Tychonema sp. BBK16 Characterisation: Lifestyle, Phylogeny and Related Phages

Cyanobacterial expansion is harmful to the environment, the ecology of Lake Baikal and the economy of nearby regions and can be dangerous to people and animals. Since 2011, the process of colonisation of the lake with potentially toxic cyanobacteria belonging to the genus Tychonema has continued. An understanding of the mechanism of successful expansion of Tychonema requires scrutiny of biological and genomic features. Tychonema sp. BBK16 was isolated from the coastal zone of Lake Baikal. The morphology of BBK16 biofilm was studied with light, scanning electron and confocal microscopy. The biofilm is based on filaments of cyanobacteria, which are intertwined like felt; there are also dense fascicles of rope-like twisted filaments that impart heterogeneity to the surface of the biofilm. Genome sequencing, intergenomic comparisons and phylogenetic analyses indicated that Tychonema sp. BBK16 represent a new species related to planktic cyanobacterium Tychonema bourrellyi, isolated from Alpine lentic freshwater. Genome investigation revealed the genes possibly responsible for the mixotrophic lifestyle. The presence of CRISPR-Cas and restriction modification defence mechanisms allowed to suggest the existence of phages infecting Tychonema sp. BBK16. Analysis of CRISPR spacers and prophage-derived regions allowed to suggest related cyanophages. Genomic analysis supported the assumption that mobile elements and horizontal transfer participate in shaping the Tychonema sp. BBK16 genome. The findings of the current research suggest that the aptitude of Tychonema sp. BBK16 for biofilm formation and, possibly, its mixotrophic lifestyle provide adaptation advantages that lead to the successful expansion of this cyanobacterium in the Baikal’s conditions of freshwater lake environments.


Introduction
The global expansion of harmful cyanobacterial blooms in eutrophic waters constitutes a serious threat to freshwater ecology and public health [1,2]. The process of colonisation of Lake Baikal by cyanobacteria, initially named Phormidium spp., was first noticed in 2011 [3]. Further studies showed that this cyanobacterium belonged to the genus Tychonema (family Microcoleaceae, order Oscillatoriales), which was new to Baikal. The potentially toxic genus Tychonema proliferates in the benthos of Lake Baikal and now prevails in the biofilm ulcers of the Baikal sponge and other biofouling. The occupation of new ecological niches by these cyanobacteria raises questions about the biological and genetic mechanisms of such an evolutionary success.
Biofilm formation is an important evolutionary step in the colonisation of new ecological niches [4,5]. Cyanobacterial mats, which are laminated biofilms, are the oldest The purpose of this study was to analyse the biofilm formed by the benthic Tychonema sp. strain BBK16 isolated in the coastal zone of Lake Baikal and to characterise the general genomics of the cyanobacterium, including the analysis of CRISPR spacers and prophagederived regions. Another intention of this research was to investigate the possibility of the mixotrophy of Tychonema sp. BBK16, using bioinformatic analysis, to reveal the mechanisms that can assist the expansion of Tychonema in the lake.

Sampling Location
Biofilm samples were obtained in 2015-2016 from the underwater part of the wooden pier located in the area of the Scientific Research Station "Bolshiye Koty" of the Limnological Institute (Bolshiye Koty Settlement, 51.883333 • N, 105.05 • E) ( Figure 1). Native biofilm macro photography was carried out using a Pentax WG-3 GPS camera (Ricoh Imaging, Tokyo, Japan). Moreover, temperate phages that insert their genome into the host chromosome can leave traces of phage infection [33][34][35][36]. The purpose of this study was to analyse the biofilm formed by the benthic Tychonema sp. strain BBK16 isolated in the coastal zone of Lake Baikal and to characterise the general genomics of the cyanobacterium, including the analysis of CRISPR spacers and prophagederived regions. Another intention of this research was to investigate the possibility of the mixotrophy of Tychonema sp. BBK16, using bioinformatic analysis, to reveal the mechanisms that can assist the expansion of Tychonema in the lake.

Sampling Location
Biofilm samples were obtained in 2015-2016 from the underwater part of the wooden pier located in the area of the Scientific Research Station "Bolshiye Koty" of the Limnological Institute (Bolshiye Koty Settlement, 51.883333° N, 105.05° E) ( Figure 1). Native biofilm macro photography was carried out using a Pentax WG-3 GPS camera (Ricoh Imaging, Japan).

Cultivation and Biofilm Visual Analysis
During cultivation, biofilm fragments were washed with sterile Z-8 mineral medium [37], crushed into smaller pieces and placed on agar plates with the same medium in an incubator. The cultivation conditions were as follows: illumination 1200 lux, the light mode consisted of alternating night and day periods 16:8, temperature 11-12 • C. Next, individual trichomes were sterilely excised from agar, suspended in a liquid medium and cultured again to obtain a unialgal culture.
Microphotographs of Tychonema sp. BBK16 were obtained using an Axio Imager light microscope (Carl Zeiss, Jena, Germany). The structure of the biofilm surface was visualised using scanning electron microscopy (SEM). First, thin sterile glasses were introduced into the liquid medium with cyanobacteria, which, after fouling, were fixed with 2% formaldehyde and dehydrated in an ethanol concentration gradient. Subsequently, the glasses with Tychonema biofouling were dried at 40 • C, coated in gold using a Balzers SCD 004 sputter-coater (Bal-Tec AG, Balzers, Liechtenstein) and examined using SEM Quanta 200 (FEI Co., Hillsboro, OR, USA). Scanning laser confocal microscopy studies were carried out on an LSM 710 microscope (Carl Zeiss) equipped with a helium-neon laser (561 nm), which causes the autofluorescence of cyanobacterial pigments. Biofilm images were obtained using ZEN 2010 software (Carl Zeiss), and 3D reconstruction was achieved with Imaris software (Bitplane Scientific Software, Zürich, Switzerland).

DNA Extraction and Sequencing
The total DNA was extracted by enzymatic lysis using lysozyme (Roche, Basel, Switzerland), proteinase K (Thermo Scientific, Waltham, MA, USA) and sodium dodecyl sulphate (VWR Life Science, Radnor, PA, USA), followed by phenol and chloroform (Medigen, Novosibirsk, Russia) extraction [38]. The NEBNextUltra DNA library prep kit for Illumina (New England BioLabs, Ipswich, MA, USA) was used for DNA library construction. DNA samples were sequenced to generate 300 bp paired-end reads using the Illumina MiSeq platform. De novo genome assembly was performed using SPAdes 3.12 [39], binning was conducted using MaxBin 2.0 [40], and the assembly was manually curated using a BLASTN [41] search with default settings against the NCBI Bacterial GenBank Database [42]. The resulting genome was deposited in GenBank (Accession # JAKJHX000000000).

Genome Annotation and Prediction of Gene Functions
The assembled genome was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP) [43] with default settings. Additionally, annotations of all genomic regions shown in this study were manually curated. Manual curation included checking the positions of open reading frames (ORFs) and functional assignment. Checking the ORF positions was conducted using Geneious Prime 2022.0.1 tools (Biomatters, Inc., Auckland, New Zealand) [44] and Gimmer 3.0.2 [45]. Gene functional assignment was performed using the BLAST search with nr/nt database and HMM-HMM motif comparison. The parameters of BLAST search were: BLASTP algorithm, E-value < 1 × 10 −5 and other parameters were default. The HMM-HMM motif comparison was performed using the HHpred server [46] and PDB, SCOPe, CATH and UniProt-SwissProt-viral databases. Pfam domains were identified using pfam_scan 1.6 tool [47] applying default settings. Clusters of orthologous groups of proteins (COGs) were identified using the eggNOG-mapper 2 server [48] applying the "Genomic" settings.

Average Nucleotide Identity Calculations and Phylogenetic Analysis
Cyanobacterial genome and gene sequences were downloaded from the NCBI GenBank. The relevance of species names was checked in Algaebase (https://www.algaebase.org, accessed on 10 January 2023). The average nucleotide identity (ANI) matrix was calculated using ANI/AAI-Matrix Genome-based distance matrix calculator (Kostas lab, Atlanta, GA, USA) and Bio-NJ clustering [49]. Alignments of 16S rDNA sequences and protein sequences were performed with MAFFT 7.48 [50] with default settings and using the L-INS-i algorithm. 16S rDNA and single protein phylogenetic trees were constructed using RAxML-NG 1.1.0 [51] and the raxmlGUI 2.0.10 graphic interface [52] with (-tree rand{10} -bs-trees 1000) settings and applying the best protein model found with ModelTest-NG 0.1.7 [53]. The robustness of the RAxML-NG 1.1.0 trees was assessed using bootstrapping and calculations of transfer bootstrap estimation (TBE) support [54].
Other details of the phylogenetic analysis using concatenated alignments were the same as described above.

Identification of CRISPR Loci and Prophage-Derived Regions
CRISPR loci were identified with MinCED [55] using "-gffFull" settings. Spacer sequences were extracted with MinCED using the "-spacers" settings. Prophage-derived regions were found using the PHASTER server [56] applying default settings. Similarity of genes of supposedly prophage origin was estimated using built-in PHASTER utilities.

Biofilm Characterisation
The fouling that was the source of strain isolation was a dense, leathery, olive-green biofilm covering the underwater surface of the wooden pier ( Figure 2a). SEM visualisation of the structure of a biofilm formed by Tychonema sp. BBK16 in a liquid medium showed that it was based on cyanobacterial filaments intertwined like felt and the presence of dense fascicles of rope-like twisted filaments that imparted heterogeneity to the biofilm surface, as observed in nature ( Figure 2b). Light microscopy showed that the trichomes of the strain were straight and that they were enclosed in thin, transparent polysaccharide sheaths, which strengthened the structure of the biofilm (Figure 2c). Since the sheaths were thin, the cells were clearly visible, even in SEM ( Figure 2b). The autofluorescence of chlorophyll and the phycobiliproteins of cyanobacteria enabled visualisation of the 3D structure of the biofilm using laser scanning microscopy without additional staining (Figure 2d). Confocal microscopy showed that the biofilm consisted of randomly intertwined threads and had a multilayer structure.

General Genomic Features and Intergenomic Comparisons
The total size of the Tychonema sp. BBK16 draft genome is 5,267,730 nucleotides (nt), which is close to the size of the genome of Tychonema bourrellyi FEM_GT703 (5,081,867 nt) isolated from a freshwater sample taken from Lake Garda [57] and less than the genome size of other Tychonema genomes contained in the NCBI GenBank Database ( Table 1). The GC-content of 44.3% is close to the GC-content of other Tychonema genomes and is slightly closer to the GC-content of T. bourrellyi FEM_GT703 (44.7%) ( Table 1). All Tychonema genomes were deposited as draft sequences. The average nucleotide identity (ANI) calculations were performed using all available genomes of Tychonema and other related cyanobacterial strains found with BLAST search using BBK16 rDNA sequences. The maximum ANI value of about 91% was found for the Tychonema sp. BBK16-T. bourrellyi FEM_GT703 pair (Supplementary Figure S1). This value is lower than the 95-96% ANI cutoff, the standard most frequently used for prokaryotic species demarcation using complete or nearly complete genomes [58][59][60]. The next highest ANI values of about 83% corresponded to other Tychonema strains and Microcoleus sp. LEGE 07076. This value is higher than the estimated prokaryotic mean demarcation boundaries of the genus, which is about 74% [61].

Phylogenetic Analyses
Phylogenetic analysis was performed using 16S rDNA and concatenated alignments of orthologous proteins. The 16S rDNA representative sequences were selected using the most similar sequences found with the BLAST search and NCBI nt databases using the sequences of Oscillatoriales species mentioned in [62] and sequences of more distantly related organisms used in [63]. The 16S rDNA phylogenetic tree ( Figure 3) places all the Tychonema strains and several Microcoleus, Phormidium and Oscillatoria strains into a clade with a TBE support of 0.81. Phylogenetic analysis using the PhyloPhlAn pipeline, which employs 400 most conserved proteins, can show better resolution and higher bootstrap support. The PhyloPhlAn phylogenetic tree ( Figure 4) using the sequences representing most of the available Tychonema/Microcoleus/Phormidium full genomic sequences and other cyanobacterial groups placed Tychonema sp. BBK16 and T. bourrellyi FEM_GT703 into a distinct clade and depicted a group of Tychonema strains as a paraphyletic group.

General Proteome Features
The Tychonema strains deposited in GenBank clearly exhibit diversity in genome size and the number of predicted proteins (Table 1). There are 4708 protein-coding sequences predicted in the BBK16 genome, which is close to the number of proteins encoded in the FEM_GT703 genome (4629) and less than in other Tychonema genomes. Comparison of the

General Proteome Features
The Tychonema strains deposited in GenBank clearly exhibit diversity in genome size and the number of predicted proteins (Table 1). There are 4708 protein-coding sequences predicted in the BBK16 genome, which is close to the number of proteins encoded in the FEM_GT703 genome (4629) and less than in other Tychonema genomes. Comparison of the distribution of clusters of orthologous groups of proteins (COGs) of Tychonema and several other cyanobacterial strains indicated that the number of orthologous groups assigned to categories V (defence mechanisms), N (cell motility), L (replication, recombination and repair), K (transcription) and to several other categories in Tychonema sp. BBK16 was similar to that in T. bourrellyi FEM_GT703, seemingly indicating the relatedness of these species ( Figure 5).
Viruses 2023, 15, x FOR PEER REVIEW 10 of 20 distribution of clusters of orthologous groups of proteins (COGs) of Tychonema and several other cyanobacterial strains indicated that the number of orthologous groups assigned to categories V (defence mechanisms), N (cell motility), L (replication, recombination and repair), K (transcription) and to several other categories in Tychonema sp. BBK16 was similar to that in T. bourrellyi FEM_GT703, seemingly indicating the relatedness of these species ( Figure 5). Transposases are of particular importance for bacteria. In freshwater, cyanobacteria provide the basis for rapid adaptation and survival in harsh freshwater environments [64]. The Tychonema sp. BBK16 genome content analysis also indicated the presence of 29 transposase-encoding genes, which is somewhat lower than in genomes of other analysed Tychonema strains (32-42 genes) and noticeably lower than in Microcoleus sp. LEGE 07076 (52 genes) ( Table 2). Interestingly, a similar situation is observed for restriction modification enzymes, which are important for protecting the cell from phages [65,66]. The Tychonema sp. BBK16 genome contains 62 genes of restriction modification enzymes, while other analysed Tychonema strains have 81-110 genes, and Microcoleus sp. LEGE 07076 has 110 such genes ( Table 2). Transposases are of particular importance for bacteria. In freshwater, cyanobacteria provide the basis for rapid adaptation and survival in harsh freshwater environments [64]. The Tychonema sp. BBK16 genome content analysis also indicated the presence of 29 transposase-encoding genes, which is somewhat lower than in genomes of other analysed Tychonema strains (32-42 genes) and noticeably lower than in Microcoleus sp. LEGE 07076 (52 genes) ( Table 2). Interestingly, a similar situation is observed for restriction modification enzymes, which are important for protecting the cell from phages [65,66]. The Tychonema sp. BBK16 genome contains 62 genes of restriction modification enzymes, while other analysed Tychonema strains have 81-110 genes, and Microcoleus sp. LEGE 07076 has 110 such genes ( Table 2).

Mixotrophy-Associated Proteins
The ability to assimilate organic nutrients detected in both marine and freshwater cyanobacteria is related to transporters needed for the uptake of organic compounds [12,67]. Pfam domain search predicted that the genome of Tychonema sp. BBK16 contains four genes to encode the proteins containing sugar-transporter-like domains. Experimentally, the uptake of sugars in cyanobacteria has been shown to be associated with the presence of GlcH permease importer, a high-affinity glucose transporter (called "Pro1404" in Prochlorococcus marinus SS120, where it was first studied) [68][69][70] and GlcP permease [71,72].
The BLAST search conducted using the Pro1404 amino acid sequence, NCBI nr database and 50 cyanobacterial genomes used in phylogenetic analysis (Figure 4) indicated the presence of genes encoding GlcH in all 50 genomes mentioned above and in numerous other cyanobacteria (E-value < 1 × 10 −5 ). Some of the 50 genomes contained two or three copies of the glcH-like gene. These results are consistent with a result published earlier using another dataset [12]. The genomes of all the analysed strains of Tychonema, Microcoleus and Kamptonema carry a single copy of the glcH-like gene.
Phylogenetic analysis using GlcH-like amino cyanobacterial acid and sequences of the closest homologues from other bacterial taxa indicate the complex evolutionary history of this protein (Supplementary Figure S2). The large clade containing Pro1404 also includes sequences from noncyanobacterial taxa, and some strains appear to carry genes resulting from duplication events.
Glucose:H+ symporters GlcP were searched in a similar way but using a sequence of experimentally studied permease from Nostoc punctiforme [72,73]. In contrast, genes encoding GlcP-like proteins have only been found in 14 of the 50 genomes mentioned above. Interestingly, no genomes of Tychonema, with the exception of BBK16, carry the glcP gene. Phylogenetic analysis indicated, with high bootstrap support (TBE 0.93), a close relatedness of Tychonema sp. BBK16, Microcoleus sp. FACHB-1, Nostoc punctiforme PCC 73102 and Pleurocapsa sp. CCALA 161 proteins ( Figure 6). The phylogeny of GlcP may indicate multiple events of horizontal transfer accompanying the evolution of GlcP, which is consistent with a result published earlier [72]. The genome of Tychonema sp. BBK16 encodes multiple amino acid ABC transporters. These are proteins that can participate in the import of amino acids, which can be a source of carbon and nitrogen [12]. A homology search revealed that most of the transporters discussed in meticulous work on mixotrophy [12], probably involved in the uptake of amino acids and other organic compounds, are encoded in the genome of BBK16 and other Tychonema strains. The genome contains the aapJQMP locus encoding the amino acid The genome of Tychonema sp. BBK16 encodes multiple amino acid ABC transporters. These are proteins that can participate in the import of amino acids, which can be a source of carbon and nitrogen [12]. A homology search revealed that most of the transporters discussed in meticulous work on mixotrophy [12], probably involved in the uptake of amino acids and other organic compounds, are encoded in the genome of BBK16 and other Tychonema strains. The genome contains the aapJQMP locus encoding the amino acid transport system (contig JAKJHX010000121.1), the phnECD locus encoding the phosphonate transport system (contig JAKJHX010000165.1), homologues of glnQ, the gene encoding glutamine transport system ATP-binding protein and homologues of proV and proW genes encoding the transporters participating in the uptake of dimethylsulfoniopropionate (DMSP).

Analysis of CRISPR Loci
Analysis of Tychonema sp. BBK16 and other cyanobacterial genomes revealed the presence of CRISPR-Cas phage defence systems (Figure 7). The analysed systems belong to class 1, which encodes multisubunit effector complexes. The organisation of loci indicated its belonging to type I and subtype I-D, typical of cyanobacteria [74,75]. The sequence search indicated that most Tychonema Cas proteins have close homologues in other cyanobacteria. Interestingly, the Tychonema sp. BBK16 CRISPR locus contains two hypothetical protein genes downstream of the cas3 gene. Other analysed cyanobacterial genomes CRISPR loci may also contain additional inserted genes, including transposases. In some genomes, the order of the cas genes may be different, presumably due to recombinations. The size of the Tychonema sp. BBK16 CRISPR arrays (2906 bp) is similar to that of Tychonema bourrellyi FEM_GT703 (2978 bp).
Viruses 2023, 15, x FOR PEER REVIEW 13 of 20 transport system (contig JAKJHX010000121.1), the phnECD locus encoding the phosphonate transport system (contig JAKJHX010000165.1), homologues of glnQ, the gene encoding glutamine transport system ATP-binding protein and homologues of proV and proW genes encoding the transporters participating in the uptake of dimethylsulfoniopropionate (DMSP).

Analysis of CRISPR Loci
Analysis of Tychonema sp. BBK16 and other cyanobacterial genomes revealed the presence of CRISPR-Cas phage defence systems (Figure 7). The analysed systems belong to class 1, which encodes multisubunit effector complexes. The organisation of loci indicated its belonging to type I and subtype I-D, typical of cyanobacteria [74,75]. The sequence search indicated that most Tychonema Cas proteins have close homologues in other cyanobacteria. Interestingly, the Tychonema sp. BBK16 CRISPR locus contains two hypothetical protein genes downstream of the cas3′ gene. Other analysed cyanobacterial genomes CRISPR loci may also contain additional inserted genes, including transposases. In some genomes, the order of the cas genes may be different, presumably due to recombinations. The size of the Tychonema sp. BBK16 CRISPR arrays (2906 bp) is similar to that of Tychonema bourrellyi FEM_GT703 (2978 bp). The CRISPR array of Tychonema sp. BBK16 contains 39 spacers. The BLAST search using NCBI GenBank Phage database indicated similarities of spacer sequences with genomic sequences of several dozens of isolated cyanophages, but there were no exact matches found. According to the results of the search, 8 spacers demonstrated higher similarity to cyanophages, and 31 spacers were more similar to other phages (Table 3). The CRISPR array of Tychonema sp. BBK16 contains 39 spacers. The BLAST search using NCBI GenBank Phage database indicated similarities of spacer sequences with genomic sequences of several dozens of isolated cyanophages, but there were no exact matches found. According to the results of the search, 8 spacers demonstrated higher similarity to cyanophages, and 31 spacers were more similar to other phages (Table 3).

Search for Prophage-Derived Regions
Search for prophages-derived regions (PDRs) in the genome of Tychonema sp. BBK16, performed with PHASTER, revealed two regions ( Figure 8). Examination of genome content indicated the absence of structural proteins and the presence of transposases in both PDRs. Homologous genes were found in Nodularia phage vB_NspS-kac65v151 (NCBI accession NC_048756, genus Ravarandavirus), Synechococcus phage S-CBP2 (NC_025455, family Autographiviridae), Synechococcus phage S-CBS2 (NC_015463, unclassified siphovirus) and other phages. The presence of transposase can indicate genetic exchanges involving phage and host genomes.

Transporter Genes in Phage Genomes
To check the assumption of the involvement of bacteriophages in the transmission of transporter genes, including genes that may be useful for a mixotrophic lifestyle, the Tychonema sp. BBK16 mixotrophy-associated proteins were used for searching the homologous sequences in phages using the NCBI nt database. In addition, all GenBank phage database sequences (about 33 thousand sequences as of January 2023) were checked for the presence of transporter annotations.
The glcH and glcP genes were not found in the genomes of isolated cyanophage, but homologues GlcH and GlcP were found to be encoded in several phage human metagenomic sequences, including sequences MK231526.1 and BK032514.1. ABC transporter ATP-binding protein genes probably related to glnQ were found in the complete genome of isolated freshwater temperate cyanophage Planktothrix phage PaV-LD [27] and partial genomic sequences of another temperate freshwater cyanophage AS-1 [76]. Interestingly, sequences with a pairwise identity of 97% and above with the Planktothrix phage PaV-LD ATP-binding protein gene were found in several Planktothrix cyanobacterial genomes (Supplementary Figure S3), indicating that Planktothrix phage PaV-LD can represent a group of related temperate phages. Twenty other complete GenBank genomes of tailed cyanophages contain the genes annotated as ABC transporters, basically encoding the

Transporter Genes in Phage Genomes
To check the assumption of the involvement of bacteriophages in the transmission of transporter genes, including genes that may be useful for a mixotrophic lifestyle, the Tychonema sp. BBK16 mixotrophy-associated proteins were used for searching the homologous sequences in phages using the NCBI nt database. In addition, all GenBank phage database sequences (about 33 thousand sequences as of January 2023) were checked for the presence of transporter annotations.
The glcH and glcP genes were not found in the genomes of isolated cyanophage, but homologues GlcH and GlcP were found to be encoded in several phage human metagenomic sequences, including sequences MK231526.1 and BK032514.1. ABC transporter ATP-binding protein genes probably related to glnQ were found in the complete genome of isolated freshwater temperate cyanophage Planktothrix phage PaV-LD [27] and partial genomic sequences of another temperate freshwater cyanophage AS-1 [76]. Interestingly, sequences with a pairwise identity of 97% and above with the Planktothrix phage PaV-LD ATP-binding protein gene were found in several Planktothrix cyanobacterial genomes (Supplementary Figure S3), indicating that Planktothrix phage PaV-LD can represent a group of related temperate phages. Twenty other complete GenBank genomes of tailed cyanophages contain the genes annotated as ABC transporters, basically encoding the phosphate ABC transporter. A total of 14,353 complete genomes of Caudoviricetes phages deposited in the NCBI Genome database contain 603 genes annotated as different genes of the ABC transporter protein.

Discussion
Benthic biofilms are highly adapted to changing environmental conditions (dramatic changes in environmental parameters and nutrient concentrations) and are absorbers of excess nutrients from the environment [77]. The trichomes of the Baikal strain tightly intertwine and form a biofilm, characteristic of many species of Oscillatoriales cyanobacteria, in which the bacteria are held together by a polysaccharide mucus that is able to withstand harsh environmental conditions that might involve nutrient deficiencies and physical stress [78]. Various types of microscopy have been used to study the morphology of biofilms. SEM enables visualisation of the surface of the film, as well as its layers in cross-section, and confocal microscopy, which examines the film's internal structure [79,80]. The structure of the Tychonema BBK16 biofilm was very similar to the 3D structures formed by its closest relatives, cyanobacteria of genera Microcoleus and Tychonema. Microcoleus and Tychonema form microbial mats in the benthos of lakes and rivers [77,81].
The genomic analysis showed a similar genome size and GC-content of Tychonema sp. BBK16 and Tychonema bourrellyi FEM_GT703. Calculations of ANI using all available Tychonema genomes and genomes of related cyanobacterial strains indicated that Tychonema sp. BBK16 may be classified as a representative of a new species. Phylogenetic analysis using concatenated alignments of orthologous protein sequences placed Tychonema sp. BBK16 and T. bourrellyi FEM_GT703 in a distinct clade and showed a complex picture of taxonomic relations between the strains classified as belonging to the Microcoleaceae family of the Oscillatoriales order. The proteome analysis also indicated close relatedness of Tychonema sp. BBK16 and T. bourrellyi FEM_GT703.
Previously, representatives of the genus Tychonema capable of forming biofilms were described in karst streams in a community with heterotrophic bacteria [82]. It has been shown that during the destruction of organic substances at the bottom of streams, a significant amount of organic matter is released, which is consumed by cyanobacteria in the lower layers of biofilms, where cells far from light are not able to photosynthesise. The bioinformatic analysis of the Tychonema sp. BBK16 genome indicated the presence of genes that can participate in the import of organic compounds. One of the sugar uptake genes, glcP, could be obtained by horizontal transfer. The Tychonema sp. BBK16 genome also contains clusters of genes of the aapJQMP amino acid transport system, the phnECD phosphonate transport system and other nutrient import genes. We hypothesise that cyanobacterial mixotrophy may be a factor for survival for Tychonema sp. BBK16 in benthic biofilms. It can be assumed that cyanobacteria of this genus are well adapted to the harsh conditions of Lake Baikal. The presence of protective polysaccharide sheaths, the ability to form dense biofilms and, probably, the capability of mixotrophy helped this species to occupy different ecological niches within the lake. We can also speculate that some genes associated with mixotrophy may be transferred by phages or other mobile elements.
It seems that there are no published isolated Tychonema phages at the moment. However, bioinformatic analysis indicated the presence of CRISPR-Cas and restriction modification phage defence systems in Tychonema sp. BBK16 genome. The Tychonema sp. BBK16 genome CRISPR-Cas locus belongs to characteristics for cyanobacteria subtype I-D of class 1 and includes a CRISPR array containing 39 spacers. Search for similar phages using the Tychonema sp. BBK16 spacers found no related cyanophages for about 80% of spacers, which can be related to the lack of genomic data on cyanophages [32]. Isolated cyanophages exhibiting similarities in genomic sequences with the CRISPR spacers of Tychonema sp. BBK16 belong to Kyanoviridae, Tamkungvirus and other phages.
Prophage predictions indicated the presence of prophage remnants in the Tychonema sp. BBK16 genome. These regions could be transferred in the genomes with the participation of transposases. Prophage-derived regions contain genes resembling cyanophage homologues in the genomes of Nodularia phage vB_NspS-kac65v151, Synechococcus phage S-CBP2 and Synechococcus phage S-CBS2.

Conclusions
Cyanobacterium Tychonema sp. BBK16 was isolated from the benthic zone of Lake Baikal. In culture, the strain formed biofilms with a felt-like interweaving of trichomes. Long fascicles of twisted trichomes were observed on the surface of the film, which caused folding. The ability to synthesise polysaccharide sheaths enabled the formation of more durable biofilms. Confocal microscopy showed the layered structure of the Tychonema biofilm. The genome of BBK16 was sequenced and analysed. The results of intergenomic comparisons and phylogenetic analyses indicate that Tychonema sp. BBK16 represent a new species related to Tychonema bourrellyi strain FEM_GT703. Genomic analysis indicated the presence of mixotrophy-associated genes. Analysis of CRISPR spacers and prophagederived regions revealed cyanophages possibly related to hypothetical Tychonema phages.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/v15020442/s1, Figure S1: ANI matrix constructed with cyanobacterial 50 genomes using ANI/AAI-Matrix Genome-based distance matrix calculator. The NCBI taxonomy is shown to the right of the organism's name, Figure S2