Genomic and Metabolic Insights into Two Novel Thiothrix Species from Enhanced Biological Phosphorus Removal Systems

Two metagenome-assembled genomes (MAGs), obtained from laboratory-scale enhanced biological phosphorus removal bioreactors, were analyzed. The values of 16S rRNA gene sequence identity, average nucleotide identity, and average amino acid identity indicated that these genomes, designated as RT and SSD2, represented two novel species within the genus Thiothrix, ‘Candidatus Thiothrix moscowensis’ and ‘Candidatus Thiothrix singaporensis’. A complete set of genes for the tricarboxylic acid cycle and electron transport chain indicates a respiratory type of metabolism. A notable feature of RT and SSD2, as well as other Thiothrix species, is the presence of a flavin adenine dinucleotide (FAD)-dependent malate:quinone oxidoreductase instead of nicotinamide adenine dinucleotide (NAD)-dependent malate dehydrogenase. Both MAGs contained genes for CO2 assimilation through the Calvin–Benson–Bassam cycle; sulfide oxidation (sqr, fccAB), sulfur oxidation (rDsr complex), direct (soeABC) and indirect (aprBA, sat) sulfite oxidation, and the branched Sox pathway (SoxAXBYZ) of thiosulfate oxidation to sulfur and sulfate. All these features indicate a chemoorganoheterotrophic, chemolithoautotrophic, and chemolithoheterotrophic lifestyle. Both MAGs comprise genes for nitrate reductase and NO-reductase, while SSD2 also contains genes for nitrite reductase. The presence of polyphosphate kinase and exopolyphosphatase suggests that RT and SSD2 could accumulate and degrade polyhosphates during the oxic-anoxic growth cycle in the bioreactors, such as typical phosphate-accumulating microorganisms.


Introduction
Filamentous colorless sulfur bacteria (FCSB) have been a classic object of microbiology since the time of S.N. Winogradsky, who used them in the research that culminated in the discovery of chemosynthesis. Nevertheless, the genomics and metabolic potential of the FCSB from the genus Thiothrix remain poorly understood [1][2][3]. The bacteria retained in the genus after the last taxonomic revision [4] include five species: T. nivea, T. fructosivorans, T. unzii, T. caldifontis and T. lacustris. Despite the fact that the genomes of T. lacustris (GCF_000621325.1), T. caldifontis (GCF_900107695.1), and T. nivea (GCF_000260135.1) are available from databases, comprehensive analysis of the main metabolic pathways of sulfur, carbon, and nitrogen has not been reported. In addition, 25 metagenome-assembled genomes (MAGs) obtained from metagenomes of natural hydrogen sulfide biotopes are currently available. Two of these genomes were attributed to certain species, T. nivea DOLJORAL78_51_81 [5,6] and T. lacustris A8 [7], and 23 others were designated as Thiothrix sp. or uncultured Thiothrix sp. [8][9][10].
Due to the feature of energy metabolism, where hydrogen sulfide is the main donor of electrons during chemosynthesis, sulfur springs were considered to be a traditional habitat for Thiothrix, where filamentous bacteria often form massive fouling. There are reports of intense growth of Thiothrix in wastewater rich in reduced sulfur compounds [11][12][13][14]. An active growth of Thiothrix in wastewater treatment plants leads to the formation of fouling that can disrupt the operation of the entire system, degrading the quality of the wastewater and increasing the treatment time [15,16]. On the other hand, the ability of Thiothrix from activated sludge to accumulate polyphosphates and participate in the removal of phosphorus from wastewater was found along with other polyphosphate accumulating organisms (PAOs) [17][18][19], such as 'Candidatus Accumulibacter phosphatis', Tetrasphaera [20,21], and Halomonas [22].
The present study aims to analyse of genomic and metabolic potential of two recently obtained Thiothrix genomes-Thiothrix sp. RT and Thiothrix sp. SSD2, recovered from metagenomes of laboratory-scale enhanced biological phosphorus removal bioreactors (EBPR). Based on the phylogenetic analysis of MAGs, we propose two new species, 'Candidatus Thiothrix moscovensis' sp.nov. and 'Candidatus Thiothrix singaporensis' sp.nov.

PAO-Enriched Laboratory Culture for Thiothrix sp. RT
Enrichment and cultivation of the PAO microbial community in a lab-scale bioreactor operated at Research Center of Biotechnology (Moscow, Russia) was reported earlier [23]. Briefly, a 15 L sequencing batch laboratory bioreactor, inoculated with an activated sludge from Luberetskya wastewater treatment plant (Moscow, Russia), was supplied with the following medium: 670 mg/L CH 3 COONa·3H 2 O, 50 mg/L NH 4 Cl, 54.3 mg/L KH 2 PO 4 , 56.7 mg/L Na 2 HPO 4 , and 8.6 mg/L yeast extract. The bioreactor was operated at room temperature in a cyclic mode. A 6 h cycle of cultivation included the stages of filling the bioreactor with fresh medium (30 min), anaerobic growth (110 min), aerobic growth (180 min), precipitation of activated sludge (30 min), and discharge of the spent medium (10 min). Under these conditions, the PAO bioreactor stably removed 75-80% of phosphorus from the medium [23].

Metagenome Sequencing and Assembly of Thiothrix sp. RT
The total DNA was extracted from 1 g of sludge sample using a DNeasy PowerSoil DNA isolation kit (Qiagen, Hilden, Germany). A total of about 1.5 µg of DNA was obtained. A metagenomic DNA sample was sequenced using the Illumina (Illumina, San Diego, CA, USA) platform. The TruSeq DNA library was prepared using NEBNext ® Ultra™ II DNA Library Prep Kit (New England Biolabs, Hitchin, UK) and sequenced on the Illumina HiSeq2500 instrument (250 nt single-end reads). Adapter sequences and low-quality reads were removed using Cutadapt [24] and Sickle (https://github.com/najoshi/sickle accessed on 10 November 2020), respectively. A total of about 50 million high-quality reads were obtained. The reads were de novo assembled into contigs using MetaSPAdes v. 3.7.1 [25]. The contigs were binned into MAGs using MetaBAT v. 2.12.1 [26]. Completeness of the MAGs and their possible contamination were estimated using the CheckM v. 1.0.13 tool [27]. The taxonomic assignment of the obtained MAGs was performed using the Genome Taxonomy Database Toolkit (GTDB-Tk) v. 1.1.1 [28] and Genome Taxonomy Database (GTDB) [29].
Gene search and annotation were performed using the RAST server 2.0 [30] followed by manual correction by the searches of predicted protein sequences against the National Center for Biotechnology Information (NCBI) databases.

Genome of Thiothrix sp. SSD2
MAG of Thiothrix sp. SSD2 was obtained from sludge from a full-scale wastewater treatment plant (PUB, Singapore) and assembled in a single contig with a total length of 4,541,730 [31]. The information about bioreactor operating conditions, samples preparation, DNA isolation, metagenome sequencing and assembly has been published [31]. Genome annotation revealed 16S rRNA gene, 43 tRNA genes and 4097 potential protein-coding genes [31]. For the present study, the Thiothrix sp. SSD2 genome was obtained from NCBI GenBank (CP059265).

Nucleotide Sequence Accession Number
The annotated genome sequence of Thiothrix sp. RT bacterium was submitted to the NCBI GenBank database and is accessible under the number JAEHNX000000000.

Assembly of the Genome of New Member of the Genus Thiothrix from EBPR Bioreactor
To obtain MAGs of microbial community members, we sequenced the metagenome of activated sludge from of the PAO bioreactor operated at Research Center of Biotechnology (Moscow, Russia) using Illumina technique. Analysis of the taxonomic affiliation of the obtained MAGs showed that one of them, designated Thiothrix sp. RT, belongs to the genus Thiothrix. Microscopic analysis of microorganisms from the PAO bioreactor also indicates the presence of cells morphologically similar to cultivated members of the genus Thiothrix: cells form rosettes, gonidia, and sheath ( Figure 1). The assembled Thiothrix sp. RT MAG consisted of 78 contigs (N50 size of 65,012 bp) with a total length of 3,693,042 bp; the CheckM-estimated completeness of this MAG as 99.8% with 0.92% possible contamination. Genome annotation revealed 16S rRNA gene, 38 tRNA genes, and 3645 potential protein-coding genes. The Thiothrix sp. RT 16S rRNA gene shares 97.9% sequence identity with Thiothrix nivea DSM 5205 and more than 99.5% identity with several environmental 16S rRNA gene sequences detected in activated sludge [34].

Phylogenetic Analysis of MAGs
Besides MAG of Thiothrix sp. RT, only one genome of Thiothrix sp. from EBPR bioreactors, Thiothrix sp. SSD2, is available [31]. Here we compared these genomes with each other and with genomes of known species of the genus Thiothrix, which have all been isolated from natural sulfidic environments.
The identity level of 16S rRNA genes of Thiothrix sp. RT and Thiothrix sp. SSD2 with genes of other members of the genus Thiothrix varies from 93.0 to 97.9%, while the 16S rRNAs of all described representatives of the genus are 93.3-98.9% identical to each other (Supplementary Materials Table S1). These values suggest assignment of Thiothrix sp. RT and Thiothrix sp. SSD2 to novel species within the same genus [35].
All species remaining in the genus Thiothrix after the last taxonomic revision [4] form two phylogenetic clusters on the tree, reconstructed on the basis of the nucleotide sequences of the 16S rRNA gene ( Figure 2). The first cluster includes Thiothrix sp. RT and Thiothrix sp. SSD2 together with Thiothrix nivea DSM 5205 and Thiothrix unzii A1. The second cluster includes T. lacustris BL T , T. caldifontis G1 T , and T. fructosivorans Q T .
The AAI values between Thiothrix sp. RT and other Thiothrix genomes were from 74.0 to 76.9%, and 74.0-78.5% AAI was detected between Thiothrix sp. SSD2 and other species (Figure 3). These values are consistent with the assignment of Thiothrix sp. RT and Thiothrix sp. SSD2 to new species of the genus Thiothrix according to the thresholds proposed for genome-based taxonomic delineation [36]. Both of the two new genomes could also be assigned to novel species according to their ANI (<80.2%) and DDH (<30.2%) values (Table S2). The obtained values of AAI, ANI and dDDH are below the threshold for species delineation [37]. Based on results of phylogenomic analysis, we propose classifying strains RT and SSD2 as 'Candidatus Thiothrix moscovensis' sp.nov. and 'Candidatus Thiothrix singaporensis' sp.nov., respectively. 'Ca. Thiothrix moscovensis' sp.nov. (mos.co.ven'sis N.L. fem. adj. moscovensis pertaining to the city of Moscow, Russia, where the EBPR bioreactor used to obtain this genome operated). 'Ca. Thiothrix singaporensis' sp.nov. (sin.ga.po.ren´sis N.L. fem. adj. singaporensis pertaining to the city of Singapore, Singapore, where the EBPR bioreactor used to obtain this genome operated).

Dissimilatory Metabolism of Sulfur Compounds
Members of the genus Thiothrix are known representatives of sulfur-oxidizing prokaryotes. Dissimilatory sulfur metabolism of the genus Thiothrix is represented by various sulfur-oxidizing complexes involved in the oxidation of hydrogen sulfide, thiosulfate, elemental sulfur, and sulfite.
Genes that encode enzymes involved in the oxidation of sulfide to elemental sulfur, sulfide:quinone oxidoreductase (SQR) and flavocytochrome c-sulfide dehydrogenase (FCSD) were identified in Thiothrix sp. RT and Thiothrix sp. SSD2.
For all members of the genus, including Thiothrix sp. RT and Thiothrix sp. SSD2, the presence of genes of the SOX multienzyme system that oxidizes thiosulfate was revealed ( Table 1, Table S3). A feature of the genus is the absence of genes for the dimeric protein SoxCD. The oxidation of thiosulfate is carried out by the branched Sox pathway (the SoxAXBYZ enzymatic complex), which oxidizes thiosulfate to sulfate and elemental sulfur, accumulated inside the cells.
Elemental sulfur can be further oxidized to sulfite using the reverse dissimilatory sulfite reductase rDsrAB. In Thiothrix sp. RT and Thiothrix sp. SSD2 genes of this pathway form one cluster consisting of 14 genes (dsrABCEFHLMKJOPRS) and 12 genes (dsrABCEFHLMKJOP), respectively ( Table 1, Table S3). An accessory gene dsrR was not found in the genome of Thiothrix sp. SSD2, while Thiothrix sp. RT contains all genes of the dsr cluster.
Genomes of Thiothrix sp. RT and Thiothrix sp. SSD2 contains genes for membrane-bound cytoplasmic sulfite:quinone oxidoreductase SoeABC, which directly oxidizes sulfite to sulfate. Genes for indirect oxidation of sulfite to sulfate (the aprAB genes encoding APS reductase (AprAB) and the sat gene encoding ATP sulfurylase (Sat)) were also found. Oxidation of sulfite to sulfate occurs via the APS intermediate. Thus, it can be assumed that Thiothrix sp. RT and Thiothrix sp. SSD2, like the well-known pure cultures of the genus Thiothrix, have pathways for the oxidation of reduced sulfur compounds, which provide the oxidation of hydrogen sulfide, elemental sulfur, sulfite, and thiosulfate.

Autotrophic CO 2 Assimilation
All genes encoding enzymes enabling autotrophic assimilation of CO 2 through the Calvin-Benson-Bassham cycle (except for the sedoheptulose-1,7-bisphosphatase) were found in all members of the genus Thiothrix, as well as in Thiothrix sp. RT and Thiothrix sp. SSD2 (Table 1, Table S3). It should be noted that the sedoheptulose-1,7-bisphosphatase activity in bacteria is often catalyzed by fructose-1,6-bisphosphatase with dual sugar specificity [38,39]. A key Calvin-Benson-Bassham cycle enzyme, RuBisCo, is represented by three types (IAc, IAq, II) in Thiothrix sp. RT and Thiothrix sp. SSD2. Another key enzyme of the Calvin-Benson-Bassham cycle, phosphoribulokinase, is encoded by two copies of the prk gene, which are phylogenetically distant from each other, which is typical for Thiothrix species.

Assimilation of N 2
Genomes of Thiothrix sp. RT and Thiothrix sp. SSD2 contain almost a complete set of nif genes required for N 2 fixation in nifASVB1XX2YENQVWMHDKZT and nifASVB1XX2B2ENQV WMHDKZTO operons. The composition of the genes of accessory nitrogenase subunits differed slightly. Genes for NifB2 which, along with NifB1, regulates the nitrogenase complex [40] and NifO which involved in synthesis of the iron-molybdenum cofactor of nitrogenase [41] were not found in the genome of Thiothrix sp. RT. The nifY gene, which promotes the maturation of the enzyme through its binding to the MoFe cofactor, was not found in the genome of Thiothrix sp. SSD2.

Dissimilatory Nitrate Reduction
Genes for the NarGHI membrane complex involved in the dissimilatory reduction of nitrate to nitrite were found in genomes of Thiothrix sp. RT and Thiothrix sp. SSD2. Genes nirBD that encode the NirBD enzyme involved in the removal of the toxic NO 2 by assimilatory reduction to NH 4 + were found in the genomes of all members of the genus Thiothrix, including Thiothrix sp. RT and Thiothrix sp. SSD2. This chain is realized in almost all representatives of the genus Thiothrix, with the exception of T. nivea, in which instead of narGHI, the periplasmic nitrate reductase encoded by napAB was found (

Phosphorus Accumulation
The accumulation of polyphosphate (poly-P) granules is known for members of the genus Thiothrix [17,43]. Both Thiothrix sp. RT and Thiothrix sp. SSD2 contained genes for polyphosphate kinase PPK (ppk), the main enzyme that catalyzes the transfer of ATP (ppk1) or GTP (ppk2) to the active site of the protein during the synthesis of the poly-P chain [44]. The ppk1 gene encoding PPK1 was found in the genomes of Thiothrix sp. RT and Thiothrix sp. SSD2. Since this reaction is reversible, in the case of PPK1, it could be used to make ATP from poly-P [44]. The gene for the exopolyphosphatase EPP (epp), which catalyzes the hydrolysis of inorganic phosphorus from the polyphosphate chain, was also found in the genomes of Thiothrix sp. RT and Thiothrix sp. SSD2. Genes for PPK and EPP are also present in other members of the genus Thiothrix.
Thus, both enzymes, PPK1 and EPP, can regulate the accumulation and degradation of polyphosphates in the cell. Genes phoURB, pstSACB which encodes systems for the regulation and transfer of inorganic phosphorus into the cell were also found in the genomes of Thiothrix sp. SSD2 and Thiothrix sp. RT. Along with participation in biosynthetic processes, polyphosphates are probably used for temporal ATP storage, which is realized through the activity of polyphosphate kinase PPK1 and exopolyphosphatase EPP in members of the genus Thiothrix.

Central Metabolic Pathways
Genome analysis of Thiothrix sp. RT and Thiothrix sp. SSD2 revealed the presence of a complete set of genes for the Embden-Meyerhof (EM) pathway, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle (Table S3).
TCA cycle has a remarkable feature in the known species of the genus Thiothrix, as well as in Thiothrix sp. SSD2 and Thiothrix sp. RT, -the absence of the mdh gene of the classic cytoplasmic nicotinamide adenine dinucleotide (NAD)-dependent malate dehydrogenase, which is found in the vast majority of prokaryotes. Instead, the mqo gene encoding membrane-bound FAD-dependent malate:quinone oxidoreductase was found in the genomes of representatives of the genus Thiothrix, including Thiothrix sp. RT and Thiothrix sp. SSD2.
Genomes of Thiothrix sp. RT and Thiothrix sp. SSD2 include all the genes of the EM pathway. The gene pckA for ATP-dependent phosphoenolpyruvate (PEP)-carboxykinase, which decarboxylates oxaloacetate to form pyruvate, was also revealed in the genomes of Thiothrix sp. SSD2 and Thiothrix sp.
RT. The genomes contain genes for pyruvate carboxylase (pyc) and decarboxylating NADP-dependent malate dehydrogenase (maeB). The two latter enzymes likely shunt the reaction of malate conversion to oxaloacetate in the TCA cycle and glyoxylate cycle in order to ensure the production of sufficient oxaloacetate for gluconeogenesis and amino acid biosynthesis.
The release to constructive metabolism from the TCA cycle occurs at the level of oxaloacetate and 2-oxoglutarate. Oxaloacetate is involved in aspartate synthesis with the participation of aspartate aminotransferase (aspB), while the conversion of 2-oxoglutarate is carried out by glutamate synthase (glt) to glutamate.

Respiratory Electron Transport Chain
Genomes of Thiothrix sp. RT and Thiothrix sp. SSD2 encode all the major components of the electron transport chain required to generate energy through oxidative phosphorylation. Complex I, NADH:quinone oxidoreductase, is encoded by the genes forming the nuoABCDEFGHIJKLM cluster and separate nuoN gene in Thiothrix sp. SSD2. The complex is encoded by two gene clusters, nuoACDEFGHIJLM and nuoBKN, in Thiothrix sp. RT.
The succinate dehydrogenase II complex includes a cytochrome b 556 subunit (sdhC), an iron-sulfur protein (sdhB), a flavoprotein subunit (sdhA), a hydrophobic membrane anchor protein (sdhD). The genes form a single sdhABCD cluster in the genome of Thiothrix sp. RT, and are located in two separate loci, sdhBCD and sdhA, in Thiothrix sp. SSD2.
Complex III, ubiquinol-cytochrome c reductase, consists of an iron-sulfur subunit (encoded by the rpi1 gene), a cytochrome b subunit (encoded by the cytB gene), and a cytochrome c 1 subunit (encoded by the cyc1 gene).
Complex IV is represented by five types of oxidases: six cbb 3 type subunits (encoded by ccoNOPGQS), six cox subunits (encoded by coxABCD11, 15), and four cytochrome d subunits of ubiquinol oxidase (encoded by cydABCDX). Conversion of ADP to ATP occurs via F-type ATP synthetase, encoded at the atpABCDEGH cluster, with a separate gene for the F-type ATP synthetase B subunit, atpF. The phosphates required for ATP synthetase to function are supplied by inorganic pyrophosphatase, encoded by the ppa gene, and polyphosphate kinase, encoded by ppk.
Overall, genome analysis of Thiothrix sp. SSD2 and Thiothrix sp. RT did not reveal any important differences in the composition of genes for metabolic pathways from other representatives of the genus Thiothrix. The scheme of potential metabolic pathways of representatives of the genus Thiothrix, based on our genome-based reconstructions, is shown in Figure 4.

Discussion
Phylogenetic analysis of Thiothrix sp. RT and Thiothrix sp. SSD2 and other members of the genus Thiothrix supported classification of the two new strains as new species 'Candidatus Thiothrix moscovensis' sp.nov. and 'Candidatus Thiothrix singaporensis' sp.nov., respectively.
The results of the analysis of Thiothrix sp. RT and Thiothrix sp. SSD2 genomes show that the studied organisms carry out the respiratory type of metabolism, and that they are facultative aerobes and facultative heterotrophs. Complete oxidation of organic substrates can occur through the TCA pathway with the formation of a transmembrane proton gradient, which is further used by ATP synthase for the synthesis of ATP. Thiothrix sp. RT and Thiothrix sp. SSD2 are potentially capable of assimilation of molecular nitrogen, anaerobic respiration on nitrates and incomplete denitrification.
Autotrophic growth in the presence of reduced sulfur compounds (hydrogen sulfide, elemental sulfur, sulfite, and thiosulfate) with the participation of enzymatic systems of dissimilatory sulfur metabolism is possible for representatives of the genus Thiothrix, including Thiothrix sp. RT and Thiothrix sp. SSD2, which ensure the lithotrophic growth of these organisms. A feature of thiosulfate oxidation is the presence of a branched Sox pathway (the SoxAXBYZ enzymatic complex) and the absence of soxCD genes, which leads to the oxidation of thiosulfate to sulfates and elemental sulfur.
A notable feature of Thiothrix sp. RT and Thiothrix sp. SSD2, as well as other representatives of the genus Thiothrix, is the absence of the classical cytoplasmic NAD-dependent malate dehydrogenase, which is found in the vast majority of prokaryotes. Instead, the mqo gene encoding a FAD-dependent malate:quinone oxidoreductase was found in the genomes of two MAGs, as well as in the genomes of other known representatives of the genus Thiothrix. The presence of only MQO, as an enzyme capable of directly oxidizing malate to oxaloacetate in TCA and in the glyoxylate cycle, is quite rare in prokaryotes [45]. In terms of energy potential (Gibbs free energy, ∆ G • '), this enzyme is much more efficient than MDH and, at the same time, its activity increases more than four times in the presence of acetate [45,46].
The two EBPR systems analyzed used a batch reactor with alternating aerobic and anaerobic conditions with acetate as a carbon and energy source. The fact that we managed to assemble MAGs representing new species of the genus Thiothrix may also be due to the fact that the samples were taken from EBPR systems. During organoheterotrophic growth, the presence of MQO in these organisms gave an advantage when growing on acetate relative to organisms with classical MDH. This capacity probably facilitated enrichment of Thiothrix species in the reactors, as well as their ability to accumulate and degrade polyphosphates as typical phosphate-accumulating microorganisms. It should be noted that the growth of a number of members of the genus Thiothrix (T. lacustris, T. caldifontis) is preferable under chemolithoheterotrophic conditions (cell biomass increases by 2-3 times). With batch cultivation under organoheterotrophic conditions other than the EBPR system, growth is ineffective and is accompanied by a slight increase in biomass, by 20-30% (our unpublished data).
Thiothrix usually grows in nature in organic-poor hydrogen sulfide biotopes or in organic-rich wastewater, but in the presence of sulfide. In this case, bacteria use a lithoautotrophic or lithoheterotrophic type of metabolism.
Periodic changes in the growth conditions realized in the EBPR system probably provided an advantage for the enrichment of new Thiothrix species that were not previously identified in natural biotopes and wastewater. There is now sufficient evidence that Thiothrix can participate in the removal of inorganic phosphorus [17][18][19]. Analysis of MAGs obtained from EBPR system metagenomes will probably be useful not only for identifying new Thiothrix species, but also for creating optimal conditions for the growth of Thiothrix as phosphate accumulating organisms in the EBPR system. In particular, the addition of nitrate could probably stimulate the growth of Thiothrix in the EBPR bioreactors, since they are able to survive anaerobic conditions by switching to anaerobic respiration on nitrates. The addition of acetate to the culture medium promotes the induction of MQO activity involved in the conversion of malate to oxaloacetate. This process is vital because oxaloacetate is an important precursor of gluconeogenesis and amino acid synthesis of the aspartate branch.

Conflicts of Interest:
The authors declare no conflict of interest.