Mycobacterium meyganense sp. nov., Mycobacterium omidense sp. nov., and Mycobacterium seymarense sp. nov.: bioremediation-capable novel species from Iranian environmental samples.

Mycobacterium meyganense sp. nov., Mycobacterium omidense sp. nov., and Mycobacterium seymarense sp. nov.: bioremediation-capable novel species from Iranian environmental samples. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mycobacterium meyganense sp. nov., Mycobacterium omidense sp. nov., and Mycobacterium seymarense sp. nov.: bioremediation-capable novel species from Iranian environmental samples. Conor J Meehan, Sari Cogneau, Mahboobeh Behruznia, Maren Diels, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8862259/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Three strains capable of bioremediation were isolated from various environmental sources throughout Iran. Physiological characterisations conformed to known Mycobacterium standards with the addition of the ability to degrade sodium sulfate and/or crude oil. Whole genome sequencing, phylogenomic analyses and average nucleotide identity comparisons to all closely related sequenced type strains of mycobacteria revealed three novel species of mycobacteria. These are hence named Mycobacterium meyganense sp. nov. (strain A16 = BCCM ITM-500916 = DSM 107075), Mycobacterium omidense sp. nov. (strain A14 = BCCM ITM-500915 = DSM 107139), and Mycobacterium seymarense sp. nov. (strain A12 = BCCM ITM-500914 = DSM 107138). bioremediation environmental Mycobacterium non-tuberculous mycobacteria phylogenomics taxogenomics Figures Figure 1 Figure 2 Introduction The genus Mycobacterium contains over 200 species that are aerobic non-motile rod-shaped bacteria, characterised by the presence of mycolic acids in their waxy hydrophobic cell wall (Magee and Ward 2015 ). This group contains several highly successful obligate pathogens such as the Mycobacterium tuberculosis complex and M. leprae and opportunistic pathogens often referred to as non-tuberculous mycobacteria (NTM) (Turenne 2019 ). Although the obligate and opportunistic pathogens have been the main focus of interest in the mycobacteria, many environmental, non-pathogenic species also exist in this group, likely many more than we have currently characterised (Pontiroli et al. 2013 ). These groups inhabit diverse environmental reservoirs, including water, soil and sediment and are often involved in saprophytic activities. Some members of this genus have also been shown to have bioremediating capabilities, specifically the ability to degrade polycyclic aromatic hydrocarbons (PAHs) (Das et al. 2015; Deng et al. 2023 ; Hennessee and Li 2016). However, most such capabilities have been found in strains without specific species definitions. A study undertaken in Iran revealed several environmental strains of mycobacteria capable of bioremediation (Azadi et al. 2017 ). Here, we demonstrate that three of these bioremediating mycobacterial strains represent novel species and present closed, complete genomes for these species, adding to the growing genomic repository for this genus. Materials and Methods Overview of strains All three isolates were derived from environmental sampling of various habitats across Iran as described elsewhere (Azadi et al. 2017 ). Isolates are from the following sources: Mycobacterium meyganense from Meygan, a salt lake in Arak city; Mycobacterium omidense from an oil well in the city of Omidieh; Mycobacterium seymarense from sediment of the Khorramabad River. Phenotypic characterisation Standard phenotypic characterisation of these isolates were undertaken previously (Azadi et al. 2017 ) and then confirmed and expanded on here by the use of a variety of conventional phenotypic and biochemical tests (Wayne 1984 ) (Table 1 ). These included acid-fast staining, colony characterization, growth rate at 22°C, and standard biochemical assays (Sharma et al. 2010 ). The growth rates of each isolate on polycyclic aromatic hydrocarbon (PAH) mix solution (1–1) (aenaphthene, acenaphthylene, anthracene, benzo (b) fluoranthene, 1,2- benz anthracene, benzo (a) pyrene, benzo (k) fluoranthene, benzo (g, h, i) perylene, chrysene, dibenz (a,h) anthracene, fluoranthene, fluorene, and indeno (1, 2, 3-cd) pyrene, phenanthrene, naphthalene, pyrene) are also described elsewhere (Azadi et al. 2017 ). Strains were deposited in the BCCM and DSMZ repositories, adding to the growing global Mycobacterium culture collection (Hazbón et al. 2018 ). Table 1 Physicochemical properties of each strain ITM-500916 T /DSM 107075 T ITM-500915 T /DSM 107139 T ITM-500914 T /DSM 107138 T Species Mycobacterium meyganense Mycobacterium omidense Mycobacterium seymarense Strain name in ( Azadi et al. 2017 ) A16 A14 A12 Source Salt lake Oil well River sedminent Colony colour White / yellowish Yellow Yellow Colony shape Rough Mucoid Mucoid Catalase + + + Growth at 22 °C + + + Growth with 3 % NaCl (w/v) + + - Nitrate reduction - + + Hydrolysis of urea - + - Acid production from : l-Rhamnose - - + d-Mannitol - + + PNB (p-nitrobenzoic acid) inhibition - - - Biodegrade sodium sulfate + - + Biodegrade crude oil - + + Genotypic characterisation of novel species Identification of strains as novel species followed the recommended workflow for minimal standards (Fig. 1 of (Riesco and Trujillo 2024 )). 16S sequence similarity Sanger sequencing of the 16S gene of each strain was undertaken as described in (Azadi et al. 2017 )). These sequences were checked against the NCBI NR database using BLASTn and default parameters (Altschul et al. 1990 ). The type strain with the highest identity to the 16S query was extracted. Genome sequencing DNA extraction suitable for long read sequencing was performed using the Genomic DNA Buffer Set (Qiagen Inc, USA). Nanopore sequencing was performed using the native barcoding kit 24 V14 (SQK-NBD114.24) according to the manufacturer's instruction on the MinION Mk1C platform with R10.4.1 flow cells. Sequence adaptors were trimmed using Porechop v0.2.4 (Wick [ 2017 ] 2023). Sequences were assembled using Flye v2.9.2 (Kolmogorov et al. 2019 ) and iteratively polished using Racon v1.5.0 (Vaser et al. 2017 ) and Medaka v1.8.0 (Oxford Nanopore [ 2017 ] 2023) to obtain a single contig for each strain. Circlator v1.5.5 was used for assembly circularisation (Hunt et al. 2015 ) and the quality of assemblies was assessed using BUSCO v5.4.7 (Simão et al. 2015 ; Manni et al. 2021 ). Complete circular assemblies were annotated using PGAP v6.4 (Tatusova et al. 2016 ). Functional characterisation was undertaken using eggNOG-mapper v2.1.12 (Cantalapiedra et al. 2021 ) with default settings and the protein sets for each strain as input. Metabolic capabilities were estimated using gapseq v1.2 (Zimmermann et al. 2021 ). Phylogenomics and taxonomic analysis To test whether these strains could be classified as novel species, a phylogenomic approach was undertaken. First all type strains for Mycobacterium species and subspecies with good quality genomes as outlined in the GTDB database (Parks et al. 2022 ) (accessed 2nd August 2023) were downloaded from NCBI (331 genomes; Online Resource 1). Average nucleotide identity (ANI) scores were calculated between the three strains and all reference type strain genomes using fastANI (v1.33) (Jain et al. 2018 ). For each of the three strains, the five Mycobacterium species with the highest ANI values were selected and a pangenome of all 18 genomes was created using Panaroo (Tonkin-Hill et al. 2020 ), with clean mode strict, invalid genes removed and merged paralogs settings, outputting a core genome alignment. A maximum likelihood phylogenomic tree was then constructed using RAxML-NG (Kozlov et al. 2019 ) v1.2.0 with 20 random starting trees and 100 bootstrap replicates (Fig. 1 ). The UBCG2 pipeline (Kim et al. 2021 ) was also used to extract a core set of 81 universal genes using Prodigal v2.6.3 (Hyatt et al. 2010 ) and HMMer v3.4 (Eddy, Sean R 2026 ) from the same set of Mycobacterium genomes, create a concatenated protein alignment using MAFFT v7.525 (Katoh and Standley 2013 ) and a ML tree using RAxML v8.2.12 (Stamatakis 2014 ). The genome of each strain was also uploaded to the Type Strain Genome Server (TYGS) on January 13th 2026 to calculate the digital DNA-DNA hybridization score (dDDH) using the d 4 formula as recommended (Meier-Kolthoff and Göker 2019) and to build a 16S tree via FASTME 2.1.6.1 (Lefort et al. 2015 ). Results Phenotypic characteristics of the proposed new species Standard phenotypic characterisation of each strain is outlined extensively in (Azadi et al. 2017 ). Additional testing was undertaken here as outlined in Table 1 . Of note, M. meyganense was found to degrade sodium sulfate, M. omidense to degrade crude oil and M. seymarense to degrade both. None of the closely related species to these strains (as outlined below) have been reported to have these abilities, indication novel phenotypic differences found in these strains. Phylogenomics and taxogenomics of the proposed new species Based on phylogenomic placement, the closest species to M. meyganense is M. stellerae (GCF_003719305.1) (Fig. 1 ; Online Resource 2). ANI analysis revealed an 83% similarity between these two species and a dDDH score of 25.8%. Analysis of the 16S sequence revealed the closest match to be M. gadium (99%; NR_115805; Online Resource 3) although the ANI between these species was only 79.8% and the dDDH was 20.7%. GapSeq analysis showed that the two species ( M. meyganense and M. stellerae ) have 489 metabolic pathways in common with a further 9 found in M. meyganense and absent in M. stellerae and 76 in the reverse (Online Resource 4). Similarly, based on phylogenomic placement, the closest species to M. omidense is M. vaccae (GCF_001655245.1) (Fig. 1 ; Online Resource 2), also by analysis of the 16S sequence (99%; KF378750; Online Resource 3). ANI analysis revealed a 92.6% similarity between these two species and a dDDH of 47.1%. These species share 419 metabolic pathways with each other and a further 16 exclusive to M. omidense and 44 only in M. vaccae (Online Resource 5). The closest species in the phylogenetic tree to M. seymarense is M. diernhoferi (GCF_019456655.1) (Fig. 1 ; Online Resource 2; Online Resource 3). ANI analysis revealed an 85.4% similarity between these two species and a dDDH of 26.6%. Analysis of the 16S sequence revealed the closest match to be M. frederiksbergense (99%; PX678563.1; Online Resource 3). There is no high-quality genome for M. frederiksbergense available. M. seymarense and M. diernhoferi share 389 metabolic pathways with a further 50 exclusive to M. seymarense and 35 only found in M. diernhoferi (Online Resource 6). Since all ANI values between the proposed novel species and closest known type strains fell below the threshold of 95% for defining new species and the dDDH also fell below the threshold of 70% (Riesco and Trujillo 2024 ), these genomic features support the creation of new Mycobacterium species (Chun et al. 2018 ), although the lack of a genome for M. frederiksbergense means further comparisons may be needed for M. seymarense . Genomic and functional characteristics of the three novel strains All genomes assembled into a single contig with no plasmids detected. BUSCO quality control showed high completeness confirming the closed status of these genomes. Resulting genome features are outlined in Table 2 . Genome sizes ranged from 5.46Mbp to 6.06Mbp which is standard for Mycobacterium species (Fedrizzi et al. 2017 ). GC content was also as expected, ranging from 66.2% to 68.7%. Table 2 Genome characteristics of each strain Strain Mycobacterium meyganense Mycobacterium omidense Mycobacterium seymarense WGS accession SAMN08578608 SAMN08578607 SAMN08578606 16S accession KU564078 KU564077 KU564076 Genome size (Mbp) 5.46 6.06 5.99 GC content (%) 66.2 68.7 67.3 Gene count 5372 5814 5829 BUSCO completeness 98.3 96.9 100 The species had 389 metabolic pathways in common with each other and a further 35 only found in M. omidense and 50 only in M. vaccae (Online Resource 4). COG category analysis showed that all genomes had similar spread of categories with 20–30% of each genome being uncharacterised (R, S and – categories) (Fig. 2 ). Interestingly, two of the strains were able to biodegrade sodium sulfate and two were able to biodegrade crude oil (Table 1 ) (Azadi et al. 2017 ). However, GapSeq metabolic pathway analysis did not clearly indicate the process by which this is undertaken. Naphthalene degradation and Alkane oxidation were not found in any species, although the octane oxidation pathway was present in all (Online Resource 7). Comparison of protein annotation categories showed no prominent differences between novel species and closest established species (Fig. 2 ). Discussion and protologues The genus Mycobacterium has many species that have been associated with bioremediation and PAH degradation in the past (Ribón 2018 ). While some have been properly named as novel species, many remain only as described strains without proper taxonomic classification. By using extensive phylogenomic and taxongenomic approaches, we have demonstrated that three such strains, previously shown to grow in the presence of crude oil or sodium sulfate (Azadi et al. 2017 ), should be classified as novel species, distinguished from closely related species in ANI, phylogenetic placement and metabolic pathway capabilities. Although comparative metabolic analyses was undertaken, the examination of these pathways did not reveal the genetic basis for the ability of these species to undertake bioremediation. This may be because the closely related species, M. diernhoferi , M. stellerae and M. vaccae , have not themselves been tested for bioremediation capabilities. Other closely related species have been shown to have bioremediation capabilities (Das et al. 2015; Deng et al. 2023 ; Hennessee and Li 2016), suggesting that this feature may be widespread in the rapid growing environmental mycobacteria. This work and others indicate that in depth investigations are needed to fully understand and exploit this property in these Mycobacterium species. The whole genome sequencing / 16S sequence accession numbers for these novel species are as follows: Mycobacterium meyganense : SAMN08578608 / KU564078; Mycobacterium omidense : SAMN08578607 / KU564077; Mycobacterium seymarense : SAMN08578606 / KU564076. Description of Mycobacterium meyganense sp. nov. Mycobacterium meyganense (mey.gan.en.se. L. neut. adj. meygan pertaining to Meygan, the salt lake in Arak city, Iran, from where the type strain originated). This species is characterized as acid-fast, non-spore-forming, non-motile short rods. Optimal growth temperature is 25°C. The cells form rough, scotochromogenic colonies, white/yellow in colour, that appear after 6–14 days on Sauton agar and Löwenstein-Jensen (LJ) medium (Table 1 ; Strain A16 in (Azadi et al. 2017 )).The organism shows a positive reaction to catalase, nitrate reduction and potassium tellurite reduction and is negative to niacin production, pyrazinamidase and urease tests. Tween 80 is not hydrolysed and growth occurs on MacConkey agar but not on LJ supplemented with NaCl 5% or PNB. The isolate can mineralize sodium sulfate, but not PAHs or crude oil. The genome of this strain is 5.46 Mbp in length, containing 5,372 predicted genes on a chromosome with no plasmid and a GC content of 66.2%. Description of Mycobacterium omidense sp. nov. Mycobacterium omidense (o.mi’den.se. L. neut. adj. omid pertaining to Omidieh city, Iran, from where the type strain originated). This species is characterized as acid-fast, non-spore-forming, non-motile short rods. The cells form smooth, scotochromogenic colonies, yellow in colour, that appear after 3 days on Sauton agar and LJ medium (Table 1 ; Strain A14 in (Azadi et al. 2017 )).). Optimal growth temperature is 25°C. The organism shows a positive reaction to urease, catalase and nitrate reduction but negative to pyrazinamidase, niacin production, and potassium tellurite reduction. Tween 80 is not hydrolyzed and growth occurs on LJ with NaCl 5% and on MacConkey agar, but not on PNB-containing LJ. The isolate is able to biodegrade PAHs and crude oil, but cannot grow in the presence of sodium sulfate. The genome of this strain is 6.06 Mbp in length, containing 5,814 predicted genes on a chromosome with no plasmid and a GC content of 68.7%. Description of Mycobacterium seymarense sp. nov. Mycobacterium seymarense (sey.mar.en.se. L. neut. adj. seymar pertaining to the Seymareh river in the Lorestan province, Iran, from where the type strain originated). This species is characterized as acid-fast, non-spore-forming, non-motile short rods. The cells form smooth scotochromogenic colonies, yellow in colour, that appear after 5 days on Sauton agar and LJ medium (Table 1 ; Strain A12 in (Azadi et al. 2017 )). Optimal growth temperature is 35°C. The organism shows a positive reaction to catalase and negative to urease, nitrate reduction, pyrazinamidase, niacin and potassium tellurite reduction. Tween 80 is hydrolyzed and growth occurs on LJ with NaCl 5% and on MacConkey agar, but not on PNB-containing LJ. The isolate is able to degrade PAHs, sodium sulfate and crude oil, showing its potential use for cleaning of petroleum hydrocarbon. The genome of this strain is 5.99 Mbp in length, containing 5,829 predicted genes on a single chromosome with no plasmid and a GC content of 67.3%. The assembly was predicted to be 100% complete by BUSCO and assembled into a single chromosome with no plasmid. Based on phylogenomic placement, the closest species to M. seymarense is M. diernhoferi. ANI analysis revealed an 85.4% similarity between these two species. Analysis of the 16S sequence revealed the closest match to be M. frederiksbergense (99.93%; PX678563.1). These genomic features support the creation of a new Mycobacterium species, although the lack of a genome for M. frederiksbergense means further comparisons may be needed. Declarations The authors declare no conflict of interest Ethical approval No human or animal related strains were collected for this study and thus no ethical approval was required. Funding The authors are grateful to the Office of Vice Chancellor for Research of Isfahan University of Medical Sciences for the support of the current study. Funding is provided by the Department of Economy, Science and Innovation of the Flemish Government and the Belgian Science Policy (Belspo). Support by the European Research Council-INTERRUPTB starting grant [nr.311725] to CJM and LR. The work is also supported by the Academy of Medical Sciences (AMS), the Wellcome Trust, the Government Department of Business, Energy and Industrial Strategy (BEIS), the British Heart Foundation and Diabetes UK and the Global Challenges Research Fund (GCRF) via a Springboard grant [SBF006\1090] to CJM. The project was supported partially by a research grant number 393409 from Isfahan University of Medical Sciences, Isfahan, Iran to HS and DA. Funds for the nanopore sequencing was provided by Nottingham Trent University internal grants. Author Contribution C.J.M. contributed to conceptualization, methodology, formal analysis, investigation, writing -original draft, writing – reviewing & editing, and funding acquisition.S.C. contributed to conceptualization, methodology, resources, data curation, writing -original draft, writing – reviewing & editingM.B. contributed to methodology, resources, data curation, writing -original draft, writing – reviewing & editing.M.D. contributed to resources, data curation and writing – reviewing & editing.L.R. contributed to resources, data curation, writing – reviewing & editing and supervision.H.S. contributed to conceptualization, methodology, resources, data curation, writing – reviewing & editing and supervision.D.A. contributed to conceptualization, formal analysis, investigation, writing -original draft, writing – reviewing & editing, supervision and funding acquisition. Data Availability The whole genome sequence and 16S gene sequence of each strain are available from NCBI with the following accessions (WGS/16S): Mycobacterium meyganense (SAMN08578608/KU564078); Mycobacterium omidense (SAMN08578607/KU564077); Mycobacterium seymarense (SAMN08578606/KU564076). References Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. ‘Basic Local Alignment Search Tool’. J Mol Biol 215 (3): 403–10. https://doi.org/10.1016/S0022-2836(05)80360-2 . Azadi, Davood, Hasan Shojaei, Sina Mobasherizadeh, and Abass Daei Naser. 2017. ‘Screening, Isolation and Molecular Identification of Biodegrading Mycobacteria from Iranian Ecosystems and Analysis of Their Biodegradation Activity’. AMB Express 7 (1): 180. https://doi.org/10.1186/s13568-017-0472-4 . Cantalapiedra, Carlos P., Ana Hernández-Plaza, Ivica Letunic, Peer Bork, and Jaime Huerta-Cepas. 2021. ‘eggNOG-Mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale’. Molecular Biology and Evolution 38 (12): 5825–29. https://doi.org/10.1093/molbev/msab293 . Chun, Jongsik, Aharon Oren, Antonio Ventosa, et al. 2018. ‘Proposed Minimal Standards for the Use of Genome Data for the Taxonomy of Prokaryotes’. International Journal of Systematic and Evolutionary Microbiology 68 (1): 461–66. https://doi.org/10.1099/ijsem.0.002516 . Das, Sarbashis, B. M. Fredrik Pettersson, Phani Rama Krishna Behra, et al. 2015. ‘Characterization of Three Mycobacterium Spp. with Potential Use in Bioremediation by Genome Sequencing and Comparative Genomics’. Genome Biology and Evolution 7 (7): 1871–86. https://doi.org/10.1093/gbe/evv111 . Deng, Yang, Tong Mou, Junhuan Wang, Jing Su, Yanchun Yan, and Yu-Qin Zhang. 2023. ‘Characterization of Three Rapidly Growing Novel Mycobacterium Species with Significant Polycyclic Aromatic Hydrocarbon Bioremediation Potential’. Frontiers in Microbiology 14 (September): 1225746. https://doi.org/10.3389/fmicb.2023.1225746 . Eddy, Sean R. 2026. ‘HMMER’. http://hmmer.org/ . Fedrizzi, Tarcisio, Conor J. Meehan, Antonella Grottola, et al. 2017. ‘Genomic Characterization of Nontuberculous Mycobacteria’. Scientific Reports 7 (1): 45258. https://doi.org/10.1038/srep45258 . Hazbón, Manzour Hernando, Leen Rigouts, Marco Schito, et al. 2018. ‘Mycobacterial Biomaterials and Resources for Researchers’. Pathogens and Disease Accepted. Hennessee, Christiane T., and Qing X. Li. 2016. ‘Effects of Polycyclic Aromatic Hydrocarbon Mixtures on Degradation, Gene Expression, and Metabolite Production in Four Mycobacterium Species’. Applied and Environmental Microbiology 82 (11): 3357–69. https://doi.org/10.1128/AEM.00100-16 . Hunt, Martin, Nishadi De Silva, Thomas D. Otto, Julian Parkhill, Jacqueline A. Keane, and Simon R. Harris. 2015. ‘Circlator: Automated Circularization of Genome Assemblies Using Long Sequencing Reads’. Genome Biology 16 (1): 294. https://doi.org/10.1186/s13059-015-0849-0 . Hyatt, Doug, Gwo-Liang Chen, Philip F. LoCascio, Miriam L. Land, Frank W. Larimer, and Loren J. Hauser. 2010. ‘Prodigal: Prokaryotic Gene Recognition and Translation Initiation Site Identification’. BMC Bioinformatics 11 (1): 119. https://doi.org/10.1186/1471-2105-11-119 . Jain, Chirag, Luis M. Rodriguez-R, Adam M. Phillippy, Konstantinos T. Konstantinidis, and Srinivas Aluru. 2018. ‘High Throughput ANI Analysis of 90K Prokaryotic Genomes Reveals Clear Species Boundaries’. Nature Communications 9 (1): 1. https://doi.org/10.1038/s41467-018-07641-9 . Katoh, Kazutaka, and Daron M. Standley. 2013. ‘MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability’. Molecular Biology and Evolution 30 (4): 772–80. https://doi.org/10.1093/molbev/mst010 . Kim, Jihyeon, Seong-In Na, Dongwook Kim, and Jongsik Chun. 2021. ‘UBCG2: Up-to-Date Bacterial Core Genes and Pipeline for Phylogenomic Analysis’. Journal of Microbiology 59 (6): 609–15. https://doi.org/10.1007/s12275-021-1231-4 . Kolmogorov, Mikhail, Jeffrey Yuan, Yu Lin, and Pavel A. Pevzner. 2019. ‘Assembly of Long, Error-Prone Reads Using Repeat Graphs’. Nature Biotechnology 37 (5): 5. https://doi.org/10.1038/s41587-019-0072-8 . Kozlov, Alexey M., Diego Darriba, Tomáš Flouri, Benoit Morel, and Alexandros Stamatakis. 2019. ‘RAxML-NG: A Fast, Scalable and User-Friendly Tool for Maximum Likelihood Phylogenetic Inference’. Bioinformatics 35 (21): 4453–55. https://doi.org/10.1093/bioinformatics/btz305 . Lefort, Vincent, Richard Desper, and Olivier Gascuel. 2015. ‘FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program’. Molecular Biology and Evolution 32 (10): 2798–800. https://doi.org/10.1093/molbev/msv150 . Magee, John G., and Alan C. Ward. 2015. ‘Mycobacterium’. In Bergey’s Manual of Systematics of Archaea and Bacteria . John Wiley & Sons, Ltd. https://doi.org/10.1002/9781118960608.gbm00029 . Manni, Mosè, Matthew R. Berkeley, Mathieu Seppey, and Evgeny M. Zdobnov. 2021. ‘BUSCO: Assessing Genomic Data Quality and Beyond’. Current Protocols 1 (12): e323. https://doi.org/10.1002/cpz1.323 . Meier-Kolthoff, Jan P., and Markus Göker. 2019. ‘TYGS Is an Automated High-Throughput Platform for State-of-the-Art Genome-Based Taxonomy’. Nature Communications 10 (1): 2182. https://doi.org/10.1038/s41467-019-10210-3 . Oxford Nanopore. (2017) 2023. Medaka . Python. June 7; Oxford Nanopore Technologies, released September 10. https://github.com/nanoporetech/medaka . Parks, Donovan H., Maria Chuvochina, Christian Rinke, Aaron J. Mussig, Pierre-Alain Chaumeil, and Philip Hugenholtz. 2022. ‘GTDB: An Ongoing Census of Bacterial and Archaeal Diversity through a Phylogenetically Consistent, Rank Normalized and Complete Genome-Based Taxonomy’. Nucleic Acids Research 50 (D1): D785–94. https://doi.org/10.1093/nar/gkab776 . Pontiroli, Alessandra, Tanya T. Khera, Brian B. Oakley, et al. 2013. ‘Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity’. PLOS ONE 8 (7): e68648. https://doi.org/10.1371/journal.pone.0068648 . Ribón, Wellman. 2018. Mycobacterium: Research and Development . BoD – Books on Demand. Riesco, Raúl, and Martha E. Trujillo. 2024. ‘Update on the Proposed Minimal Standards for the Use of Genome Data for the Taxonomy of Prokaryotes’. International Journal of Systematic and Evolutionary Microbiology 74 (3): 006300. https://doi.org/10.1099/ijsem.0.006300 . Sharma, Babita, Nita Pal, Bharti Malhotra, and Leela Vyas. 2010. ‘Evaluation of a Rapid Differentiation Test for Mycobacterium Tuberculosis from Other Mycobacteria by Selective Inhibition with P-Nitrobenzoic Acid Using MGIT 960’. Journal of Laboratory Physicians 2 (2): 89. https://doi.org/10.4103/0974-2727.72157 . Simão, Felipe A., Robert M. Waterhouse, Panagiotis Ioannidis, Evgenia V. Kriventseva, and Evgeny M. Zdobnov. 2015. ‘BUSCO: Assessing Genome Assembly and Annotation Completeness with Single-Copy Orthologs’. Bioinformatics 31 (19): 3210–12. https://doi.org/10.1093/bioinformatics/btv351 . Stamatakis, Alexandros. 2014. ‘RAxML Version 8: A Tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies’. Bioinformatics 30 (9): 1312–13. https://doi.org/10.1093/bioinformatics/btu033 . Tatusova, Tatiana, Michael DiCuccio, Azat Badretdin, et al. 2016. ‘NCBI Prokaryotic Genome Annotation Pipeline’. Nucleic Acids Research 44 (14): 6614–24. https://doi.org/10.1093/nar/gkw569 . Tonkin-Hill, Gerry, Neil MacAlasdair, Christopher Ruis, et al. 2020. ‘Producing Polished Prokaryotic Pangenomes with the Panaroo Pipeline’. Genome Biology 21 (1): 180. https://doi.org/10.1186/s13059-020-02090-4 . Turenne, Christine Y. 2019. ‘Nontuberculous Mycobacteria: Insights on Taxonomy and Evolution’. Infection, Genetics and Evolution 72 (January). https://doi.org/10.1016/J.MEEGID.2019.01.017 . Vaser, Robert, Ivan Sović, Niranjan Nagarajan, and Mile Šikić. 2017. ‘Fast and Accurate de Novo Genome Assembly from Long Uncorrected Reads’. Genome Research 27 (5): 737–46. https://doi.org/10.1101/gr.214270.116 . Wayne, LG. 1984. Mycobacterial Speciation. in The Mycobacteria: A Sourcebook . Edited by GP Kubica and LG Wayne. Marcel Dekker, New York, N.Y. Wick, Ryan. (2017) 2023. Porechop . C++. February 13, released September 9. https://github.com/rrwick/Porechop . Zimmermann, Johannes, Christoph Kaleta, and Silvio Waschina. 2021. ‘Gapseq: Informed Prediction of Bacterial Metabolic Pathways and Reconstruction of Accurate Metabolic Models’. Genome Biology 22 (1): 81. https://doi.org/10.1186/s13059-021-02295-1 . Additional Declarations No competing interests reported. Supplementary Files NovelSpeciesMycoIranSupplementaryTables.xlsx OnlineResource2.png OnlineResource3.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8862259","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594766227,"identity":"fdbdff7b-cde5-4602-a4a3-a0258bb2309e","order_by":0,"name":"Conor J Meehan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYFADCSD+AOcdIFIL4wwGAxK1MPMQo4W/vffg4wqGbXL80s3PHtvu+JPYwH74ATPPGTxmnzmXbHiG4bax5Jxj5sa5ZwwSG3jSDJh5buDWYiCRYybZwHA7ccONBDPp3DagFoYcoAs/ENZSv/9G+jdpS5AW/jfEaUkAMaQZQVokQLbgcZjEmTPGhg0Gtw1n3Mgpk+xtMzZuk3hmcHAOHu/zt/cYPmyouC3PPyN9m8TPNjnZfv7khw/eHMOtBeo8JDYbA5EROQpGwSgYBaMANwAAV9dL01/u2UwAAAAASUVORK5CYII=","orcid":"","institution":"Nottingham Trent University","correspondingAuthor":true,"prefix":"","firstName":"Conor","middleName":"J","lastName":"Meehan","suffix":""},{"id":594766232,"identity":"a990c171-243e-4071-9cc6-7f73147b4cac","order_by":1,"name":"Sari Cogneau","email":"","orcid":"","institution":"Institute of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sari","middleName":"","lastName":"Cogneau","suffix":""},{"id":594766236,"identity":"c70120f3-f6b6-4373-8003-b538480f09c3","order_by":2,"name":"Mahboobeh Behruznia","email":"","orcid":"","institution":"Nottingham Trent University","correspondingAuthor":false,"prefix":"","firstName":"Mahboobeh","middleName":"","lastName":"Behruznia","suffix":""},{"id":594766237,"identity":"4f74cd62-2b26-45cb-a818-f6b3a06fd5e2","order_by":3,"name":"Maren Diels","email":"","orcid":"","institution":"Institute of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Maren","middleName":"","lastName":"Diels","suffix":""},{"id":594766240,"identity":"0222a218-05b9-4e72-95b9-6421895b74c3","order_by":4,"name":"Leen Rigouts","email":"","orcid":"","institution":"Institute of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Leen","middleName":"","lastName":"Rigouts","suffix":""},{"id":594766241,"identity":"a4d6dceb-5c57-4a3f-9be5-810a2769957f","order_by":5,"name":"Hasan Shojaei","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hasan","middleName":"","lastName":"Shojaei","suffix":""},{"id":594766244,"identity":"601c6eb9-02b0-460e-a131-25590bb57dcd","order_by":6,"name":"Davood Azadi","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Davood","middleName":"","lastName":"Azadi","suffix":""}],"badges":[],"createdAt":"2026-02-12 12:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8862259/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8862259/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103248412,"identity":"4d1b48a9-1898-40f1-afa6-a0d62f994352","added_by":"auto","created_at":"2026-02-23 15:31:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":137124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenomic tree of strains including closely related rapidly growing mycobacteria.\u003c/strong\u003e A core genome alignment of 120 genes output from Panaroo was used to construct a Maximum Likelihood tree using RAxML-NG. Species proposed in this paper are highlighted in red. Bootstrap support percentage values are shown on nodes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8862259/v1/b7bf0625cffddc4ad5749a8d.png"},{"id":103248411,"identity":"0fc9437c-513b-4e4c-9c90-0d086507c71b","added_by":"auto","created_at":"2026-02-23 15:31:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProportion of COG categories per annotated genome of each species.\u003c/strong\u003e The spread of predicted COG category (x-axis) proportions in the genome of each of the three proposed novel species (\u003cem\u003eM. meyganense\u003c/em\u003e: dark blue, 1\u003csup\u003est\u003c/sup\u003e in order; \u003cem\u003eM. omidense\u003c/em\u003e: grey, 3\u003csup\u003erd\u003c/sup\u003e in order; \u003cem\u003eM. seymarense\u003c/em\u003e: light blue, 5\u003csup\u003eth\u003c/sup\u003e in order) and their closest phylogenomic species (\u003cem\u003eM. stellerae \u003c/em\u003e(GCF_003719305.1): orange, 2\u003csup\u003end\u003c/sup\u003e in order; \u003cem\u003eM. vaccae\u003c/em\u003e (GCF_001655245.1): yellow, 4\u003csup\u003eth\u003c/sup\u003e in order; \u003cem\u003eM. diernohoferi \u003c/em\u003e(GCF_019456655.1): green, 6\u003csup\u003eth\u003c/sup\u003e in order respectively) are shown. COG category single letter code meanings are outlined on the COG website: https://www.ncbi.nlm.nih.gov/research/cog\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8862259/v1/50c1fc78fad6c500bcec6b60.png"},{"id":104289522,"identity":"27d92c40-c6cd-46e9-adf7-35716e2a1b2c","added_by":"auto","created_at":"2026-03-10 06:26:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1214521,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8862259/v1/11ebd066-e413-4163-a28c-d8b93437572c.pdf"},{"id":103248413,"identity":"141e7429-1197-4ad4-bf02-f70a7f1aa6eb","added_by":"auto","created_at":"2026-02-23 15:31:18","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":78828,"visible":true,"origin":"","legend":"","description":"","filename":"NovelSpeciesMycoIranSupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8862259/v1/b3181f3b0d4419b6588f7257.xlsx"},{"id":103248409,"identity":"90149c0b-c914-4dbe-84f6-0d65bb5bd974","added_by":"auto","created_at":"2026-02-23 15:31:17","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40918,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource2.png","url":"https://assets-eu.researchsquare.com/files/rs-8862259/v1/ff587e1ce4c27026b5e27c7e.png"},{"id":103248410,"identity":"e8e7e9a1-2516-4f52-b85c-6ecd3da24c3f","added_by":"auto","created_at":"2026-02-23 15:31:17","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":113331,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource3.png","url":"https://assets-eu.researchsquare.com/files/rs-8862259/v1/e24ffd091a5ef0c1450253eb.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mycobacterium meyganense sp. nov., Mycobacterium omidense sp. nov., and Mycobacterium seymarense sp. nov.: bioremediation-capable novel species from Iranian environmental samples.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus \u003cem\u003eMycobacterium\u003c/em\u003e contains over 200 species that are aerobic non-motile rod-shaped bacteria, characterised by the presence of mycolic acids in their waxy hydrophobic cell wall (Magee and Ward \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This group contains several highly successful obligate pathogens such as the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e complex and \u003cem\u003eM. leprae\u003c/em\u003e and opportunistic pathogens often referred to as non-tuberculous mycobacteria (NTM) (Turenne \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although the obligate and opportunistic pathogens have been the main focus of interest in the mycobacteria, many environmental, non-pathogenic species also exist in this group, likely many more than we have currently characterised (Pontiroli et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These groups inhabit diverse environmental reservoirs, including water, soil and sediment and are often involved in saprophytic activities. Some members of this genus have also been shown to have bioremediating capabilities, specifically the ability to degrade polycyclic aromatic hydrocarbons (PAHs) (Das et al. 2015; Deng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hennessee and Li 2016). However, most such capabilities have been found in strains without specific species definitions.\u003c/p\u003e \u003cp\u003eA study undertaken in Iran revealed several environmental strains of mycobacteria capable of bioremediation (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Here, we demonstrate that three of these bioremediating mycobacterial strains represent novel species and present closed, complete genomes for these species, adding to the growing genomic repository for this genus.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOverview of strains\u003c/h2\u003e \u003cp\u003eAll three isolates were derived from environmental sampling of various habitats across Iran as described elsewhere (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Isolates are from the following sources: \u003cem\u003eMycobacterium meyganense\u003c/em\u003e from Meygan, a salt lake in Arak city; \u003cem\u003eMycobacterium omidense\u003c/em\u003e from an oil well in the city of Omidieh; \u003cem\u003eMycobacterium seymarense\u003c/em\u003e from sediment of the Khorramabad River.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhenotypic characterisation\u003c/h3\u003e\n\u003cp\u003eStandard phenotypic characterisation of these isolates were undertaken previously (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and then confirmed and expanded on here by the use of a variety of conventional phenotypic and biochemical tests (Wayne \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These included acid-fast staining, colony characterization, growth rate at 22\u0026deg;C, and standard biochemical assays (Sharma et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The growth rates of each isolate on polycyclic aromatic hydrocarbon (PAH) mix solution (1\u0026ndash;1) (aenaphthene, acenaphthylene, anthracene, benzo (b) fluoranthene, 1,2- benz anthracene, benzo (a) pyrene, benzo (k) fluoranthene, benzo (g, h, i) perylene, chrysene, dibenz (a,h) anthracene, fluoranthene, fluorene, and indeno (1, 2, 3-cd) pyrene, phenanthrene, naphthalene, pyrene) are also described elsewhere (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Strains were deposited in the BCCM and DSMZ repositories, adding to the growing global \u003cem\u003eMycobacterium\u003c/em\u003e culture collection (Hazb\u0026oacute;n et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of each strain\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eITM-500916 \u003csup\u003eT\u003c/sup\u003e /DSM 107075 \u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eITM-500915 \u003csup\u003eT\u003c/sup\u003e /DSM 107139 \u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eITM-500914\u003csup\u003eT\u003c/sup\u003e/DSM 107138 \u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpecies\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMycobacterium meyganense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMycobacterium omidense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMycobacterium seymarense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStrain name in (\u003c/b\u003eAzadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSource\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSalt lake\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOil well\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRiver sedminent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eColony colour\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWhite / yellowish\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eColony shape\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMucoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMucoid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCatalase\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrowth at 22\u0026thinsp;\u0026deg;C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGrowth with 3 % NaCl (w/v)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNitrate reduction\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydrolysis of urea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcid production from\u003c/b\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003el-Rhamnose\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ed-Mannitol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePNB (p-nitrobenzoic acid) inhibition\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBiodegrade sodium sulfate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBiodegrade crude oil\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eGenotypic characterisation of novel species\u003c/h3\u003e\n\u003cp\u003eIdentification of strains as novel species followed the recommended workflow for minimal standards (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e of (Riesco and Trujillo \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e16S sequence similarity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSanger sequencing of the 16S gene of each strain was undertaken as described in (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)). These sequences were checked against the NCBI NR database using BLASTn and default parameters (Altschul et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The type strain with the highest identity to the 16S query was extracted.\u003c/p\u003e\n\u003ch3\u003eGenome sequencing\u003c/h3\u003e\n\u003cp\u003eDNA extraction suitable for long read sequencing was performed using the Genomic DNA Buffer Set (Qiagen Inc, USA). Nanopore sequencing was performed using the native barcoding kit 24 V14 (SQK-NBD114.24) according to the manufacturer's instruction on the MinION Mk1C platform with R10.4.1 flow cells. Sequence adaptors were trimmed using Porechop v0.2.4 (Wick [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e] 2023). Sequences were assembled using Flye v2.9.2 (Kolmogorov et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and iteratively polished using Racon v1.5.0 (Vaser et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and Medaka v1.8.0 (Oxford Nanopore [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e] 2023) to obtain a single contig for each strain. Circlator v1.5.5 was used for assembly circularisation (Hunt et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and the quality of assemblies was assessed using BUSCO v5.4.7 (Sim\u0026atilde;o et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Manni et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Complete circular assemblies were annotated using PGAP v6.4 (Tatusova et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Functional characterisation was undertaken using eggNOG-mapper v2.1.12 (Cantalapiedra et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) with default settings and the protein sets for each strain as input. Metabolic capabilities were estimated using gapseq v1.2 (Zimmermann et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhylogenomics and taxonomic analysis\u003c/h3\u003e\n\u003cp\u003eTo test whether these strains could be classified as novel species, a phylogenomic approach was undertaken. First all type strains for \u003cem\u003eMycobacterium\u003c/em\u003e species and subspecies with good quality genomes as outlined in the GTDB database (Parks et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (accessed 2nd August 2023) were downloaded from NCBI (331 genomes; Online Resource 1). Average nucleotide identity (ANI) scores were calculated between the three strains and all reference type strain genomes using fastANI (v1.33) (Jain et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For each of the three strains, the five \u003cem\u003eMycobacterium\u003c/em\u003e species with the highest ANI values were selected and a pangenome of all 18 genomes was created using Panaroo (Tonkin-Hill et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with clean mode strict, invalid genes removed and merged paralogs settings, outputting a core genome alignment. A maximum likelihood phylogenomic tree was then constructed using RAxML-NG (Kozlov et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) v1.2.0 with 20 random starting trees and 100 bootstrap replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe UBCG2 pipeline (Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was also used to extract a core set of 81 universal genes using Prodigal v2.6.3 (Hyatt et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and HMMer v3.4 (Eddy, Sean R \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2026\u003c/span\u003e) from the same set of \u003cem\u003eMycobacterium\u003c/em\u003e genomes, create a concatenated protein alignment using MAFFT v7.525 (Katoh and Standley \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and a ML tree using RAxML v8.2.12 (Stamatakis \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe genome of each strain was also uploaded to the Type Strain Genome Server (TYGS) on January 13th 2026 to calculate the digital DNA-DNA hybridization score (dDDH) using the d\u003csub\u003e4\u003c/sub\u003e formula as recommended (Meier-Kolthoff and G\u0026ouml;ker 2019) and to build a 16S tree via FASTME 2.1.6.1 (Lefort et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic characteristics of the proposed new species\u003c/h2\u003e \u003cp\u003eStandard phenotypic characterisation of each strain is outlined extensively in (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additional testing was undertaken here as outlined in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Of note, \u003cem\u003eM. meyganense\u003c/em\u003e was found to degrade sodium sulfate, \u003cem\u003eM. omidense\u003c/em\u003e to degrade crude oil and \u003cem\u003eM. seymarense\u003c/em\u003e to degrade both. None of the closely related species to these strains (as outlined below) have been reported to have these abilities, indication novel phenotypic differences found in these strains.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenomics and taxogenomics of the proposed new species\u003c/h3\u003e\n\u003cp\u003eBased on phylogenomic placement, the closest species to \u003cem\u003eM. meyganense\u003c/em\u003e is \u003cem\u003eM. stellerae\u003c/em\u003e (GCF_003719305.1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Online Resource 2). ANI analysis revealed an 83% similarity between these two species and a dDDH score of 25.8%. Analysis of the 16S sequence revealed the closest match to be \u003cem\u003eM. gadium\u003c/em\u003e (99%; NR_115805; Online Resource 3) although the ANI between these species was only 79.8% and the dDDH was 20.7%. GapSeq analysis showed that the two species (\u003cem\u003eM. meyganense\u003c/em\u003e and \u003cem\u003eM. stellerae\u003c/em\u003e) have 489 metabolic pathways in common with a further 9 found in \u003cem\u003eM. meyganense\u003c/em\u003e and absent in \u003cem\u003eM. stellerae\u003c/em\u003e and 76 in the reverse (Online Resource 4).\u003c/p\u003e \u003cp\u003eSimilarly, based on phylogenomic placement, the closest species to \u003cem\u003eM. omidense\u003c/em\u003e is \u003cem\u003eM. vaccae\u003c/em\u003e (GCF_001655245.1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Online Resource 2), also by analysis of the 16S sequence (99%; KF378750; Online Resource 3). ANI analysis revealed a 92.6% similarity between these two species and a dDDH of 47.1%. These species share 419 metabolic pathways with each other and a further 16 exclusive to \u003cem\u003eM. omidense\u003c/em\u003e and 44 only in \u003cem\u003eM. vaccae\u003c/em\u003e (Online Resource 5).\u003c/p\u003e \u003cp\u003eThe closest species in the phylogenetic tree to \u003cem\u003eM. seymarense\u003c/em\u003e is \u003cem\u003eM. diernhoferi\u003c/em\u003e (GCF_019456655.1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Online Resource 2; Online Resource 3). ANI analysis revealed an 85.4% similarity between these two species and a dDDH of 26.6%. Analysis of the 16S sequence revealed the closest match to be \u003cem\u003eM. frederiksbergense\u003c/em\u003e (99%; PX678563.1; Online Resource 3). There is no high-quality genome for \u003cem\u003eM. frederiksbergense\u003c/em\u003e available. \u003cem\u003eM. seymarense\u003c/em\u003e and \u003cem\u003eM. diernhoferi\u003c/em\u003e share 389 metabolic pathways with a further 50 exclusive to \u003cem\u003eM. seymarense\u003c/em\u003e and 35 only found in \u003cem\u003eM. diernhoferi\u003c/em\u003e (Online Resource 6).\u003c/p\u003e \u003cp\u003eSince all ANI values between the proposed novel species and closest known type strains fell below the threshold of 95% for defining new species and the dDDH also fell below the threshold of 70% (Riesco and Trujillo \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), these genomic features support the creation of new \u003cem\u003eMycobacterium\u003c/em\u003e species (Chun et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), although the lack of a genome for \u003cem\u003eM. frederiksbergense\u003c/em\u003e means further comparisons may be needed for \u003cem\u003eM. seymarense\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenomic and functional characteristics of the three novel strains\u003c/h2\u003e \u003cp\u003eAll genomes assembled into a single contig with no plasmids detected. BUSCO quality control showed high completeness confirming the closed status of these genomes. Resulting genome features are outlined in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Genome sizes ranged from 5.46Mbp to 6.06Mbp which is standard for \u003cem\u003eMycobacterium\u003c/em\u003e species (Fedrizzi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). GC content was also as expected, ranging from 66.2% to 68.7%.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenome characteristics of each strain\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMycobacterium meyganense\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMycobacterium omidense\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMycobacterium seymarense\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWGS accession\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSAMN08578608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSAMN08578607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSAMN08578606\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16S accession\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKU564078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKU564077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKU564076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGenome size (Mbp)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGC content (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGene count\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5829\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBUSCO completeness\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe species had 389 metabolic pathways in common with each other and a further 35 only found in \u003cem\u003eM. omidense\u003c/em\u003e and 50 only in \u003cem\u003eM. vaccae\u003c/em\u003e (Online Resource 4). COG category analysis showed that all genomes had similar spread of categories with 20\u0026ndash;30% of each genome being uncharacterised (R, S and \u0026ndash; categories) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, two of the strains were able to biodegrade sodium sulfate and two were able to biodegrade crude oil (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, GapSeq metabolic pathway analysis did not clearly indicate the process by which this is undertaken. Naphthalene degradation and Alkane oxidation were not found in any species, although the octane oxidation pathway was present in all (Online Resource 7). Comparison of protein annotation categories showed no prominent differences between novel species and closest established species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion and protologues","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003cp\u003eThe genus \u003cem\u003eMycobacterium\u003c/em\u003e has many species that have been associated with bioremediation and PAH degradation in the past (Rib\u0026oacute;n \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While some have been properly named as novel species, many remain only as described strains without proper taxonomic classification. By using extensive phylogenomic and taxongenomic approaches, we have demonstrated that three such strains, previously shown to grow in the presence of crude oil or sodium sulfate (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), should be classified as novel species, distinguished from closely related species in ANI, phylogenetic placement and metabolic pathway capabilities.\u003c/p\u003e \u003cp\u003eAlthough comparative metabolic analyses was undertaken, the examination of these pathways did not reveal the genetic basis for the ability of these species to undertake bioremediation. This may be because the closely related species, \u003cem\u003eM. diernhoferi\u003c/em\u003e, \u003cem\u003eM. stellerae\u003c/em\u003e and \u003cem\u003eM. vaccae\u003c/em\u003e, have not themselves been tested for bioremediation capabilities. Other closely related species have been shown to have bioremediation capabilities (Das et al. 2015; Deng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hennessee and Li 2016), suggesting that this feature may be widespread in the rapid growing environmental mycobacteria. This work and others indicate that in depth investigations are needed to fully understand and exploit this property in these \u003cem\u003eMycobacterium\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eThe whole genome sequencing / 16S sequence accession numbers for these novel species are as follows: \u003cem\u003eMycobacterium meyganense\u003c/em\u003e: SAMN08578608 / KU564078; \u003cem\u003eMycobacterium omidense\u003c/em\u003e: SAMN08578607 / KU564077; \u003cem\u003eMycobacterium seymarense\u003c/em\u003e: SAMN08578606 / KU564076.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eMycobacterium meyganense\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycobacterium meyganense\u003c/em\u003e (mey.gan.en.se. L. neut. adj. \u003cem\u003emeygan\u003c/em\u003e pertaining to Meygan, the salt lake in Arak city, Iran, from where the type strain originated).\u003c/p\u003e \u003cp\u003eThis species is characterized as acid-fast, non-spore-forming, non-motile short rods. Optimal growth temperature is 25\u0026deg;C. The cells form rough, scotochromogenic colonies, white/yellow in colour, that appear after 6\u0026ndash;14 days on Sauton agar and L\u0026ouml;wenstein-Jensen (LJ) medium (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Strain A16 in (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)).The organism shows a positive reaction to catalase, nitrate reduction and potassium tellurite reduction and is negative to niacin production, pyrazinamidase and urease tests. Tween 80 is not hydrolysed and growth occurs on MacConkey agar but not on LJ supplemented with NaCl 5% or PNB. The isolate can mineralize sodium sulfate, but not PAHs or crude oil.\u003c/p\u003e \u003cp\u003eThe genome of this strain is 5.46 Mbp in length, containing 5,372 predicted genes on a chromosome with no plasmid and a GC content of 66.2%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eMycobacterium omidense\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycobacterium omidense\u003c/em\u003e (o.mi\u0026rsquo;den.se. L. neut. adj. \u003cem\u003eomid\u003c/em\u003e pertaining to Omidieh city, Iran, from where the type strain originated).\u003c/p\u003e \u003cp\u003eThis species is characterized as acid-fast, non-spore-forming, non-motile short rods. The cells form smooth, scotochromogenic colonies, yellow in colour, that appear after 3 days on Sauton agar and LJ medium (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Strain A14 in (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)).). Optimal growth temperature is 25\u0026deg;C. The organism shows a positive reaction to urease, catalase and nitrate reduction but negative to pyrazinamidase, niacin production, and potassium tellurite reduction. Tween 80 is not hydrolyzed and growth occurs on LJ with NaCl 5% and on MacConkey agar, but not on PNB-containing LJ. The isolate is able to biodegrade PAHs and crude oil, but cannot grow in the presence of sodium sulfate.\u003c/p\u003e \u003cp\u003eThe genome of this strain is 6.06 Mbp in length, containing 5,814 predicted genes on a chromosome with no plasmid and a GC content of 68.7%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eMycobacterium seymarense\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycobacterium seymarense\u003c/em\u003e (sey.mar.en.se. L. neut. adj. \u003cem\u003eseymar\u003c/em\u003e pertaining to the Seymareh river in the Lorestan province, Iran, from where the type strain originated).\u003c/p\u003e \u003cp\u003eThis species is characterized as acid-fast, non-spore-forming, non-motile short rods. The cells form smooth scotochromogenic colonies, yellow in colour, that appear after 5 days on Sauton agar and LJ medium (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Strain A12 in (Azadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)). Optimal growth temperature is 35\u0026deg;C. The organism shows a positive reaction to catalase and negative to urease, nitrate reduction, pyrazinamidase, niacin and potassium tellurite reduction. Tween 80 is hydrolyzed and growth occurs on LJ with NaCl 5% and on MacConkey agar, but not on PNB-containing LJ. The isolate is able to degrade PAHs, sodium sulfate and crude oil, showing its potential use for cleaning of petroleum hydrocarbon.\u003c/p\u003e \u003cp\u003eThe genome of this strain is 5.99 Mbp in length, containing 5,829 predicted genes on a single chromosome with no plasmid and a GC content of 67.3%. The assembly was predicted to be 100% complete by BUSCO and assembled into a single chromosome with no plasmid. Based on phylogenomic placement, the closest species to \u003cem\u003eM. seymarense\u003c/em\u003e is \u003cem\u003eM. diernhoferi.\u003c/em\u003e ANI analysis revealed an 85.4% similarity between these two species. Analysis of the 16S sequence revealed the closest match to be \u003cem\u003eM. frederiksbergense\u003c/em\u003e (99.93%; PX678563.1). These genomic features support the creation of a new \u003cem\u003eMycobacterium\u003c/em\u003e species, although the lack of a genome for \u003cem\u003eM. frederiksbergense\u003c/em\u003e means further comparisons may be needed.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e\u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eNo human or animal related strains were collected for this study and thus no ethical approval was required.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors are grateful to the Office of Vice Chancellor for Research of Isfahan University of Medical Sciences for the support of the current study. Funding is provided by the Department of Economy, Science and Innovation of the Flemish Government and the Belgian Science Policy (Belspo). Support by the European Research Council-INTERRUPTB starting grant [nr.311725] to CJM and LR. The work is also supported by the Academy of Medical Sciences (AMS), the Wellcome Trust, the Government Department of Business, Energy and Industrial Strategy (BEIS), the British Heart Foundation and Diabetes UK and the Global Challenges Research Fund (GCRF) via a Springboard grant [SBF006\\1090] to CJM. The project was supported partially by a research grant number 393409 from Isfahan University of Medical Sciences, Isfahan, Iran to HS and DA. Funds for the nanopore sequencing was provided by Nottingham Trent University internal grants.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.J.M. contributed to conceptualization, methodology, formal analysis, investigation, writing -original draft, writing \u0026ndash; reviewing \u0026amp; editing, and funding acquisition.S.C. contributed to conceptualization, methodology, resources, data curation, writing -original draft, writing \u0026ndash; reviewing \u0026amp; editingM.B. contributed to methodology, resources, data curation, writing -original draft, writing \u0026ndash; reviewing \u0026amp; editing.M.D. contributed to resources, data curation and writing \u0026ndash; reviewing \u0026amp; editing.L.R. contributed to resources, data curation, writing \u0026ndash; reviewing \u0026amp; editing and supervision.H.S. contributed to conceptualization, methodology, resources, data curation, writing \u0026ndash; reviewing \u0026amp; editing and supervision.D.A. contributed to conceptualization, formal analysis, investigation, writing -original draft, writing \u0026ndash; reviewing \u0026amp; editing, supervision and funding acquisition.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe whole genome sequence and 16S gene sequence of each strain are available from NCBI with the following accessions (WGS/16S): Mycobacterium meyganense (SAMN08578608/KU564078); Mycobacterium omidense (SAMN08578607/KU564077); Mycobacterium seymarense (SAMN08578606/KU564076).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAltschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. \u0026lsquo;Basic Local Alignment Search Tool\u0026rsquo;. \u003cem\u003eJ Mol Biol\u003c/em\u003e 215 (3): 403\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0022-2836(05)80360-2\u003c/span\u003e\u003cspan address=\"10.1016/S0022-2836(05)80360-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzadi, Davood, Hasan Shojaei, Sina Mobasherizadeh, and Abass Daei Naser. 2017. \u0026lsquo;Screening, Isolation and Molecular Identification of Biodegrading Mycobacteria from Iranian Ecosystems and Analysis of Their Biodegradation Activity\u0026rsquo;. \u003cem\u003eAMB Express\u003c/em\u003e 7 (1): 180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13568-017-0472-4\u003c/span\u003e\u003cspan address=\"10.1186/s13568-017-0472-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCantalapiedra, Carlos P., Ana Hern\u0026aacute;ndez-Plaza, Ivica Letunic, Peer Bork, and Jaime Huerta-Cepas. 2021. \u0026lsquo;eggNOG-Mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale\u0026rsquo;. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e 38 (12): 5825\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msab293\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msab293\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChun, Jongsik, Aharon Oren, Antonio Ventosa, et al. 2018. \u0026lsquo;Proposed Minimal Standards for the Use of Genome Data for the Taxonomy of Prokaryotes\u0026rsquo;. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e 68 (1): 461\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/ijsem.0.002516\u003c/span\u003e\u003cspan address=\"10.1099/ijsem.0.002516\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas, Sarbashis, B. M. Fredrik Pettersson, Phani Rama Krishna Behra, et al. 2015. \u0026lsquo;Characterization of Three Mycobacterium Spp. with Potential Use in Bioremediation by Genome Sequencing and Comparative Genomics\u0026rsquo;. \u003cem\u003eGenome Biology and Evolution\u003c/em\u003e 7 (7): 1871\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/gbe/evv111\u003c/span\u003e\u003cspan address=\"10.1093/gbe/evv111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng, Yang, Tong Mou, Junhuan Wang, Jing Su, Yanchun Yan, and Yu-Qin Zhang. 2023. \u0026lsquo;Characterization of Three Rapidly Growing Novel Mycobacterium Species with Significant Polycyclic Aromatic Hydrocarbon Bioremediation Potential\u0026rsquo;. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e 14 (September): 1225746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2023.1225746\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2023.1225746\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEddy, Sean R. 2026. \u0026lsquo;HMMER\u0026rsquo;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hmmer.org/\u003c/span\u003e\u003cspan address=\"http://hmmer.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFedrizzi, Tarcisio, Conor J. Meehan, Antonella Grottola, et al. 2017. \u0026lsquo;Genomic Characterization of Nontuberculous Mycobacteria\u0026rsquo;. \u003cem\u003eScientific Reports\u003c/em\u003e 7 (1): 45258. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep45258\u003c/span\u003e\u003cspan address=\"10.1038/srep45258\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHazb\u0026oacute;n, Manzour Hernando, Leen Rigouts, Marco Schito, et al. 2018. \u0026lsquo;Mycobacterial Biomaterials and Resources for Researchers\u0026rsquo;. \u003cem\u003ePathogens and Disease\u003c/em\u003e Accepted.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHennessee, Christiane T., and Qing X. Li. 2016. \u0026lsquo;Effects of Polycyclic Aromatic Hydrocarbon Mixtures on Degradation, Gene Expression, and Metabolite Production in Four Mycobacterium Species\u0026rsquo;. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e 82 (11): 3357\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00100-16\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00100-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunt, Martin, Nishadi De Silva, Thomas D. Otto, Julian Parkhill, Jacqueline A. Keane, and Simon R. Harris. 2015. \u0026lsquo;Circlator: Automated Circularization of Genome Assemblies Using Long Sequencing Reads\u0026rsquo;. \u003cem\u003eGenome Biology\u003c/em\u003e 16 (1): 294. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13059-015-0849-0\u003c/span\u003e\u003cspan address=\"10.1186/s13059-015-0849-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyatt, Doug, Gwo-Liang Chen, Philip F. LoCascio, Miriam L. Land, Frank W. Larimer, and Loren J. Hauser. 2010. \u0026lsquo;Prodigal: Prokaryotic Gene Recognition and Translation Initiation Site Identification\u0026rsquo;. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e 11 (1): 119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2105-11-119\u003c/span\u003e\u003cspan address=\"10.1186/1471-2105-11-119\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, Chirag, Luis M. Rodriguez-R, Adam M. Phillippy, Konstantinos T. Konstantinidis, and Srinivas Aluru. 2018. \u0026lsquo;High Throughput ANI Analysis of 90K Prokaryotic Genomes Reveals Clear Species Boundaries\u0026rsquo;. \u003cem\u003eNature Communications\u003c/em\u003e 9 (1): 1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-018-07641-9\u003c/span\u003e\u003cspan address=\"10.1038/s41467-018-07641-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh, Kazutaka, and Daron M. Standley. 2013. \u0026lsquo;MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability\u0026rsquo;. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e 30 (4): 772\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/mst010\u003c/span\u003e\u003cspan address=\"10.1093/molbev/mst010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, Jihyeon, Seong-In Na, Dongwook Kim, and Jongsik Chun. 2021. \u0026lsquo;UBCG2: Up-to-Date Bacterial Core Genes and Pipeline for Phylogenomic Analysis\u0026rsquo;. \u003cem\u003eJournal of Microbiology\u003c/em\u003e 59 (6): 609\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12275-021-1231-4\u003c/span\u003e\u003cspan address=\"10.1007/s12275-021-1231-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolmogorov, Mikhail, Jeffrey Yuan, Yu Lin, and Pavel A. Pevzner. 2019. \u0026lsquo;Assembly of Long, Error-Prone Reads Using Repeat Graphs\u0026rsquo;. \u003cem\u003eNature Biotechnology\u003c/em\u003e 37 (5): 5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41587-019-0072-8\u003c/span\u003e\u003cspan address=\"10.1038/s41587-019-0072-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozlov, Alexey M., Diego Darriba, Tom\u0026aacute;š Flouri, Benoit Morel, and Alexandros Stamatakis. 2019. \u0026lsquo;RAxML-NG: A Fast, Scalable and User-Friendly Tool for Maximum Likelihood Phylogenetic Inference\u0026rsquo;. \u003cem\u003eBioinformatics\u003c/em\u003e 35 (21): 4453\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btz305\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btz305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLefort, Vincent, Richard Desper, and Olivier Gascuel. 2015. \u0026lsquo;FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program\u0026rsquo;. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e 32 (10): 2798\u0026ndash;800. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msv150\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msv150\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagee, John G., and Alan C. Ward. 2015. \u0026lsquo;Mycobacterium\u0026rsquo;. In \u003cem\u003eBergey\u0026rsquo;s Manual of Systematics of Archaea and Bacteria\u003c/em\u003e. John Wiley \u0026amp; Sons, Ltd. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/9781118960608.gbm00029\u003c/span\u003e\u003cspan address=\"10.1002/9781118960608.gbm00029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManni, Mos\u0026egrave;, Matthew R. Berkeley, Mathieu Seppey, and Evgeny M. Zdobnov. 2021. \u0026lsquo;BUSCO: Assessing Genomic Data Quality and Beyond\u0026rsquo;. \u003cem\u003eCurrent Protocols\u003c/em\u003e 1 (12): e323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cpz1.323\u003c/span\u003e\u003cspan address=\"10.1002/cpz1.323\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeier-Kolthoff, Jan P., and Markus G\u0026ouml;ker. 2019. \u0026lsquo;TYGS Is an Automated High-Throughput Platform for State-of-the-Art Genome-Based Taxonomy\u0026rsquo;. \u003cem\u003eNature Communications\u003c/em\u003e 10 (1): 2182. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-019-10210-3\u003c/span\u003e\u003cspan address=\"10.1038/s41467-019-10210-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOxford Nanopore. (2017) 2023. \u003cem\u003eMedaka\u003c/em\u003e. Python. June 7; Oxford Nanopore Technologies, released September 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/nanoporetech/medaka\u003c/span\u003e\u003cspan address=\"https://github.com/nanoporetech/medaka\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParks, Donovan H., Maria Chuvochina, Christian Rinke, Aaron J. Mussig, Pierre-Alain Chaumeil, and Philip Hugenholtz. 2022. \u0026lsquo;GTDB: An Ongoing Census of Bacterial and Archaeal Diversity through a Phylogenetically Consistent, Rank Normalized and Complete Genome-Based Taxonomy\u0026rsquo;. \u003cem\u003eNucleic Acids Research\u003c/em\u003e 50 (D1): D785\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkab776\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkab776\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePontiroli, Alessandra, Tanya T. Khera, Brian B. Oakley, et al. 2013. \u0026lsquo;Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity\u0026rsquo;. \u003cem\u003ePLOS ONE\u003c/em\u003e 8 (7): e68648. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0068648\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0068648\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRib\u0026oacute;n, Wellman. 2018. \u003cem\u003eMycobacterium: Research and Development\u003c/em\u003e. BoD \u0026ndash; Books on Demand.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiesco, Ra\u0026uacute;l, and Martha E. Trujillo. 2024. \u0026lsquo;Update on the Proposed Minimal Standards for the Use of Genome Data for the Taxonomy of Prokaryotes\u0026rsquo;. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e 74 (3): 006300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/ijsem.0.006300\u003c/span\u003e\u003cspan address=\"10.1099/ijsem.0.006300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma, Babita, Nita Pal, Bharti Malhotra, and Leela Vyas. 2010. \u0026lsquo;Evaluation of a Rapid Differentiation Test for Mycobacterium Tuberculosis from Other Mycobacteria by Selective Inhibition with P-Nitrobenzoic Acid Using MGIT 960\u0026rsquo;. \u003cem\u003eJournal of Laboratory Physicians\u003c/em\u003e 2 (2): 89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4103/0974-2727.72157\u003c/span\u003e\u003cspan address=\"10.4103/0974-2727.72157\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSim\u0026atilde;o, Felipe A., Robert M. Waterhouse, Panagiotis Ioannidis, Evgenia V. Kriventseva, and Evgeny M. Zdobnov. 2015. \u0026lsquo;BUSCO: Assessing Genome Assembly and Annotation Completeness with Single-Copy Orthologs\u0026rsquo;. \u003cem\u003eBioinformatics\u003c/em\u003e 31 (19): 3210\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btv351\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btv351\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStamatakis, Alexandros. 2014. \u0026lsquo;RAxML Version 8: A Tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies\u0026rsquo;. \u003cem\u003eBioinformatics\u003c/em\u003e 30 (9): 1312\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btu033\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btu033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTatusova, Tatiana, Michael DiCuccio, Azat Badretdin, et al. 2016. \u0026lsquo;NCBI Prokaryotic Genome Annotation Pipeline\u0026rsquo;. \u003cem\u003eNucleic Acids Research\u003c/em\u003e 44 (14): 6614\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkw569\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkw569\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTonkin-Hill, Gerry, Neil MacAlasdair, Christopher Ruis, et al. 2020. \u0026lsquo;Producing Polished Prokaryotic Pangenomes with the Panaroo Pipeline\u0026rsquo;. \u003cem\u003eGenome Biology\u003c/em\u003e 21 (1): 180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13059-020-02090-4\u003c/span\u003e\u003cspan address=\"10.1186/s13059-020-02090-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurenne, Christine Y. 2019. \u0026lsquo;Nontuberculous Mycobacteria: Insights on Taxonomy and Evolution\u0026rsquo;. \u003cem\u003eInfection, Genetics and Evolution\u003c/em\u003e 72 (January). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.MEEGID.2019.01.017\u003c/span\u003e\u003cspan address=\"10.1016/J.MEEGID.2019.01.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaser, Robert, Ivan Sović, Niranjan Nagarajan, and Mile Šikić. 2017. \u0026lsquo;Fast and Accurate de Novo Genome Assembly from Long Uncorrected Reads\u0026rsquo;. \u003cem\u003eGenome Research\u003c/em\u003e 27 (5): 737\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gr.214270.116\u003c/span\u003e\u003cspan address=\"10.1101/gr.214270.116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWayne, LG. 1984. \u003cem\u003eMycobacterial Speciation. in The Mycobacteria: A Sourcebook\u003c/em\u003e. Edited by GP Kubica and LG Wayne. Marcel Dekker, New York, N.Y.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWick, Ryan. (2017) 2023. \u003cem\u003ePorechop\u003c/em\u003e. C++. February 13, released September 9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rrwick/Porechop\u003c/span\u003e\u003cspan address=\"https://github.com/rrwick/Porechop\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmermann, Johannes, Christoph Kaleta, and Silvio Waschina. 2021. \u0026lsquo;Gapseq: Informed Prediction of Bacterial Metabolic Pathways and Reconstruction of Accurate Metabolic Models\u0026rsquo;. \u003cem\u003eGenome Biology\u003c/em\u003e 22 (1): 81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13059-021-02295-1\u003c/span\u003e\u003cspan address=\"10.1186/s13059-021-02295-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bioremediation, environmental, Mycobacterium, non-tuberculous mycobacteria, phylogenomics, taxogenomics","lastPublishedDoi":"10.21203/rs.3.rs-8862259/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8862259/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThree strains capable of bioremediation were isolated from various environmental sources throughout Iran. Physiological characterisations conformed to known \u003cem\u003eMycobacterium\u003c/em\u003e standards with the addition of the ability to degrade sodium sulfate and/or crude oil. Whole genome sequencing, phylogenomic analyses and average nucleotide identity comparisons to all closely related sequenced type strains of mycobacteria revealed three novel species of mycobacteria. These are hence named \u003cem\u003eMycobacterium meyganense\u003c/em\u003e sp. nov. (strain A16\u0026thinsp;=\u0026thinsp;BCCM ITM-500916\u0026thinsp;=\u0026thinsp;DSM 107075), \u003cem\u003eMycobacterium omidense\u003c/em\u003e sp. nov. (strain A14\u0026thinsp;=\u0026thinsp;BCCM ITM-500915\u0026thinsp;=\u0026thinsp;DSM 107139), and \u003cem\u003eMycobacterium seymarense\u003c/em\u003e sp. nov. (strain A12\u0026thinsp;=\u0026thinsp;BCCM ITM-500914\u0026thinsp;=\u0026thinsp;DSM 107138).\u003c/p\u003e","manuscriptTitle":"Mycobacterium meyganense sp. nov., Mycobacterium omidense sp. nov., and Mycobacterium seymarense sp. nov.: bioremediation-capable novel species from Iranian environmental samples.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 15:31:12","doi":"10.21203/rs.3.rs-8862259/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c5eeaed-b9a8-488f-8fb2-c41469e532c4","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-10T06:25:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 15:31:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8862259","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8862259","identity":"rs-8862259","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}