A Metagenome from a Steam Vent in Los Azufres Geothermal Field Shows an Abundance of Thermoplasmatales archaea and Bacteria from the Phyla Actinomycetota and Pseudomonadota

Los Azufres National Park is a geothermal field that has a wide number of thermal manifestations; nevertheless, the microbial communities in many of these environments remain unknown. In this study, a metagenome from a sediment sample from Los Azufres National Park was sequenced. In this metagenome, we found that the microbial diversity corresponds to bacteria (Actinomycetota, Pseudomonadota), archaea (Thermoplasmatales and Candidatus Micrarchaeota and Candidatus Parvarchaeota), eukarya (Cyanidiaceae), and viruses (Fussellovirus and Caudoviricetes). The functional annotation showed genes related to the carbon fixation pathway, sulfur metabolism, genes involved in heat and cold shock, and heavy-metal resistance. From the sediment, it was possible to recover two metagenome-assembled genomes from Ferrimicrobium and Cuniculiplasma. Our results showed that there are a large number of microorganisms in Los Azufres that deserve to be studied.


Introduction
One characteristic of many geothermal fields is the presence of steam vents, i.e., fumaroles, that consist of permanent emissions of steam and gases from the subsoil due to magmatic activity or groundwater geothermal heating [1]. Consequently, the steam has temperatures above 70 • C, wet conditions, and a concentration of minerals that form deposits as a result of the transport and evaporation of water in the walls of the caves or vents [2]. As a consequence, the walls of the vents have an important amount of minerals that provide conditions for the support of microbial life. Even though limitations of culturing thermophilic and acidophilic microorganisms exist, the use of independent culture approaches such as metagenomics helps us study in greater detail the diversity of microorganisms that inhabit the walls of the vents [3,4].
Mexico has multiple geothermal fields that have been recently explored in the Trans-Mexican Volcanic Belt, an orographic formation with high volcanic activity due to the activity of the Cocos and Rivera plates [5]. Los Azufres geothermal field is located in the state of Michoacán, Mexico, in the Trans-Mexican Volcanic Belt region, and it hosts a large number of thermal manifestations;for instance, hot springs, fumaroles, and steam vents that can reach temperatures above 90 • C and pH values below 4 [6]. The high temperatures of

Phylogenomic Analysis of Thermoplasmatales archaea and Ferrimicrobium sp. AZ2-2013
Two phylogenomic trees were constructed using MAGs that have contamination less than 6.0 and completeness greater than 80%. Open reading frames (ORFs) from each bin were searched using Prokka v.1.14.6 [28]. Model organisms and type strains of Thermoplasmatales and Actinobacteria were selected from the NCBI database. The construction of the phylogenomic tree was carried out by PhyloPhlan v.0.99 [39].

Metagenome Assembly and Annotation
The metagenome has 46,728,050 reads with a length between 35 and 301 bp and an average quality per read of 37 (Phred Quality Score). After the quality trimming 43,034,606 with a length between 30 and 266 and an average quality per read of 37 (Phred Quality Score) remained. The metagenome has 97,177 contigs (N50, 6617) containing 144,161,343 bp and a GC content of 55.52%. The metagenome annotation found 212,400 protein coding genes. These protein coding genes were classified using Clusters of Orthologous Groups (COGs) ( Table 1). Only 37.58% of the total reads (8,087,196) were classified by Kaiju, and 62.42% of the total reads remained unclassified. According to the analysis of microbial diversity (Figure 2), the most prevalent phylum was Ca. Thermoplasmatota, which is followed by the bacterial phyla Actinomycetota, Pseudomonadota, and Cyanobacteriota.

Metagenome Genes Involved in Metabolic Pathway and Resistances
Genes involved in the carbon fixation pathways in prokaryotes were found. These genes correspond to the pathways of the reductive citric acid cycle, reductive acetylcoenzyme A, dicarboxylate/hydroxybutyrate cycle, 3-hydroxypropionate bicycle, and hydroxypropionate/hydroxybutyrate cycle. However, some genes involved in the 3hydroxypropionate bicycle and hydroxypropionate/hydroxybutyrate cycle were not found. The enzymes sulfur oxygenase/reductase (SOR) and sulfide:quinone oxidoreductase (SQR) were found. All the genes involved in dissimilatory/assimilatory nitrate reduction and assimilatory sulfate reduction were found. In addition, genes related

Metagenome Viral Sequences
Two viral genomes were assembled. The first viral genome (Vaz-01) belonged to

Binning, Taxonomic Classification, and MAGs Annotation
Thirty-two bins were obtained from the sediment metagenome, of which fifteen belonged to Archaea, thirteen belonged to Bacteria, and four belonged to Eukarya. Thirteen archaea were identified from the order Thermoplasmatales ("alphabet plasmas" Cuniculiplasma and Ferroplasma) and two archaea from the phylum Candidatus Micrarchaeota (Microcaldota) and Nanoarchaeota (Parvarchaeota order). Ten bacteria were identified related to Actinomycetota, one from the order Acidimricrobiales (Ferrimicrobium genus), another from the order Corynebacteriales (Mycobacterium genus), and eight unidentified. Furthermore, we identified three bacteria from the phylum Pseudomonadota, one from the order Acidithiobacillales (Acidithiobacillus caldus) and two unidentified. Finally, we recovered four bins of Eukarya belonging to the family Cyanidiaceae. The statistical information for each bin is shown in Table 2. The MAGs functional diversity analysis (Figure 3) showed that the COGs categories with a great number of genes were related to translation, ribosomal structure, and biogenesis. The analysis also revealed that the MAGs related to bacteria had a great number of genes involved in energy production and conversion, carbohydrate metabolism, amino acid metabolism, and lipid metabolism, whereas the MAGs related to archaea only had a great number of genes related to amino acid metabolism.

Phylogenomic Analysis of Thermoplasmatales archaea and Ferrimicrobium sp. AZ2-2013
The phylogenomic analysis of Thermoplasmatales archaea found in Los Azufres showed a wide diversity of these organisms (Figure 4). According to the phylogenomic analysis of Actinomycetota ( Figure 5), the close relationship between BinB008 and Ferrimicrobium acidiphilum DSM19497, BinB003 and Mycobacterium marinum MMA1, and BinB006 and Miltoncostaea marina SCSIO 60955 was revealed. We decided to study BinB008 more deeply because it is one of the highest quality (completeness of 96.58% and a contamination of 1.38%). We propose here the next name Ferrimicrobium sp. AZ2-2013′ to account for it. The enzyme sulfate sulfur oxygenase/reductase was present only in BinA002 and BinA010. All the genes involved in the assimilatory sulfate reduction pathway were present in BinB003 and BinB011. Other high-quality bacterial MAGs (BinB006, BinB009, and BinB012) did not contain the gene sulfite reductase (SiR), which is essential for the assimilatory sulfate reduction pathway. The entire metabolic pathway for the assimilatory nitrate reduction pathway was not present in any high-quality MAGs. The ferredoxinnitrite reductase gene (NirA) was the only gene present.
Genes for heavy metal resistance were found. High-quality MAGs have the genes arsenate reductase (arsC) and arsenite transporters (arsA and arsB), but BinA010 and BinB004 lacked the genes for arsenic resistance. Archaeal MAGs and BinB004 lacked genes involved in mercuric resistance. Others had one of the genes for mercuric reductase (merA) and mercuric ion transport protein (merC). BinB004, BinB009, BinB011, and BinB012 had genes related to the cobalt-zinc-cadmium efflux system protein (czcD). Genes related to resistance to nickel and chromium were not present in any high-quality MAGs.
There were numerous genes for heat shock proteins (Hsp20, Hsp33, and Hsp90) in highquality MAGs BinB002, BinB003, BinB004, BinB011, and BinB012. Finally, only BinA010 did not have the machinery for repairing heat-induced protein damage that involves the chaperones DnaJ, DnaK, and the protein GrpE.

Phylogenomic Analysis of Thermoplasmatales archaea and Ferrimicrobium sp. AZ2-2013
The phylogenomic analysis of Thermoplasmatales archaea found in Los Azufres showed a wide diversity of these organisms (Figure 4). According to the phylogenomic analysis of Actinomycetota ( Figure 5), the close relationship between BinB008 and Ferrimicrobium acidiphilum DSM19497, BinB003 and Mycobacterium marinum MMA1, and BinB006 and Miltoncostaea marina SCSIO 60955 was revealed. We decided to study BinB008 more deeply because it is one of the highest quality (completeness of 96.58% and a contamination of 1.38%). We propose here the next name 'Ferrimicrobium sp. AZ2-2013 to account for it.  . Phylogenomic tree of Thermoplasmatales archaea. The phylogenomic tree shows the predicted evolutionary relationships of genomes from the order Thermoplasmatales against genomes of Thermoplasmatales in NCBI database. MAGs recovered in this study are shown in bold letters. Aciduliprofundum boonei T469 was selected as an outgroup. Phylogenomic tree was generated using maximum likelihood model, and numbers at the branch points represent SH-like local support values. The scale bar represents the estimated number of amino acid changes per site.

MAG Annotation and Comparative Genomic Analysis
The MAG related to Ferrimicrobium sp. (Table 2)

60% (DDH) against
Ferrimicrobium acidiphilum DSM19497 strain T23 (GCF_000949255.1); the size of this genome was smaller compared to F. acidiphilum (3.08 Mb) [37]. This was the first genome of the genus Ferrimicrobium reported in Los Azufres. The annotation of this genome revealed many similarities between this genome and Ferrimicrobium acidiphilum DSM19497. There are genes related to the Calvin-Benson-Bassham cycle (RuBisCo), carboxysome, and tricarboxylic acid cycle and genes involved in iron and sulfur oxidation. Also, there are genes related to stress responses (heat shock and acid stress) and heavy metal resistance, especially arsenic and mercury.

Figure 5.
Phylogenomic tree of Actinobacteria. The phylogenomic tree shows the predicted evolutionary relationships of genomes from the phylum Actinomycetota against genomes of Actinomycetota in NCBI database. MAGs recovered in this study are shown in bold letters. Sulfobacillus thermosulfidooxidans strain ZJ was selected as an outgroup. Phylogenomic tree was generated using maximum likelihood model, and numbers at the branch points represent SH-like local support values. The scale bar represents the estimated number of amino acid changes per site.

MAG Annotation and Comparative Genomic Analysis
The MAG related to Ferrimicrobium sp. (Table 2) had 60 contigs containing 2,544,086 bp (N50 value of 60560), and it had a coverage of 6.49X and G + C content of 57.97. This MAG was called Ferrimicrobium sp. AZ2-2013. This MAG had values of 77.07% (ANI), 76.12% (AAI), and 19.10% (DDH) against the genome of Ferrimicrobium acidiphilum DSM19497 (GCF_000745905.1) and values of 77.16% (ANI), 76.06% (AAI), and 19.60% (DDH) against Ferrimicrobium acidiphilum DSM19497 strain T23 (GCF_000949255.1); the size of this genome was smaller compared to F. acidiphilum (3.08 Mb) [37]. This was the first genome of the genus Ferrimicrobium reported in Los Azufres. The annotation of this genome revealed many similarities between this genome and Ferrimicrobium acidiphilum DSM19497. There are genes related to the Calvin-Benson-Bassham cycle (RuBisCo), carboxysome, and tricarboxylic acid cycle and genes involved in iron and sulfur oxidation. Also, there are genes related to stress responses (heat shock and acid stress) and heavy metal resistance, especially arsenic and mercury. There are genes involved in the citric acid cycle, but the enzymes 2-oxoglutarate dehydrogenase and fumarase were absent. In the synthesis of amino acids, Cuniculiplasma sp. AZ1-2013 lacked the genes for the synthesis of histidine, leucine, isoleucine, proline, and valine. Furthermore, there is a gene related to the heat shock protein HtpX. Notably, there are no genes related to metal resistance.

Discussion
The microbial diversity found in sediments from Los Azufres corresponds principally to phyla Ca. Thermoplasmatota, Actinomycetota, Pseudomonadota, and Cyanobacteriota. The most prevalent microorganisms in the sediment sample from the Los Azufres geothermal area are Thermoplasmatales archaea. Only 0.060% (12,993) (Supplementary Table S1) of the total reads from this metagenome belonged to Sulfolobales. This low abundance may be due to the type of sample. Sulfolobales archea were found in water samples from an acid solfatara [15] and in a yellow sediment sample (sulfur crystal) [18]. Thirteen MAGs associated with Thermoplasmatales were discovered by us, and phylogenomic research reveals that these archaea are very diverse. Thermoplasmatales archaea are present in acid mine drainage sites all over the world, including Parys Mountain, United Kingdom [42,43], Los Rueldos, Spain [44], and Richmond mine at Iron Mountain, United States [45,46]. Additionally, Thermoplasmatales were discovered in water samples from the Kamchatka Peninsula and Kunashir Island in Russia [47] as well as the Tenorio Volcano National Park in Costa Rica [48]. However, the temperature in Parys Mountain, UK, is between 8 and 18 • C. Thermoplasmatales were discovered to be the most prevalent microorganisms (62%) in sediment samples from Parys Mountain, UK, according to Korzhenkov et al., 2019 [42], despite the low temperatures. To further understand why Thermoplasmatales archaea are present in low temperatures, a comparative genomic analysis between the genomes discovered in Parys Mountain, UK, and genomes discovered in thermal environments is essential.
It is important to note that bacteria from the phyla Actinomycetota and Pseudomonadota were also discovered in the Parys Mountain in the UK [42,43] and Tenorio Volcano National Park in Costa Rica [48]. Additionally, algae from the Chlorophyceae family were present in Parys Mountain [43], whereas acidophilic algae from the Cyanidiaceae family were found in the Los Azufres geothermal area.
Finally, we obtained viral sequences that are relevant to understanding the interactions between microbial communities. Viruses from the class Caudoviricetes have the capacity to infect both archaea and bacteria [49]. The same study mentioned that members of this class can infect cells from the order Thermoplamatales. However, many of the viruses in this taxonomic group remain unknown and relate to marine and anoxic environments. In the case of Fusellovirus, previous studies in Los Azufres reported the presence of viral sequences related to this group that infect archaea, and it is believed that this group has a key role in the gene exchange between archaea populations. As well, the presence of these viruses is common in geothermal environments [50].
According to the functional analysis, we detected that the microbial community in Los Azufres has the ability to obtain energy and resources from several sources, and bacteria have the possibility to use carbon through carbon fixation. In addition, the microbial community is capable of assimilating nitrates and sulfates. Similar adaptations have been detected in other steam vents in Mexico [3] and hot springs located in Costa Rica [48] and Malaysia [51]. Our results showed that bacteria are possible candidates for sulfate assimilation processes. Our analysis offered a vision of the versatility of the microbial community to obtain energy from several sources. The presence of genes related to heat and cold shock shows the flexibility of the microbial community to face changes in temperature. Consequently, the microbial community has been exposed to abrupt temperature changes. Furthermore, the weather in Los Azufres varies each season, reaching low temperatures. For this reason, the microbial community has developed not only strategies to live in high temperatures but also adaptations to survive in low temperatures during the winter.
The presence of metal resistance genes is related to the presence of those metals in the environment that the microbial community inhabits; gene prediction in steam vents located in Paricutin volcano showed the presence of similar adaptations to metal resistance [3]. The heavy metals in steam vents and hot springs in Los Azufres may be due to the constant geological activity therein. For this reason, the microbial community has developed adaptations to survive and manage the metal high concentration.
In this study, we obtained the first genome related to the genus Ferrimicrobium from Mexico. The bacterial genus Ferrimicrobium was reported for the first time in the United Kingdom in a sample obtained from an abandoned sulfur mine [52,53]. There are reports of this genus in an acid river in Argentina [54], and acid mine drainage in Finland [55], Turkey [56], and Russia [57]. According to Johnson et al. [52] and Li et al. [58], Ferrimicrobium is able to oxidize ferrous iron and is typical of acidic environments, and it probably plays a role in the sulfur and iron biogeochemical cycle in Los Azufres due to its capacity to oxidize iron and sulfur. Furthermore, Ferrimicrobium sp. AZ2-2013 has genes related to metal resistance for arsenic and mercuric; heavy metal resistance was reported previously by Johnson et al. [52], but not for these metals.
In addition, one genome of archaea related to Cuniculiplasma divulgatum was obtained here. Cuniculiplasma divulgatum was found for the first time in Spain and the United Kingdom [59]. Golyshina et al. [60] found that Cuniculiplasma has interactions with Ca. Micrarchaeota. Also, Golyshina et al. [61] found the presence of acidophilic algae and proposed that these algae are primary producers in these environments and Cuniculiplasma, being heterotrophic, could take advantage of the organic compounds produced by algae. In this metagenome, we found the presence of an acidophilic algae belonging to the genera Cyanidiaceae and also an archaeon related to Ca. Micrarchaeota, and it is possible that this archaeon had interactions with Cuniculiplasma sp. AZ1-2013. Cuniculiplasma sp. AZ1-2013 had high values of ANI, AAI, and DDH against Cuniculiplasma divulgatum PM4, indicating that Cuniculiplasma sp. AZ1-2013 belongs to the species Cuniculiplasma divulgatum. Cuniculiplasma sp. AZ1-2013 lacks the same genes related to the citric acid cycle and synthesis of amino acids as Cuniculiplasma divulgatum PM4.
Our study expanded the knowledge of the microbial community in Los Azufres geothermal field and showed a wide variety of archaea and bacteria that were successfully assembled in MAGs. We consider it necessary to study viruses found in Los Azufres to understand their role in the ecosystem.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cimb45070370/s1, Table S1: Microbial diversity of sediment sample at the taxonomic level of order.