, 1980). Bootstrap analysis based on 1000 replicates was also conducted in order to obtain confidence levels for the branches (Felsenstein, 1985).

The phylogenetic analysis of A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T was carried out using the type strain genomes server pipeline (TYGS, https://tygs.dsmz.de/) (Meier-Kolthoff and Göker 2019). The digital DNA-DNA hybridization (dDDH) value between the draft genome sequences of A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T was calculated using Formula 2 of the online Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/distcalc2.php) (Meier-Kolthoff et al., 2013). Average nucleotide identity (ANI) values were calculated for evaluating the genetic relationship between A. karvacharensis K1^T and A A. kestanbolensis NCIMB 13971^T by using the orthoANIu algorithm and an online ANI calculator (www. ezbiocloud.net/tools/ani) (Lee et al., 2016; Yoon et al., 2017b). A phylogenetic tree based on whole-genome sequences was constructed using the TYGS web server (https://tygs.dsmz.de/) (Meier-Kolthoff and Göker 2019). The amino acid identity (AAI) value was calculated with CompareM (https://github.com/dparks1134/CompareM).

The phylogenetic analysis based on whole genome sequences has clarified the taxonomic inconsistence of prokaryotic taxa (Orata et al. 2018). In this study, the taxonomic relationship of A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T was re-evaluated by using whole-genome phylogenetic analysis. A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T were isolated from hot spring in Karvachar Nagorno-Karabakh and Turkey, respectively.

In the original article, Panosyan et al. (2021) stated that the 16S rRNA sequence of A. karvacharensis K1^T showed the highest similarity to A. flavithermus DSM 2641T (99.81%) and less similarity to other species of the genus Anoxybacillus. Phylogenetic tree based on 16S rRNA gene sequences in the original article showed that A. karvacharensis K1^T, A. flavithermus DSM 2641^T and A. kestanbolensis NCIMB 13971^T clustered together. However, in the original article Panosyan et al. determined only A. flavithermus DSM 2641^T as the nearest neighbor based on 16S rRNA gene sequences similarity and phylogenetic tree, they did not determine the taxonomic relationship between A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T.

In the present study, we determined whole-genome sequencing of A. kestanbolensis NCIMB 13971^T and obtained the 16S rRNA gene sequence from the genome of A. kestanbolensis NCIMB 13971^T (deposited under accession number ON331912). We noticed that the 16S rRNA gene sequence obtained from the genome of A. kestanbolensis NCIMB 13971^T had 99.93% similarity with A. karvacharensis K1^T. Also, in the present sturdy, we reconstructed phylogenetic trees based on 16S rRNA gene sequences by using the obtained the 16S rRNA gene sequence from the genome of A. kestanbolensis NCIMB 13971^T (Fig. 1, 2, 3). We determined that A. karvacharensis K1^T formed a cluster with A. kestanbolensis NCIMB 13971^T in the phylogenetic trees.

Further, in the phylogenomic tree (Fig. 4) A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T formed a robust branch different from other type strains of this genus with high bootstrap resampling values of 100%. The ANI value between A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T was 96.95% which was greater than the threshold value (95–96%) for species demarcation (Richter and Rosselló-Móra 2009), confirming that A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T were highly phylogenetically closely related. The calculated AAI value between the A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T was 97.6% and this value is also clearly above the suggested cut-offs for species delineation (AAI > 95%) (Luo et al., 2014), confirming that they belong to the same species. Meanwhile, digital DNA–DNA hybridization (DDH) analyses indicated that A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T exhibited 98.8% dDDH value which is higher than the cut-off (70%) used to classify bacterial strains to the same species (Wayne et al., 1987).

Taken together, the present results indicate that A. karvacharensis K1^T and A. kestanbolensis NCIMB 13971^T were considered to belong to the same species. Thus, based on the phylogenetic analysis based on whole genome sequences and rule 42 of the Bacteriological Code (Parker et al., 2019), we propose that A. karvacharensis K1^T Panosyan et al. 2021 should be reclassified as a later heterotypic synonym of A. kestanbolensis Dulger et al. 2004. The type strain is NCIMB 13971^T (= K4^T = NCCB 100051T) and K1^T (= DSM 106524^T = KCTC 15807^T) is an additional strain of A. kestanbolensis.

Acknowledgements This study was supported by Karadeniz Technical University (KTU BAP FAT-2019-7822).

Author Contributions KIB designed the study. KIB, HIB and SC performed genome analysis. KIB, HIB, AOB analysed the data and wrote the manuscript. All authors read and approved the final manuscript.

Conflict of interest The authors declare that there is no conflict of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

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Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004

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