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. 2022 Apr 29;1(2):198–209. doi: 10.1002/mlf2.12009

Comparative genomic analysis of Thermus provides insights into the evolutionary history of an incomplete denitrification pathway

Jian‐Yu Jiao 1, # , Zheng‐Han Lian 1, # , Meng‐Meng Li 1, # , Nimaichand Salam 1, En‐Min Zhou 2, Lan Liu 1, Hong Ming 3, Guoxing Nie 4, Wensheng Shu 5, Guoping Zhao 6, Brian P Hedlund 7,8,✉, Wen‐Jun Li 1,9,✉
PMCID: PMC10989939  PMID: 38817678

Impact statement

For decades, Thermus was always considered to be aerobic. However, recent studies have suggested that the denitrification abilities of Thermus species may be widely underestimated. In the present study, we used comparative genomic analysis to investigate the evolutionary history of the denitrification pathway in Thermus and other members of the phylum Deinococcota. We revealed incomplete denitrification pathways to be common in Thermus and showed they are inherited mostly vertically, which further supports the importance of Thermus as a significant denitrifier in hydrothermal environments.

Keywords: comparative genomics, denitrification, evolutionary history, Thermus

Abstract

Biological denitrification is a crucial process in the nitrogen biogeochemical cycle, and Thermus has been reported to be a significant heterotrophic denitrifier in terrestrial geothermal environments. However, neither the denitrification potential nor the evolutionary history of denitrification genes in the genus Thermus or phylum Deinococcota is well understood. Here, we performed a comparative analysis of 23 Thermus genomes and identified denitrification genes in 15 Thermus strains. We confirmed that Thermus harbors an incomplete denitrification pathway as none of the strains contain the nosZ gene. Ancestral character state reconstructions and phylogenetic analyses showed that narG, nirS, and norB genes were acquired by the last common ancestor of Thermales and were inherited vertically. In contrast, nirK of Thermales was acquired via two distinct horizontal gene transfers from Proteobacteria to the genus Caldithermus and from an unknown donor to the common ancestor of all known Thermus species except Thermus filiformis. This study expands our understanding of the genomic potential for incomplete denitrification in Thermus, revealing a largely vertical evolutionary history of the denitrification pathway in the Thermaceae, and supporting the important role for Thermus as an important heterotrophic denitrifier in geothermal environments.

INTRODUCTION

Thermus species are members of the family Thermaceae and are generally Gram‐strain‐negative, high‐G + C‐content, nonmotile, rod‐shaped, and obligately aerobic or facultatively anaerobic. The genus Thermus belongs to the phylum Deinococcota, along with the genera Calidithermus, Marinithermus, Meiothermus, Oceanithermus, Rhabdothermus, Vulcanithermus, Deinococcus, Deinobacterium, and Truepera. To date, the genus Thermus comprises 19 validly published species names under the International Code of Nomenclature of Prokaryotes (ICNP) 1 , and members of this genus are archetypal thermophilic bacteria that are readily isolated from terrestrial geothermal environments, especially circumneutral pH or alkaline hot springs 2 . Thermus species are well known for their biotechnological applications, such as the production of thermostable enzymes (e.g., Taq DNA polymerase) 3 . Several species of Thermus were reported to have incomplete denitrification pathways with corresponding experimental evidence for the production of nitrite or nitrous oxide 4 , 5 .

Denitrification is a crucial component of the nitrogen cycle in geothermal systems. Complete biological denitrification is a respiratory process that reduces nitrogenous oxides to dinitrogen and is normally catalyzed by nitrate reductase (NarGHI or NapAB), nitrite reductase (NirK or NirS), nitric oxide reductase (NorBC), and nitrous oxide reductase (NosZ) under oxygen‐limiting or anaerobic conditions 2 . Previous studies have shown that denitrification is highly active in hot springs and that members of the genus Thermus play a significant role as heterotrophic denitrifiers in hot spring environments 4 , 6 . However, the distribution of denitrification genes within Thermus and the evolutionary history of the Thermus denitrification pathway have not been investigated.

Currently, only a few strains of the genus Thermus have been reported to be incomplete denitrifiers, and there is no consensus in the literature on the nature of denitrification in the genus Thermus. Furthermore, considering the high plasticity of Thermus genomes 7 , natural competence to transform DNA 8 , and abundance of insertion sequence (IS) elements and prophages on Thermus genomes, including the plasmid‐borne nitrate conjugative element in Thermus thermophilus HB8 and NAR1 5 , 9 , 10 , 11 , we hypothesize that denitrification genes might be obtained through various horizontal gene transfer (HGT) events. If so, the distribution and evolutionary origins of denitrification genes in various Thermus species might be highly variable and responsive to selective forces in individual geothermal systems, including the supply of oxidized nitrogen species.

To test our hypothesis, we performed a comparative genome analysis of Thermus species and analyzed three aspects of the evolutionary history of the Thermus denitrification pathway viz. (1) the presence of genes involved in denitrification pathway in a representative of all Thermus species; (2) phylogenetic analysis of major denitrification proteins; and (3) investigation of gene gain and loss events in Thermus genomes.

RESULTS AND DISCUSSION

Phylogenetic analysis shows monophyly of the genus Thermus

The genus Thermus presently harbors 19 species, most of which can be isolated from terrestrial geothermal environments. A phylogenetic dendrogram indicating the phylogenetic relationships within the phylum Deinococcota was generated based on a concatenated alignment of conserved positions based on 120 bacterial marker genes, as implemented by Genome Taxonomy Database‐Toolkit (GTDB‐Tk) 12 . The phylogenomic tree showed that the genus Thermus is monophyletic within the family Thermaceae with high bootstrap support (Figure 1A). Both average nucleotide identity (ANI) and average amino acid identity (AAI) values showed large genomic divergences among the Thermus genomes (Figure 1B,C and Tables S1–S2). However, among the 23 Thermus strains, Thermus thermophilus and Thermus parvatiensis formed a monophyletic clade, and both ANI and AAI values between these two species were higher than 95%, indicating they belong to the same species 13 , 14 . Given the priority of the species T. thermophilus, we suggest that T. parvatiensis is a later heterotypic synonym of T. thermophilus. The independence of all other Thermus species was confirmed using a 95% ANI species boundary 15 . The evolutionary distances between Thermus  filiformis ATCC 43280T and the other Thermus strains were high, although T. filiformis ATCC 43280T was monophyletic with other members of the genus Thermus with high bootstrap support (Figure 1 and Tables S1–S2).

Figure 1.

Figure 1

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Phylogenetic inference of Thermus strains. (A) Phylogenetic placement of Thermus strains. Multiple sequence alignments of 120 bacterial marker genes were generated by GTDB‐Tk and IQ‐Tree was used to construct the phylogenomic tree. Red stars illustrate that the strains were isolated or the genomes were sequenced previously. (B) Average nucleotide identity (ANI) values shared among the Thermus species. (C) Average amino acid identity (AAI) values shared among the Thermus species. The ANI and AAI values are presented in Tables S1–S2, respectively.

Comparative genome analysis of Thermus shows similar genomic features

The properties and statistics of the finished and draft Thermus genomes are summarized in Table 1. Twenty‐three Thermus genomes with 20 different species names ranged in size from 2.02 to 2.56 Mbp, with G + C content between 64.81% and 69.50%. The number of protein‐coding genes ranged from 2224 to 2749, while the number of predicted transfer RNA (tRNAs), encoding almost all 20 amino acids, ranged from 43 to 53. The estimated completeness of all the genomes based on conserved marker genes was more than 99% (except Thermus amyloliquefaciens YIM 77409T at 97.81% and T. parvatiensis RL at 95.76%). The genome sizes of Thermus species were smaller than those of most members within the phylum Deinococcota, and the coding density of Thermus was higher compared with most other species (Table S3). The smaller genome sizes of Thermus reflect a well‐documented pattern of genomic reduction among thermophiles 16 . Mobile genetic elements such as IS elements and genomic islands (GI) were also detected. Each Thermus genome contains a large number of IS elements (Table S4), and almost all the genomes (22/23) contain a GI (Table S5). However, no denitrification genes were detected close to any of the mobile genetic elements, suggesting that the denitrification genes might not be transferred through these two mobilomes. The detection of CRISPR‐Cas system indicated their bacterial immune system for protection against alien DNA.

Table 1.

General features of the Thermus genomes.

Strain Completeness (%) Contamination (%) Genome size (bp) Scaffolds N 50 (scaffolds) GC (%) Coding density (%) Predicted genes Unique genes tRNAs rRNA copies CRISPR
Thermus amyloliquefaciens YIM 77409T 97.81 0 21,60,855 6 20,55,291 67.44 92.61 2315 129 48 2 5
Thermus antranikianii DSM 12462T 100.00 0 21,65,150 33 2,18,058 64.81 94.46 2265 41 47 1 0
Thermus aquaticus Y51MC23 100.00 0 23,38,641 5 21,58,963 68.04 93.15 2501 145 53 2 10
Thermus arciformis CGMCC 1.6992T 100.00 0 24,42,297 88 58,107 68.79 93.70 2643 105 47 2 12
Thermus brockianus GE‐1 99.36 0 23,88,273 3 20,35,182 66.90 93.27 2530 132 47 2 9
Thermus caldifontis YIM 73026T 100.00 0 21,63,786 41 1,93,267 64.85 94.27 2275 52 46 1 4
Thermus caldilimi YIM 78456T 99.58 0 24,47,659 1 24,47,659 65.14 93.87 2639 155 47 2 0
Thermus caliditerrae YIM 77777 100.00 0.42 22,18,114 4 20,46,548 67.22 93.83 2285 76 50 2 5
Thermus composti JCM 19902T 100.00 0 20,80,570 64 82,383 67.70 94.64 2247 54 45 1 3
Thermus filiformis ATCC 43280T 99.15 0.42 23,86,081 40 5,51,922 69.01 92.76 2410 188 47 2 15
Thermus igniterrae ATCC 700962T 99.58 0.42 22,25,983 74 62,194 68.80 94.94 2331 52 43 2 10
Thermus islandicus DSM 21543T 99.15 0 22,63,010 67 2,41,998 68.35 93.75 2412 145 47 2 2
Thermus oshimai DSM 12092T 100.00 0 22,60,954 24 2,00,705 68.74 94.80 2355 93 48 1 15
Thermus parvatiensis RL 95.76 0 20,16,098 2 18,72,821 68.53 90.78 2463 189 47 2 2
Thermus scotoductus SA‐01 100.00 0 23,55,186 2 23,46,803 64.89 94.25 2458 80 48 2 5
Thermus sediminis L198 99.15 0 21,60,271 4 20,28,157 68.21 92.46 2287 149 48 2 5
Thermus tengchongensis YIM 77401 100.00 2.54 25,62,314 5 22,24,342 66.40 92.57 2749 179 47 2 5
Thermus tenuipuniceus YIM 76954T 100.00 0 22,61,036 66 51,325 65.54 94.02 2375 76 47 1 5
Thermus thermamylovorans CFH 72773T 99.58 0 22,50,808 54 1,71,757 69.50 94.05 2336 89 45 1 11
Thermus thermophilus HB27 99.58 0 21,27,482 2 18,94,877 69.41 93.86 2224 43 48 2 10
T. thermophilus HB8T 99.58 0 21,16,056 3 18,49,742 69.50 93.80 2224 38 48 2 11
T. thermophilus JL‐18 100.00 0 23,11,212 3 19,02,595 68.98 94.11 2459 51 49 2 6
T. thermophilus SG0.5JP17‐16 100.00 0.42 23,03,227 2 18,63,201 68.62 93.81 2451 98 48 2 8
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The genetic variability among Thermus genomes can be further determined from the distribution of the conserved (core) and species‐specific (unique) genes. The pan‐genome of the Thermus genomes comprised 6992 gene clusters including 1027 homologous gene clusters in the core genome (Figure 2A). Comparative genome analysis revealed that each of the Thermus species contains a small number (38–189) of unique genes in their genomes (Table 1).

Figure 2.

Figure 2

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Pan‐ and core‐genome evolution and functional annotations of Thermus genomes. (A) Pan‐ and core‐genome evolution of Thermus. (B) Categorization of the function of each protein‐coding gene was based on COG categories. J: Translation, ribosomal structure, and biogenesis; K: Transcription; L: Replication, recombination, and repair; B: Chromatin structure and dynamics; Z: Cytoskeleton; D: Cell‐cycle control, cell division, chromosome partitioning; V: Defense mechanisms; T: Signal transduction mechanisms; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; U: Intracellular trafficking, secretion, and vesicular transport; O: Posttranslational modification, protein turnover, chaperones; C: Energy production and conversion; G: Carbohydrate transport and metabolism; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; H: Coenzyme transport and metabolism; I: Lipid transport and metabolism; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport, and catabolism; R: General function prediction only; S: Function unknown.

The properties and statistics of genes annotated into the cluster of orthologous groups (COGs) functional categories are shown in Figure 2B and Table S6. The most abundant COGs in the Thermus genomes are assigned to general function prediction only (COG category R), followed closely by CDSs dedicated to amino acid transport and metabolism (COG category E). These two categories are in higher proportions compared with other COG categories in all Thermus genomes. Notably, for an overall comparison among the genomes of Thermus strains, all the genomes contain a similar gene number in each COG category, and almost half of the gene clusters (1027) are conserved in all genomes, which suggests that Thermus genomes are more stable than generally considered.

Thermus incomplete denitrification pathways evolve through a combination of horizontal and vertical processes

Nitrogen oxides can act as the terminal electron acceptors for respiration in the absence of oxygen. Previous studies have reported that the members of Thermus, such as Thermus scotoductus SE‐1T, Thermus tenuipuniceus YIM 76954T, and a variety of T. thermophilus and Thermus oshimai strains can grow anaerobically with nitrate as electron acceptors 17 , 18 . In total, 15 of the examined genomes contained annotated denitrification genes (Figure 3), and the genes coding for denitrification enzymes were located close to genes for cytochrome c oxidase (Figures S1–S2), supporting their potential roles in respiration. Furthermore, the synteny narGHI, nirS, and norBC are conserved, suggesting that these genes evolve as an evolutionary unit (Figures S1–S2). Fourteen of the Thermus genomes contain the genes coding for nitrate reductase. Additionally, narGHI was prevalent in other genera of Thermaceae but was absent from the genera Truepera and Deinococcus, which belong to the related families Treuperaceae and Deinococcaceae. Furthermore, phylogenetic analysis of NarG showed that the Thermales nitrate reductases were clustered in a single clade (Figure 4), indicating the narG genes, overall, were inherited vertically.

Figure 3.

Figure 3

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Gain events of denitrification genes in the phylum Deinococcota. The Bayesian tree was determined by MrBayes 19 , and gene gain events were calculated by using COUNT software 20 . The black circles represent nodes with posterior possibilities higher than 0.9. The presence or absence of genes related to denitrification genes in the phylum Deinococcota is shown on the right of the Bayesian tree. The gain of denitrification genes in Deinococcota is marked.

Figure 4.

Figure 4

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Consistency between the phylogenomic tree of Deinococcota and the phylogenetic tree of NarG. (A) The phylogenomic tree of Deinococcota was constructed as in Figure 1A. (B) The NarG sequences listed in Table S7 were aligned using MUSCLE with 100 iterations. The NarG tree was constructed using IQ‐Tree with the parameters (‐alrt 1000 ‐bb 1000 ‐nt AUTO). The best‐fit model (LG + R9) was determined by ModelFinder.

Nitrite reduction (NO2 − to NO) is often a rate‐limiting process in denitrification 21 . Although 14 genomes contain nitrite reductases, the enzymes for nitrite reduction are different in different Thermus species. Thermus species can catalyze nitrite reduction by two types of nitrite reductases, NirK or NirS. Five Thermus strains were shown to contain nirK, which encodes the Cu‐containing nitrite reductase (Figure 3), whereas nirS, encoding the isofunctional tetraheme cytochrome cd1‐containing nitrite reductase, was present in 12 strains. This finding indicates that nirS‐type nitrite reductases are more prevalent in Thermus species. A previous study reported the distinct roles of nirK and nirS in the genus Thermus: nirS was expressed higher in comparison to nirK under oxic and stable conditions. In contrast, nirK was expressed over a wider range of nitrite concentrations 21 . Three Thermus genomes, T. oshimai DSM 12092T, T. scotoductus SA‐01, and Thermus antranikianii DSM 12462T, harbor two types of nitrite reductases, suggesting that they might be able to utilize nitrite more effectively over a wide concentration range and could use nitrite as an electron acceptor under oxic conditions 21 .

The phylogeny of the NirK from Thermales (Figure 5) revealed three major clades, representing the three genera Thermus, Calidithermus, and Deinococcus, which suggests a more complex evolutionary history compared with other denitrification genes. The NirK of the genera Calidithermus and Deinococcus were clearly within a clade found in Proteobacteria, indicating potential HGT events from Proteobacteria to these two genera. However, the five Thermus NirK proteins were monophyletic, and their sister lineages contained multiple phyla (e.g., Spirochaetes, Firmicutes, Acidobacteria, Crenarchaeota, etc.), suggesting a different evolutionary origin. In contrast, NirS was only present in the order Thermales and formed a monophyletic clade (Figure 6). Furthermore, the ancestral character state reconstruction of NirS (Figure 3) showed that the last common ancestor of Thermales likely encoded NirS for nitrite reduction. Our results suggest that the Thermales acquired nirS before their divergence, and that nirS was subsequently inherited vertically with multiple gene losses; in contrast, nirK was acquired from different donors.

Figure 5.

Figure 5

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Discrepancy between the phylogenomic tree of Deinococcota and the phylogenetic tree of NirK. (A) The phylogenomic tree of Deinococcota was constructed as in Figure 1A. (B) The NirK protein sequences listed in Table S8 were aligned using MUSCLE with 100 iterations. The NirK tree was constructed using IQ‐Tree with the parameters (‐alrt 1000 ‐bb 1000 ‐nt AUTO). The best‐fit model (WAG + F + R10) was determined by ModelFinder. Red star illustrates the strains from the family Deinococcaceae.

Figure 6.

Figure 6

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Consistency between the phylogenomic tree of Deinococcota and the phylogenetic tree of NirS. (A) The phylogenomic tree of Deinococcota was constructed as in Figure 1A. (B) The NirS protein sequences listed in Table S9 were aligned using MUSCLE with 100 iterations. The NirS tree was constructed using IQ‐Tree with the parameters (‐alrt 1000 ‐bb 1000 ‐nt AUTO). The best‐fit model (LG + F + R10) was determined by ModelFinder.

The genes norBC, coding for the nitric oxide reductase, were also detected in the order Thermales. In particular, nine Thermus genomes and two Oceanithermus genomes contain norBC (Figure 3). However, no norBC was detected in the families Deinococcaceae and Trueperaceae (Figure 3). The phylogenetic analysis showed that the NorB of Thermales formed a single clade (Figure 7), which indicates that norBC was present in the common ancestor of the order and was inherited vertically but with multiple gene loss events.

Figure 7.

Figure 7

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Consistency between the phylogenomic tree of Deinococcota and the phylogenetic tree of NorB. (A) The phylogenomic tree of Deinococcota was constructed as in Figure 1A. (B) The NorB protein sequences listed in Table S10 were aligned using MUSCLE with 100 iterations. The NorB tree was constructed using IQ‐Tree with the parameters (‐alrt 1000 ‐bb 1000 ‐nt AUTO). The best‐fit model (LG + R10) was determined by ModelFinder.

No nosZ gene coding for nitrous‐oxide reductase was detected in the Thermus genomes (Figure 3), which is consistent with the incomplete denitrification phenotype commonly reported in Thermus strains 4 , 5 . However, one nosZ gene was detected in the genome of the type strain of Deinococcus ficus CC‐FR2‐10T. The NosZ phylogenetic tree showed that it might have been horizontally transferred from Firmicutes (Figure S3). The nitrous‐oxide reductase is the most sensitive enzyme in denitrification to oxygen 22 , 23 , and the absence of nosZ in Thermus might reflect the niche of Thermus in relatively oxidized habitats within thermal environments. All known Thermus species are aerobic and tolerant of atmospheric concentrations of oxygen, despite the low solubility of oxygen at high temperatures. Furthermore, most geothermal springs are sourced with ammonium as the dominant source of dissolved organic nitrogen 24 , and aerobic chemolithotrophic ammonia oxidation is thought to be the main process delivering oxidized nitrogen as a source for denitrifiers 6 ; the frequent coupling of nitrification and denitrification might select for oxygen tolerance and therefore select against nitrous‐oxide reductase, consistent with high nitrous oxide fluxes measured in some geothermal systems 4 .

In total, the phylogenetic analyses of Thermus denitrification proteins were not always consistent with phylogenomic trees (Figures 1A and 4, 5, 6, 7), suggesting some elements of nonvertical evolution. In addition to the complex evolution of NirK, the denitrification enzymes (NarG, NirS, and NorB) from T. oshimai DSM 12092T consistently branched in the middle of the clade of the Thermus enzymes. This position contrasts with the phylogenomic analyses in which T. oshimai DSM 12092T is deep branching within the genus Thermus (Figure 1A). The reason may be due to a recent HGT event leading to the gain of the denitrification gene cluster in T. oshimai DSM 12092T from a Thermus donor, as suggested by a previous study 25 , or due to the low phylogenetic resolution of single genes at the highest and lowest taxonomic ranks 26 .

The emerging view of the evolution of denitrification genes in Thermales is one in which the last common ancestor of the order gained an incomplete denitrification gene cluster containing narGHI, nirS, and norBC, which was generally vertically inherited, but with multiple gene losses, particularly for nirS and norBC. A completely separate evolution is inferred for NirK. Although the genera Thermus, Calidithermus, and Deinococcus (Deinococcales) contain nirK, phylogenetic analysis of NirK showed that these proteins are not closely related among the genera and were thus gained through independent HGT events. Further back, we infer that the last common ancestor of the Deinococcota did not contain genes for complete denitrification, and the Thermales acquired these genes early in their evolutionary history, allowing them to adapt to low‐oxygen or anoxic conditions where nitrification or other processes provide oxidized nitrogen species to support denitrification.

In summary, we first analyzed the distribution and evolution of denitrification genes in Deinococcota genomes and provided insights into the evolutionary history of the Thermus denitrification pathway. Similar to other studies, our results show that the Thermus genomes contain an incomplete denitrification pathway; more than half of the investigated Thermus genomes contain one or more denitrification genes, and none of them contain the nosZ. The phylogenetic analysis revealed distinct evolutionary histories of narGHI, nirS, and norBC versus nirK, and demonstrated that the last common ancestor of the Thermales acquired these genes early in their evolutionary history, and these genes were largely inherited vertically. Our results support the incomplete denitrification pathway in Thermus and suggest that this pathway allowed it to adapt to the low‐oxygen or anoxic conditions that are common in geothermal environments, which also expand our knowledge of the evolution of the denitrification pathway in Thermus and support the importance of Thermus as a significant heterotrophic denitrifier in thermal environments.

MATERIALS AND METHODS

Genome sequencing, assembly, and annotation

The genomes of T. tenuipuniceus YIM 76954T, T. sediminis L198, T. caliditerrae YIM 77777, T. tengchongensis YIM 77401, Thermus caldifontis YIM 73026T, T. amyloliquefaciens YIM 77409T, T. thermophilus JL‐18, T. caldilimi YIM 78456T, and T. thermamylovorans CFH 72773T were sequenced and assembled as reported earlier 2 , 17 , 27 , 28 , 29 , 30 , 31 , 32 . Other reference genomes of Thermus species were downloaded from the NCBI database (Table S3). The habitat of these strains was present in Table S3, and the completeness and contamination of each genome were calculated using CheckM 33 . Putative protein‐coding sequences (CDSs) of each genome were predicted using Prodigal 34 with the “‐p single” parameter, and the CDSs were annotated against eggNOG, KEGG, and NCBI‐nr databases using DIAMOND 35 by applying E‐values < 1e−5. The predicted CDSs were also uploaded to KEGG Automatic Annotation Server (KAAS) 36 with “bidirectional best hit” and “for prokaryotes” parameters. The tRNAs and rRNAs were identified by tRNAscan‐SE version 2.0.2 37 and RNAmmer version 1.2 38 , respectively. Detection of the CRISPR‐Cas system in the genomes of Thermus was done using the CRISPRCasFinder tool (https://crisprcas.i2bc.paris-saclay.fr) 39 . The ViroBLAST (http://indra.mullins.microbiol.washington.edu/viroblast/viroblast.php) 40 was used to identify the targets of the spacers, while the nonsimilar spacers were analyzed using BLAST against the NT database. The ISsaga software (http://issaga.biotoul.fr/issaga_index.php) 41 was used to determine the IS elements, and the IslandPath‐DIMOB 42 was used to predict GI, to provide the evidence of horizontal transfer regions.

Phylogenetic analysis

A reference genomic dataset of the genus Thermus was established by downloading type strain genomes from NCBI (Table S3). The genomes with estimated completeness >95% and contamination <5% were kept for phylogenetic analysis. The phylogeny of Thermus was generated as mentioned in our previous study 43 , 44 . Briefly, GTDB‐Tk software 12 was used to generate the multiple sequence alignments (MSAs) with 120 bacterial marker genes, and IQ‐Tree 45 was used for calculating a maximum‐likelihood phylogeny for MSAs with parameters (‐alrt 1000 ‐bb 1000 ‐nt AUTO). The best‐fit model (LG + F + R10) determined by ModelFinder 46 was chosen according to Akaike Information Criterion (AIC), Corrected Akaike Information Criterion (Corrected AIC), and Bayesian Information Criterion (BIC).

For phylogeny based on genes of the denitrification pathway, datasets were derived from the NCBI reference genomes. The genomes were annotated as the methods above. The NarG protein sequences with length >1000 aa and <1300 aa were retained for this study. Reference NirK protein datasets with sequences longer than 400 aa were kept for further analysis. Similarly, full‐length NirS (>400 aa), NorB with the length of 450–850 aa, and NosZ with the average length of 662 aa were kept. Finally, representative sequences for each dataset (Tables S7–S11) were aligned using MUSCLE 47 with 100 iterations. Phylogenetic trees were constructed using IQ‐Tree 45 with the parameters mentioned above. All trees were visualized and annotated using iTOL 48 .

Gene content comparison

Pan‐ and core‐genome analysis was performed using the Roary pipeline 49 at 40% minimum sequence identity. The ANI among the genomes of genus Thermus was calculated by using the pyANI 50 with BLAST method. Orthologous proteins were identified based on reciprocal BLAST best hits of predicted amino acid sequences (E‐value < 1e−5), and AAI of each pair of genomes was calculated as the mean identity of all orthologous proteins. All plots were generated with R 51 with the ggplot2 package 52 . A Bayesian tree based on MSAs was constructed with the parameters (ngen = 3000000 Nruns = 2 Nchains = 4 diagnfreq = 1000 relburnin = yes burninfrac = 0.25 samplefreq = 100 printfreq = 100) 43 by using MrBayes software 19 . The evolutionary history of the phylum Deinococcota was inferred by COUNT 20 as described previously 43 , 53 .

AUTHOR CONTRIBUTIONS

Wen‐Jun Li and Brian P. Hedlund jointly conceived the study. Jian‐Yu Jiao conceptualized the research goals under the supervision of Wen‐Jun Li and Brian P. Hedlund, Jian‐Yu Jiao, Zheng‐Han Lian, Meng‐Meng Li, En‐Min Zhou, Lan Liu, and Hong Ming performed the bioinformatics analyses. Jian‐Yu Jiao and Zheng‐Han Lian prepared the figures and tables. Jian‐Yu Jiao, Zheng‐Han Lian, Meng‐Meng Li, Nimaichand Salam, Brian P. Hedlund, and Wen‐Jun Li wrote the manuscript. All authors revised and approved the final manuscript.

ETHICS STATEMENT

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

CONFLICT OF INTERESTS

The authors declare that they have no conflict of interests.

Supporting information

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ACKNOWLEDGMENTS

We thank Xue‐Qin Jia for collecting the data about the isolate information of Deinococcota strains. We are grateful to the staff from the Yunnan Tengchong Volcano and Spa Tourist Attraction Development Corporation for their assistance. This study was supported by funding from the National Natural Science Foundation of China (Nos. 91951205, 92051108, 31850410475, and 31970122), the National Science and Technology Fundamental Resources Investigation Program of China (2021FY100900), and the U.S. National Science Foundation (DEB 1557042 and DEB 1841658).

Jiao J‐Y, Lian Z‐H, Li M‐M, Salam N, Zhou E‐M, Liu L, et al. Comparative genomic analysis of Thermus provides insights into the evolutionary history of an incomplete denitrification pathway. mLife. 2022;1:198–209. 10.1002/mlf2.12009

Edited by Meng Li, Shenzhen University, China

Contributor Information

Brian P. Hedlund, Email: brian.hedlund@unlv.edu.

Wen‐Jun Li, Email: liwenjun3@mail.sysu.edu.cn.

DATA AVAILABILITY

All the genomes described in this article have been deposited in NCBI, and the accession numbers were provided in Table S3. The Thermus information can also be found in eLMSG (an eLibrary of Microbial Systematics and Genomics, https://www.biosino.org/elmsg/index) under accession numbers MSG069325, MSG066408, MSG019978, MSG066894, MSG067219, MSG066285, MSG068958, MSG065983, MSG068209, MSG065453, MSG067283, MSG007907. The R codes have been deposited in Github (https://github.com/lianzhh-pub/Rcode-mLife2021).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting information.

MLF2-1-198-s007.pdf (480.5KB, pdf)

Supporting information.

MLF2-1-198-s003.pdf (473.4KB, pdf)

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MLF2-1-198-s009.pdf (631.3KB, pdf)

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MLF2-1-198-s006.xlsx (16.1KB, xlsx)

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MLF2-1-198-s014.xlsx (15.5KB, xlsx)

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MLF2-1-198-s001.xlsx (31.1KB, xlsx)

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MLF2-1-198-s005.xlsx (105KB, xlsx)

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MLF2-1-198-s012.xlsx (14.2KB, xlsx)

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MLF2-1-198-s010.xlsx (12.7KB, xlsx)

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MLF2-1-198-s004.xlsx (89.9KB, xlsx)

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MLF2-1-198-s011.xlsx (83.4KB, xlsx)

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MLF2-1-198-s013.xlsx (225.8KB, xlsx)

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MLF2-1-198-s015.xlsx (328KB, xlsx)

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MLF2-1-198-s008.docx (14.1KB, docx)

Data Availability Statement

All the genomes described in this article have been deposited in NCBI, and the accession numbers were provided in Table S3. The Thermus information can also be found in eLMSG (an eLibrary of Microbial Systematics and Genomics, https://www.biosino.org/elmsg/index) under accession numbers MSG069325, MSG066408, MSG019978, MSG066894, MSG067219, MSG066285, MSG068958, MSG065983, MSG068209, MSG065453, MSG067283, MSG007907. The R codes have been deposited in Github (https://github.com/lianzhh-pub/Rcode-mLife2021).

Articles from mLife are provided here courtesy of Wiley

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