History of the Study of the Genus Thiothrix: From the First Enrichment Cultures to Pangenomic Analysis

Representatives of the genus Thiothrix are filamentous, sulfur-oxidizing bacteria found in flowing waters with counter-oriented sulfide and oxygen gradients. They were first described at the end of the 19th century, but the first pure cultures of this species only became available 100 years later. An increase in the number of described Thiothrix species at the beginning of the 21st century shows that the classical phylogenetic marker, 16S rRNA gene, is not informative for species differentiation, which is possible based on genome analysis. Pangenome analysis of the genus Thiothrix showed that the core genome includes genes for dissimilatory sulfur metabolism and central metabolic pathways, namely the Krebs cycle, Embden–Meyerhof–Parnas pathway, glyoxylate cycle, Calvin–Benson–Bassham cycle, and genes for phosphorus metabolism and amination. The shell part of the pangenome includes genes for dissimilatory nitrogen metabolism and nitrogen fixation, for respiration with thiosulfate. The dispensable genome comprises genes predicted to encode mainly hypothetical proteins, transporters, transcription regulators, methyltransferases, transposases, and toxin–antitoxin systems.


Introduction
The first attempts to describe bacteria from the genus Thiothrix were made by Rabenhorst as early as 1865, when he described the first member of this genus as Beggiatoa nivea [1]. Winogradsky, in turn, based on studies of key features of enrichment culture, named a new genus, Thiothrix [2]. The genus Thiothrix belongs to the class Gammaproteobacteria, order Thiotrichales, family Thiotrichaceae.
Habitats of Thiothrix vary from natural sulfidic waters, irrigation systems, and activated sludge in wastewater treatment plants to ectosymbionts of invertebrates in deep-sea hydrotherms. The influx of H 2 S into the growth zone of these bacteria occurs from sulfidic springs, either from the near-bottom layers of sediments (in the shallow waters of lakes, in ponds, sea littorals, etc.) or from hydrothermal vents [3,4]. The hydrogen sulfide concentration can vary significantly-from tens of micrograms to several milligrams per litre. In nature, Thiothrix forms powerful foulings, visible to the naked eye.
Currently, the genus Thiothrix includes aerobic and facultative anaerobic, attached, filamentous, non-motile bacteria. They are capable of auto-and heterotrophic growth and are characterized by a respiratory type of metabolism. During autotrophic growth, CO 2 fixation occurs through the Calvin-Benson-Bassham cycle. Their ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) belongs to types IAq, IAc, and II. All genomes contain genes encoding all enzymes of the Krebs cycle, with the exception of malate dehydrogenase 2 of 11 (MDH), which is functionally replaced by malate:quinone oxidoreductase (MQO). Thiothrix spp. are capable of organotrophic growth, as well as lithotrophic growth in the presence of reduced sulfur compounds. During lithotrophic growth in the presence of hydrogen sulfide and thiosulfate, elemental sulfur is accumulated intracellularly. Hydrogen sulfide is oxidized to sulfur by the sulfide:quinone oxidoreductase (SQR) and flavocytochrome c-sulfide dehydrogenase (FCSD). Thiosulfate is oxidized by the branched sulfur-oxidizing system (SOX) pathway without SoxCD with the formation of sulfur and sulfate. Sulfite is oxidized via direct (membrane-bound cytoplasmic sulfite:quinone oxidoreductase (Soe-ABC) and indirect (adenosine phosphosulfate reductase (AprAB) and ATP sulfurylase (Sat)) oxidation pathways (Figure 1). fixation occurs through the Calvin-Benson-Bassham cycle. Their ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) belongs to types IAq, IAc, and II. All genomes contain genes encoding all enzymes of the Krebs cycle, with the exception of malate dehydrogenase (MDH), which is functionally replaced by malate:quinone oxidoreductase (MQO). Thiothrix spp. are capable of organotrophic growth, as well as lithotrophic growth in the presence of reduced sulfur compounds. During lithotrophic growth in the presence of hydrogen sulfide and thiosulfate, elemental sulfur is accumulated intracellularly. Hydrogen sulfide is oxidized to sulfur by the sulfide:quinone oxidoreductase (SQR) and flavocytochrome c-sulfide dehydrogenase (FCSD). Thiosulfate is oxidized by the branched sulfur-oxidizing system (SOX) pathway without SoxCD with the formation of sulfur and sulfate. Sulfite is oxidized via direct (membrane-bound cytoplasmic sulfite:quinone oxidoreductase (SoeABC) and indirect (adenosine phosphosulfate reductase (AprAB) and ATP sulfurylase (Sat)) oxidation pathways ( Figure 1). Before 1965, eleven morphotypes of the genus Thiothrix were described mainly in natural marine and freshwater habitats containing hydrogen sulfide [5][6][7]. These microorganisms are differentiated based solely on the diameter of the filaments and the characteristics of the habitat. Subsequently, obtaining pure cultures made it possible to reveal that the morphology of the genus Thiothrix is variable [8]. The final invalidity of using phenotypic characters for the taxonomy of the genus Thiothrix was confirmed by Howarth et al., 1999 [9]. In 1983, Larkin and Shinabarger isolated the first pure culture for a representative of this genus [5]. Based on Winogradsky's description, Shinabarger suggested that the culture he received was Thiothrix nivea. The strain JP2 with a validly published Figure 1. Scheme of dissimilation sulfur metabolism of the genus Thiothrix. FccAB, flavocytochrome csulfide dehydrogenase; SqrA/F, sulfide:quinone oxidoreductase; SoxAXBYZ, SOX multienzyme system; MQ, menaquinone; rDsrABCEFHEMPKJOL, dissimilatory sulfite reductase; AprABM, APS reductase; Sat, ATP sulfurylase; SoeABC, membrane-bound cytoplasmic sulfite:quinone oxidoreductase; S 0 , sulfur globule; APS, adenosine 5 -phosphosulfate; R−S n+1 H, thiol compound; H−S n+1 , polysulfide.
Before 1965, eleven morphotypes of the genus Thiothrix were described mainly in natural marine and freshwater habitats containing hydrogen sulfide [5][6][7]. These microorganisms are differentiated based solely on the diameter of the filaments and the characteristics of the habitat. Subsequently, obtaining pure cultures made it possible to reveal that the morphology of the genus Thiothrix is variable [8]. The final invalidity of using phenotypic characters for the taxonomy of the genus Thiothrix was confirmed by Howarth et al., 1999 [9]. In 1983, Larkin and Shinabarger isolated the first pure culture for a representative of this genus [5]. Based on Winogradsky's description, Shinabarger suggested that the culture he received was Thiothrix nivea. The strain JP2 with a validly published name Thiothrix nivea JP2 T (=ATCC 35100 T = DSM 5205 T ) is the only established neotype of the species [5].
The genus Thiothrix was significantly expanded by Howarth in 1999 [9]. Four new species were included in the genus: Thiothrix fructosivorans, Thiothrix unzii, Thiothrix defluvii, and Thiothrix eikelboomii. The last two species were assigned to the Eikelboom type 021N group within the genus Thiothrix, and the species T. nivea, T. fructosivorans, and T. unzii were assigned to the T. nivea group [9]. Comparative analysis of the 16S rRNA gene sequence of members of the Eikelboom type 021N and the T. nivea groups showed low similarity (90-91%). However, the notable phenotypic similarity between the Eikelboom type 021N group and the Thiothrix nivea group did not allow division into new genera at that time.
The increase in the number of species of the genus Thiothrix has set the task of searching for new phylogenetic markers. Pure cultures of the genus Thiothrix isolated from various biotopes (hydrogen sulfide springs, wastewater treatment plants, the White Sea littoral, activated sludge treatment systems, freshwater lakes, groundwater, invertebrate ectosymbionts, etc.) have a similar morphotype, but a rather variable metabolism. The phylogeny based on the 16S rRNA gene sequences does not always correspond to the phylogenetic diversity of the representatives of this group [8]. Some strains assigned to the same species based on the 16S rRNA gene were reclassified as separate species after determination of whole-genome sequences (T. lacustris BL T , 'Thiothrix litoralis' AS T , and 'Thiothrix winogradskyi' CT3 T ) ( Figure 2). the species [5].
The genus Thiothrix was significantly expanded by Howarth in 1999 [9]. Four new species were included in the genus: Thiothrix fructosivorans, Thiothrix unzii, Thiothrix defluvii, and Thiothrix eikelboomii. The last two species were assigned to the Eikelboom type 021N group within the genus Thiothrix, and the species T. nivea, T. fructosivorans, and T. unzii were assigned to the T. nivea group [9]. Comparative analysis of the 16S rRNA gene sequence of members of the Eikelboom type 021N and the T. nivea groups showed low similarity (90-91%). However, the notable phenotypic similarity between the Eikelboom type 021N group and the Thiothrix nivea group did not allow division into new genera at that time.
The increase in the number of species of the genus Thiothrix has set the task of searching for new phylogenetic markers. Pure cultures of the genus Thiothrix isolated from various biotopes (hydrogen sulfide springs, wastewater treatment plants, the White Sea littoral, activated sludge treatment systems, freshwater lakes, groundwater, invertebrate ectosymbionts, etc.) have a similar morphotype, but a rather variable metabolism. The phylogeny based on the 16S rRNA gene sequences does not always correspond to the phylogenetic diversity of the representatives of this group [8]. Some strains assigned to the same species based on the 16S rRNA gene were reclassified as separate species after determination of whole-genome sequences (T. lacustris BL T , 'Thiothrix litoralis' AS T , and 'Thiothrix winogradskyi' CT3 T ) ( Figure 2).  However, the 16S rRNA gene can be successfully used to identify Thiothrix at the genus level since the levels of 16S rRNA gene sequence identity between representatives of the genus Thiothrix exceed 94%, while with members of other genera, this value is below 91%.
Thiothrix defluvii and Thiothrix flexilis were reclassified as representatives of the new genus Thiofilum within the family Thiofilaceae with the proposed names Thiofilum flexile and Thiofilum defluvii. Thiothrix eikelboomii and Thiothrix disciformis were placed in the new genus Thiolinea within the new family Thiolineaceae with the proposed names 'Thiolinea eikelboomii' for Thiothrix eikelboomii. However, the reclassification of Thiolinea eikelboomii is currently only formal due to the lack of cultures in two international collections, as required for species validation. T. caldifontis, T. lacustris, T. nivea, T. unzii, and T. fructosivorans remained in the genus Thiothrix [15].
The development of genomics and metagenomics methods made it possible to obtain complete genome sequences and use them for phylogenetic studies, which, in turn, contributed to the development of a new genome-based taxonomic system of prokaryotes [16]. The wholegenome comparison has higher accuracy and resolution than taxonomy based on individual phylogenetic markers. Whole-genome sequences of isolates T. fructosivorans Q T , T. unzii A1 T , Thiothrix litoralis AS T , 'Thiothrix subterranea' Ku-5 T [8], and 'Thiothrix winogradskyi' CT3 T [17], as well as metagenome-assembled genomes (MAGs) of 'Candidatus Thiothrix anitrata' A52, Candidatus Thiothrix moscovensis RT [18,19], and Candidatus Thiothrix singaporensis SSD2 [19,20], were obtained during the last three years. Just recently, 'Candidatus Thiothrix sulfatifontis' KT was obtained from the fouling of a hydrogen sulfide source [17].
The main characteristics of the obtained genomes are shown in Table 1. Several MAGs were obtained from the Svalliden-Norrby groundwater metagenome, Oskarshamn, Sweden. When analyzing the obtained MAGs, we found that the genome assemblies GCA_018813855.1, GCA_018822845.1, and GCA_018825285.1 represented mem-bers of the genus Thiothrix. ANI values between the three genomes were 99.8-100%, which indicates that all assemblies represented the same species. For analysis of the pangenome assembly, GCA_018813855.1 (Modern_marine.mb.207), designated as MAG of Thiothrix sp. 207, was chosen. ANI and dDDH values between MAG of Thiothrix sp. 207 and other representatives of the genus (76-79% and 20-26%, respectively) indicated that this genome represented a novel Candidatus species (Figure 2).

Pangenome Analysis
Based on the genome sequences of eight isolates and five MAGs, an updated phylogeny of the genus Thiothrix was proposed (Figure 3).

A52
GCF_017901155. Several MAGs were obtained from the Svalliden-Norrby groundwater metagenome, Oskarshamn, Sweden. When analyzing the obtained MAGs, we found that the genome assemblies GCA_018813855.1, GCA_018822845.1, and GCA_018825285.1 represented members of the genus Thiothrix. ANI values between the three genomes were 99.8-100%, which indicates that all assemblies represented the same species. For analysis of the pangenome assembly, GCA_018813855.1 (Modern_marine.mb.207), designated as MAG of Thiothrix sp. 207, was chosen. ANI and dDDH values between MAG of Thiothrix sp. 207 and other representatives of the genus (76-79% and 20-26%, respectively) indicated that this genome represented a novel Candidatus species (Figure 2).

Pangenome Analysis
Based on the genome sequences of eight isolates and five MAGs, an updated phylogeny of the genus Thiothrix was proposed (Figure 3).  The genus Thiothrix accommodates lithotrophic sulfur-oxidizing bacteria possessing various systems for the oxidation of reduced sulfur compounds. Bacteria from the genus Thiothrix are able to use a number of reduced inorganic sulfur compounds as an energy source, for example, hydrogen sulfide, sulfur, sulfite, and thiosulfate. Pangenome analysis showed the presence of two types of SQR (SqrF and SqrA) in the core genome. The FCSD complex is also present in all Thiothrix genomes but due to sequence divergence appeared to be among the auxiliary genes. Genes of the Sox complex involved in thiosulfate oxidation (soxAXBYZ) and of the rDsr system of elemental sulfur oxidation to sulfite (dsrABCEFHEMPKJOL) are present in the core genome. The genes encoding enzyme for direct (SoeABC) and indirect (Sat and AprAB) sulfite oxidation are also present in all species. The genus Thiothrix accommodates lithotrophic sulfur-oxidizing bacteria possessing various systems for the oxidation of reduced sulfur compounds. Bacteria from the genus Thiothrix are able to use a number of reduced inorganic sulfur compounds as an energy source, for example, hydrogen sulfide, sulfur, sulfite, and thiosulfate. Pangenome analysis showed the presence of two types of SQR (SqrF and SqrA) in the core genome. The FCSD complex is also present in all Thiothrix genomes but due to sequence divergence appeared to be among the auxiliary genes. Genes of the Sox complex involved in thiosulfate oxidation (soxAXBYZ) and of the rDsr system of elemental sulfur oxidation to sulfite (dsrABCEFHEMP-KJOL) are present in the core genome. The genes encoding enzyme for direct (SoeABC) and indirect (Sat and AprAB) sulfite oxidation are also present in all species.
The core genome contains genes hyaABC encoding type I membrane-bound respiratory H 2uptake [NiFe]-hydrogenase, as well as hypABCDEF genes involved in hydrogenase maturation, which indicates the potential for lithotrophic growth in the presence of molecular hydrogen.
All members of the genus Thiothrix can assimilate carbon dioxide through the Calvin-Benson-Bassham cycle. The genes responsible for the regeneration and reduction of the substrate in the Calvin-Benson-Bassham cycle were found in the core genome. RuBisCO in the genus Thiothrix is represented by three types: IAq, IAc, and II. Previous pangenome analysis has shown that RuBisCO of the IAq type is common to all species, in contrast to IAc and II types [8]. However, the addition of 'Thiothrix winogradskyi' CT3 T into the pangenome analysis excluded type IAq RuBisCO from the core genome, since its genome does not contain this type of RuBisCO. All species, except for 'Ca. Thiothrix anitrata' A52, encode RuBisCO of type II, and all species except T. fructosivorans Q T , T. lacustris BL T , and MAG of Thiothrix sp. 207 encode type IAc ( Figure 5).
As for phosphoribulokinase in the genus Thiothrix, the prk gene is represented by two phylogenetically different copies. The first copy has a high level of sequence similarity among all representatives of Thiothrix and was found in the core genome. The second copy of prk is also present in all Thiothrix; however, the amino acid sequence identity with the first type of phosphoribulokinase is below the 70% threshold used to determine the genes of the core genome. The genes for the tricarboxylic acid cycle, Embden-Meyerhof-Parnas pathway, the glyoxylate cycle, and phosphorus metabolism are mainly included in the core genome except for phosphate transport system permease (pstB), glucokinase (glk), dihydrolipoamide dehydrogenase of pyruvate dehydrogenase complex (pdhD), aconitate hydratase (acnB), and 6-phosphogluconolactonase (pgl), the level of similarity of which is below 70%. The genes of the first, second, and third complexes of the electron transport chain, as well as ATPase (complex V), are present in the core genome. The genes of the fourth respiratory chain complex were found in the core genome (cox15, ccoO, ccoP, ccoG, ccoQ, ccoS). Genes cyoE, cox11, coxBACD, cydABCDX, and ccoN are also present in all species but fell into the auxiliary genome due to higher sequence divergence. The cytochrome ubiquinol oxidase is present in all Thiothrix species except for 'Thiothrix winogradskyi' CT3 T .
The core genome contains genes hyaABC encoding type I membrane-bound respiratory H2-uptake [NiFe]-hydrogenase, as well as hypABCDEF genes involved in hydrogenase maturation, which indicates the potential for lithotrophic growth in the presence of molecular hydrogen.
All members of the genus Thiothrix can assimilate carbon dioxide through the Calvin-Benson-Bassham cycle. The genes responsible for the regeneration and reduction of the substrate in the Calvin-Benson-Bassham cycle were found in the core genome. Ru-BisCO in the genus Thiothrix is represented by three types: IAq, IAc, and II. Previous pangenome analysis has shown that RuBisCO of the IAq type is common to all species, in contrast to IAc and II types [8]. However, the addition of 'Thiothrix winogradskyi' CT3 T into the pangenome analysis excluded type IAq RuBisCO from the core genome, since its genome does not contain this type of RuBisCO. All species, except for 'Ca. Thiothrix anitrata' A52, encode RuBisCO of type II, and all species except T. fructosivorans Q T , T. lacustris BL T , and MAG of Thiothrix sp. 207 encode type IAc ( Figure 5). Figure 5. Distribution of genes related to nitrogen metabolism and carbon fixation in Thiothrix genomes. Genes: rbcL, the large subunit of RuBisCO; narG, membrane-bound nitrate reductase; napAB, periplasmic nitrate reductase; nasA, assimilatory nitrate reductase; nirS, dissimilatory nitrite reductase; nirBD, ammonia-forming assimilatory nitrite reductase; norBC, nitric oxide reductase; nosZ, nitrous oxide reductase; nif-cluster, nitrogenase genes; phsABC, thiosulfate reductase. Colored blocks indicate the presence of a gene.
As for phosphoribulokinase in the genus Thiothrix, the prk gene is represented by two phylogenetically different copies. The first copy has a high level of sequence similarity among all representatives of Thiothrix and was found in the core genome. The second copy of prk is also present in all Thiothrix; however, the amino acid sequence identity with the first type of phosphoribulokinase is below the 70% threshold used to determine the genes of the core genome. The genes for the tricarboxylic acid cycle, Embden-Meyerhof-Parnas pathway, the glyoxylate cycle, and phosphorus metabolism are mainly included in the core genome except for phosphate transport system permease (pstB), glucokinase (glk), dihydrolipoamide dehydrogenase of pyruvate dehydrogenase complex (pdhD), aconitate hydratase (acnB), and 6-phosphogluconolactonase (pgl), the level of similarity of which is below 70%. The genes of the first, second, and third complexes of the electron transport chain, as well as ATPase (complex V), are present in the core genome. The genes of the fourth respiratory chain complex were found in the core genome (cox15, ccoO, ccoP, ccoG, Figure 5. Distribution of genes related to nitrogen metabolism and carbon fixation in Thiothrix genomes. Genes: rbcL, the large subunit of RuBisCO; narG, membrane-bound nitrate reductase; napAB, periplasmic nitrate reductase; nasA, assimilatory nitrate reductase; nirS, dissimilatory nitrite reductase; nirBD, ammonia-forming assimilatory nitrite reductase; norBC, nitric oxide reductase; nosZ, nitrous oxide reductase; nif -cluster, nitrogenase genes; phsABC, thiosulfate reductase. Colored blocks indicate the presence of a gene. Pangenome analysis of the genus Thiothrix revealed that variable part of the genome that comprises genes responsible for anaerobic respiration in the presence of nitrogen compounds and nitrogen fixation. The distribution of genes responsible for different stages of nitrate reduction is irregular ( Figure 5). The gene encoding nitrate reductase (Nar) is present in all Thiothrix species, except for 'Ca. Thiothrix anitrata' A52, 'Ca. Thiothrix sulfatifontis' KT, and T. nivea JP2 T . Moreover, T. lacustris BL T , T. litoralis AS T , T. fructosivorans Q T , 'Thiothrix subterranea' Ku-5 T , T. caldifontis G1 T , and 'Thiothrix winogradskyi' CT3 T encode one form of NarG, Ca. Thiothrix moscovensis RT, and the MAG of Thiothrix sp. 207 encode another form, and T. unzii A1 T encodes both forms. Such a distribution pattern is probably the result of horizontal transfer with the subsequent loss of one of the two narG genes [8]. The T. nivea JP2 T genome contains genes for periplasmic nitrate reductase NapAB instead of Nar, while the MAG of Thiothrix sp. 207 contains both nar and nap genes. The gene for assimilatory nitrate reductase (nasA) was found in all members of the genus, except for 'Ca. Thiothrix anitrata' A52.
Nitrite could be reduced to ammonium by the assimilatory nitrite reductase NirBD, which is encoded in the genomes of all species, with the exception of 'Ca. Thiothrix anitrata' A52. The gene encoding dissimilatory nitrite reductase (nirS), involved in the reduction of nitrite to NO, was not found in 'Ca. Thiothrix anitrata' A52, 'Ca. Thiothrix sulfatifontis' KT, T. nivea JP2 T , Ca. Thiothrix moscovensis RT, 'Thiothrix subterranea' Ku-5 T , or T. lacustris BL T . Nitric oxide reductase NorBC catalyses the reduction of NO to N 2 O. 'Ca. Thiothrix anitrata' A52, 'Ca. Thiothrix sulfatifontis' KT, T. nivea JP2 T , 'Thiothrix subterranea' Ku-5 T , and T. lacustris BL T lacked these genes. Analysis of the MAG of Thiothrix sp. 207 revealed the nosZ gene encoding nitrous oxide reductase, which enables the reduction of N 2 O to molecular nitrogen. This gene was most likely acquired via horizontal transfer from other gammaproteobacteria. Thiothrix sp. 207 therefore could be capable of complete denitrification.
Classical amination is represented by the genes for glutamine synthetase (glnA), glutamate synthase (gltBD), and aspartate aminotransferase (aspB) in the core genome.
Previously, respiration in the presence of thiosulfate as a terminal electron acceptor was not described for any Thiothrix species. Genome analysis revealed that all analyzed members of the genus Thiothrix, except for Ca. Thiothrix moscovensis RT and 'Thiothrix winogradskyi' CT3, contain phsABC genes encoding thiosulfate reductase of the molybdopterin oxidoreductase family ( Figure 5). Verification of genomic data, in particular the determination of the ability for anaerobic respiration in the presence of thiosulfate as an electron acceptor and lactate as an electron donor, as well as the determination of the products of the conversion of thiosulfate to hydrogen sulfide and sulfite, proved that most representatives of the genus Thiothrix are capable of dissimilatory thiosulfate reduction. A high sequence similarity of the deduced amino acid sequences of PhsA (>80%) most likely indicates the loss of the phs genes in Ca. Thiothrix moscovensis RT and 'Thiothrix winogradskyi' CT3 [17].
Thus, representatives of the genus Thiothrix differ significantly in their ability for anaerobic respiration in the presence of various electron acceptors ( Figure 5). Such metabolic diversity can serve to adapt to changing environmental conditions.
The study of the composition of the species-specific genes in the genus Thiothrix has expanded our understanding of the metabolic potential of some species. Although most of such genes were predicted to encode hypothetical proteins with unknown functions, the presence of nosZ genes in the MAG of Thiothrix sp. 207 indicates that it could reduce N 2 O to molecular nitrogen, a trait absent in other Thiothrix species.

Environmental Distribution and Ecological Functions
The first species of the genus Thiothrix were isolated from sulfidic springs, and for a long time, they were considered limited to such habitats. A common feature of these habitats is the low organic content and the simultaneous presence of hydrogen sulfide and oxygen, which is consistent with the ability of all members of the genus Thiothrix to grow chemolithoautotrophically, obtaining energy from the aerobic oxidation of reduced sulfur compounds.
Later, 16S rRNA gene sequences assigned to Thiothrix were found by molecular profiling of microbial communities from organic-rich habitats, including the rhizosphere of the aquatic plant Eichhornia crassipes [21], the larvae of an aquatic insect Chironomus ramosus [22], and in activated sludge from wastewater treatment plants and laboratory bioreactors simulating wastewater treatment systems [23][24][25][26][27]. In all of the above works, the taxonomic composition of the microbiome of a plant, animal, or bioreactor was studied. In some of them [22,23], the community metagenome was also sequenced and the genes of certain metabolic pathways were analyzed. However, in these communities, the relative abundance of Thiothrix did not exceed a few percent. It can be assumed that in these habitats, the source of hydrogen sulfide can be either microbial sulfate reduction or decomposition of protein substrates under anaerobic conditions. It is possible that, in these ecosystems, Thiothrix grow organotrophically, receiving energy from the oxidation of organic substances rather than hydrogen sulfide. Although no cultured isolates have been obtained from these habitats to date, analysis of Thiothrix MAGs obtained from metagenomes of activated sludge from wastewater treatment plants did not reveal any significant peculiarities in their metabolic capabilities [17]. Representatives of the genus Thiothrix are much more widespread in nature than previously thought, but they become the dominant group only under specific conditions of organic-poor waters, simultaneously containing hydrogen sulfide and oxygen.
Bacteria of the genus Thiothrix form ectosymbiosis with marine and freshwater amphipods. For the first time, ectosymbiosis of bacteria from the genus Thiothrix with invertebrates was found in marine amphipods, Urothoe poseidonis [28]. However, the best studied is the ectosymbiosis of bacteria from the genus Thiothrix with freshwater amphipods of the genus Niphargus. Examples of such symbiosis were found in sulfide-rich waters in underground caves in Italy and Romania [29,30]. It has been suggested that a similar symbiosis may be much more widespread in European groundwater [30]. Thiothrix filaments are attached to the base of hairs on amphipod appendages. Bacteria from the genus Thiothrix have not been shown to be involved in the detoxification of hydrogen sulfide for the host organism, so the ecological role of this ectosymbiosis is not yet clear [31].

Comparison with Phylogenetically Related Genera Thiolinea and Thiofilum
The genera Thiolinea and Thiofilum are the closest phylogenetic relatives of the genus Thiothrix (Figure 3), initially described as members of that genus. Determination of genome sequences for representatives of these two genera ('Thiolinea eikelboomii', Thiolinea disciformis, and Thiofilum flexile) allowed a comparative genome-based analysis of the main metabolic pathways.
First of all, members of all three genera have the same enzyme systems for the oxidation of sulfur and hydrogen sulfide under aerobic conditions. Members of the genus Thiolinea, such as Thiothrix, are capable of autotrophic carbon fixation through the Calvin cycle. Their genomes contain genes encoding RuBisCO types IAq and II [15]. The phylogenetically more distant Thiofilum flexile lacks the RuBisCO genes and therefore is capable of only lithoheterotrophic but not lithoautotrophic growth.
Like different Thiothrix species, the genomes of Thiolinea and Thiofilum encode different sets of genes to reduce nitrogen compounds. In the genomes of Thiofilum flexile and Thiolinea disciformis, genes for denitrification systems (narG, nirS, norBC, nosZ) were not found, while the 'Thiolinea eikelboomii' genome contains only genes for dissimilatory Nar-type nitrate reductase. Unlike most Thiothrix species, none of the members of Thiolinea and Thiofilum have genes of the nif -cluster, which enables nitrogen fixation.
As noted above, one of the features of the genus Thiothrix is the presence of a FADdependent membrane-bound malate:quinone oxidoreductase instead of the typical NADdependent cytoplasmic malate dehydrogenase. The mqo genes are also present in the genomes of Thiolinea and Thiofilum flexile, but the latter also contains the mdh gene. It is likely that the acquisition of mqo occurred in a common ancestor of all three genera, and mdh was subsequently lost in the Thiolinea/Thiothrix lineage.

Conclusions
A pangenome analysis of the genus Thiothrix revealed that the genes of dissimilatory sulfur metabolism are the most conserved part of the genome. The ability to carry out the interconversions of reduced sulfur compounds is probably a key characteristic for these bacteria, which once again indicates their lithotrophic type of metabolism.
Thiothrix thrives as a surface-attached organism in running water, which contains H 2 S and O 2 in fairly high concentrations. In such habitats, Thiothrix filaments exist under conditions in which the concentrations of these substances (oxygen and hydrogen sulfide) could change significantly in a short period of time. As a rule, the O 2 concentration in Thiothrix growth zones can vary from full to 10% of saturation. In such frequently changing environmental conditions, bacteria have developed an adaptation strategy through changes at the genomic level.
Significant differences in the inventory of genes for nitrogen metabolism, RuBisCO gene profiles [8], and phsABC thiosulfate reductase genes, and the discovery of new speciesspecific genes (nosZ), indicate a dynamic process of gene acquisition via horizontal transfer and lineage-specific gene losses that allows bacteria to adapt to variable environmental conditions. Thus, thiosulfate and oxidized forms of nitrogen, which can be used as electron acceptors during anaerobic respiration, are not always available in different ecosystems and can be interchanged; the presence of various RuBisCO forms can be explained by the adaptation to different concentrations of CO 2 and O 2 , which vary in a wide range.