Genome-Based Phylogeny of the Genus Thiothrix: Comparison
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Representatives of the genus Thiothrix are filamentous, sulfur-oxidizing bacteria, capable of lithoauto-, lithohetero- and organoheterotrophic growth. They are often 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.

  • Thiothrix
  • phylogeny
  • pangenome
  • metagenome-assembled genome

1. 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 H2S 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][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, CO2 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).
Figure 1. Scheme of dissimilation sulfur metabolism of the genus Thiothrix. FccAB, flavocytochrome c-sulfide 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; S0, sulfur globule; APS, adenosine 5′-phosphosulfate; R−Sn+1H, thiol compound; H−Sn+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][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 JP2T (=ATCC 35100T = DSM 5205T) is the only established neotype of the species [5].
At the end of the 20th century, several new isolates were obtained: Thiothrix ramosa [10], Thiothrix arctophila [11], and Thiothrix sp. CT3 [12]. Unfortunately, two proposed species, T. ramosa [10] and T. arctophila [11], are absent in international collections and were lost (Dubinina, personal communication).
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.
Two extra representatives of Eikelboom type 021N, Thiothrix disciformis and Thiothrix flexilis, and two species from the Thiothrix nivea group, Thiothrix lacustris and Thiothrix caldifontis, were described in later years [13,14][13][14].
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 BLT, ‘Thiothrix litoralis’ AST, and ‘Thiothrix winogradskyi’ CT3T) (Figure 2).
Figure 2. Heatmap of 16S rRNA gene sequence similarity and pairwise ANI values (%) for Thiothrix genomes. T. lacustris BLT, (GCF_000621325.1); Thiothrix litoralis AST (GCF_017901135.1); ‘Thiothrix subterranea’ Ku-5T (GCF_016772315.1); ‘Ca. Thiothrix sulfatifontis’ KT (GCA_022828425.1); T. caldifontis G1T (GCF_900107695.1); ‘Thiothrix winogradskyi’ CT3T (GCF_021650945.1); T. fructosivorans QT (GCA_017349355.1); T. unzii A1T (GCA_017901175.1); ‘Ca. Thiothrix anitrata’ A52 (GCF_017901155.1); Ca. Thiothrix moscovensis RT (GCA_016292235.1); T. nivea JP2T (GCF_000260135.1); Ca. Thiothrix singaporensis SSD2 (GCA_013693955.1); MAG of Thiothrix sp. 207 (GCA_018813855.1). Note that 16S rRNA gene is missing in MAG of Thiothrix sp. 207.
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%.

2. Genome-Based Phylogeny

The determination of complete genome sequences for T. disciformis, T. eikelboomii, T. flexilis, T. caldifontis, T. lacustris, and T. nivea has enabled a more accurate phylogenetic analysis. In 2018, Boden and Scott undertook a multi-phase study which included morphological, biochemical, physiological, and genomic properties, and gene-based phylogeny to reclassify Thiothrix species. The 16S rRNA gene (rrs), recombination protein A (recA), polynucleotide nucleotide transferase (pnp), translation initiation factor IF-2 (infB), glyceraldehyde-3-phosphate dehydrogenase (gapA), glutamyl-tRNA synthetase (glnS), elongation factor EF-G (fusA), and concatenated sequences of 53 ribosomal proteins allowed the distribution of Thiothrix species between three different families: Thiolineaceae, Thiofilaceae, and Thiotrichaceae [15].
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 whole-genome comparison has higher accuracy and resolution than taxonomy based on individual phylogenetic markers. Whole-genome sequences of isolates T. fructosivorans QT, T. unzii A1T, Thiothrix litoralis AST, ‘Thiothrix subterranea’ Ku-5T [8], and ‘Thiothrix winogradskyi’ CT3T [17], as well as metagenome-assembled genomes (MAGs) of ‘Candidatus Thiothrix anitrata’ A52, Candidatus Thiothrix moscovensis RT [18,19][18][19], and Candidatus Thiothrix singaporensis SSD2 [19[19][20],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.
Table 1.
The general properties of
Thiothrix
genomes.
Species Genome

Assembly		 Isolate or MAG Size

(MB)		 Contigs G + C

Content

(mol %)		 Protein-Coding Genes 16S rRNA Genes tRNA Genes Plasmids *
‘T. winogradskyi’ CT3T

(DSM 12730T)		 GCA_021650935.1 Isolate 4.38 3 51.4 4292 5 66 2
‘Ca. Thiothrix sulfatifontis’ KT GCA_022828425.1 MAG 3.69 1 51.5 3729 2 47 NA
T. lacustris BLT (DSM 21227T) GCF_000621325.1 Isolate 3.72 56 51.3 3537 2 40 U
T. litoralis AST

(DSM 113264T)		 GCF_017901135.1 Isolate 4.28 1 52.8 4045 3 44 0
‘T. subterranea’ Ku-5T (VKM B-3544T) GCF_016772315.1 Isolate 4.08 4 51.1 3885 4 46 3
T. caldifontis G1T (DSM 21228T) GCF_900107695.1 Isolate 3.94 72 50.6 3752 1 42 U
T. unzii A1T

(ATCC 49747T)		 GCA_017901175.1 Isolate 3.72 8 50.8 3626 2 45 7
T. nivea JP2T

(DSM 5205T)		 GCF_000260135.1 Isolate 4.69 15 54.9 4327 2 44 U
T. fructosivorans QT (ATCC 49748T) GCA_017349355.1 Isolate 4.36 6 51.3 3616 2 44 5
Ca. Thiothrix moscovensis RT GCA_016292235.1 MAG 3.69 78 53.6 3483 1 38 NA
Ca. Thiothrix singaporensis SSD2 GCA_013693955.1 MAG 4.54 1 55.6 4097 2 43 NA
‘Ca. Thiothrix anitrata’ A52 GCF_017901155.1 MAG 3.55 1 50.1 3387 2 45 NA
Thiothrix sp. 207 GCA_018813855.1 MAG 3.93 136 54.6 3675 0 40 NA
* U, unknown because the assembly consisted of multiple contigs; NA, not applicable for MAGs.
Several MAGs were obtained from the Svalliden-Norrby groundwater metagenome, Oskarshamn, Sweden. When analyzing the obtained MAGs, wresearchers 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).

References

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  13. Aruga, S.; Kamagata, Y.; Kohno, T.; Hanada, S.; Nakamura, K.; Kanagawa, T. Characterization of filamentous Eikelboom type 021N bacteria and description of Thiothrix disciformis sp. nov. and Thiothrix flexilis sp. nov. Int. J. Syst. Evol. Microbiol. 2002, 52, 1309–1316.
  14. Chernousova, E.; Gridneva, E.; Grabovich, M.; Dubinina, G.; Akimov, V.; Rossetti, S.; Kuever, J. Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov., gammaproteobacteria isolated from sulfide springs. Int. J. Syst. Evol. Microbiol. 2009, 59, 3128–3135.
  15. Boden, R.; Scott, K.M. Evaluation of the genus Thiothrix Winogradsky 1888 (Approved Lists 1980) emend. Aruga et al. 2002: Reclassification of Thiothrix disciformis to Thiolinea disciformis gen. nov., comb. nov., and of Thiothrix flexilis to Thiofilum flexile gen. nov., comb nov., with emended description of Thiothrix. Int. J. Syst. Evol. Microbiol. 2018, 68, 2226–2239.
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  18. Mardanov, A.V.; Gruzdev, E.V.; Smolyakov, D.D.; Rudenko, T.S.; Beletsky, A.V.; Gureeva, M.V.; Markov, N.D.; Berestovskaya, Y.Y.; Pimenov, N.V.; Ravin, N.V.; et al. Genomic and metabolic insights into two novel Thiothrix species from enhanced biological phosphorus removal systems. Microorganisms 2020, 8, 2030.
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