In Silico Analysis of the Metabolic Potential and Niche Specialization of Candidate Phylum "Latescibacteria" (WS3)

The “Latescibacteria” (formerly WS3), member of the Fibrobacteres–Chlorobi–Bacteroidetes (FCB) superphylum, represents a ubiquitous candidate phylum found in terrestrial, aquatic, and marine ecosystems. Recently, single-cell amplified genomes (SAGs) representing the “Latescibacteria” were obtained from the anoxic monimolimnion layers of Sakinaw Lake (British Columbia, Canada), and anoxic sediments of a coastal lagoon (Etoliko lagoon, Western Greece). Here, we present a detailed in-silico analysis of the four SAGs to gain some insights on their metabolic potential and apparent ecological roles. Metabolic reconstruction suggests an anaerobic fermentative mode of metabolism, as well as the capability to degrade multiple polysaccharides and glycoproteins that represent integral components of green (Charophyta and Chlorophyta) and brown (Phaeophycaea) algae cell walls (pectin, alginate, ulvan, fucan, hydroxyproline-rich glycoproteins), storage molecules (starch and trehalose), and extracellular polymeric substances (EPSs). The analyzed SAGs also encode dedicated transporters for the uptake of produced sugars and amino acids/oligopeptides, as well as an extensive machinery for the catabolism of all transported sugars, including the production of a bacterial microcompartment (BMC) to sequester propionaldehyde, a toxic intermediate produced during fucose and rhamnose metabolism. Finally, genes for the formation of gas vesicles, flagella, type IV pili, and oxidative stress response were found, features that could aid in cellular association with algal detritus. Collectively, these results indicate that the analyzed “Latescibacteria” mediate the turnover of multiple complex organic polymers of algal origin that reach deeper anoxic/microoxic habitats in lakes and lagoons. The implications of such process on our understanding of niche specialization in microbial communities mediating organic carbon turnover in stratified water bodies are discussed.


Introduction
Over the past few decades, small subunit ribosomal RNA (SSU or 16S rRNA) gene-based surveys have prompted a drastic reevaluation of the scope of phylum level diversity within the domain Bacteria. Current taxonomic outlines indicate that the majority of recognized bacterial phyla (54.1% using SILVA database [1], 65.48% using Greengenes database [2]) have no pure culture representatives (candidate phyla). Many of these candidate phyla, so-called microbial dark matter (MDM) are globally distributed and display significant levels of intra-phylum level diversity [3][4][5][6][7]. Recent advances in cell sorting and whole genome amplification and assembly have facilitated the acquisition of single-cell amplified genomes (SAGs) derived from numerous candidate phyla [8][9][10][11][12][13][14][15][16]. Metabolic reconstruction with these SAGs provides a unique opportunity to uncover the ecological and biogeochemical roles played by these enigmatic microbial groups.
However, little is known about the biological capabilities of this phylum, and no systematic attempts have been made to reconstruct its metabolic potential. Thus, we here present a detailed analysis of the metabolic and physiological capabilities, and putative ecological roles of four "Latescibacteria" SAGs obtained from two different aquatic environments. Our analysis suggests that the "Latescibacteria" recovered from Sakinaw Lake and Etoliko lagoon transform algal detritus sinking from sunlit surface waters into fermentation products with the potential to contribute to microbial food webs in anaerobic waters below.
Detailed analysis was conducted on S-E07, which has the highest estimated genome completion (73.02%) among the "Latescibacteria" SAGs. The closely related S-B13 (94% 16S rRNA gene sequence similarity to SAG S-E07) with 57.1% estimated genome completion was used to confirm shared gene content and fill pathway holes when needed. Only general metabolic features for SAG S-D10 (94% 16S rRNA gene sequence similarity to S-E07, and 96% to S-B13) are discussed, given its low percentage of estimated genome completeness (38.2%). Due to the observed differences between the 3 Sakinaw Lake SAGs, and the Etoliko lagoon SAG E-K07 (85-86% 16S rRNA gene sequence similarity to Sakinaw Lake SAGs), as well as its low estimated genome completion (23.02%), analysis of SAG E-K07 was restricted to identifying variation in conserved genes or pathways between "Latescibacteria" SAGs from two distinct locations.

Genome annotation, general genomic features, and metabolic reconstruction
The IMG platform (http://img.jgi.doe.gov) was used for genome functional annotation. Detailed metabolic reconstruction of relevant pathways was performed with both KEGG [30] and Metacyc [31] databases. As part of the IMG annotation pipeline, CRISPR elements are detected with CRT [32] and PILERCR [33]. Predictions from both methods are concatenated and in case of overlapping elements, the shorter one is removed. Overall annotation followed procedures outlined in [13]: In brief, proteases, peptidases, and protease inhibitors were identified with Blastp against the Merops database [34]. Transporters were identified with the transporter classification database (TCDB) [35]. dbCAN HMMs [36] were used to identify carbohydrate active enzymes (CAZymes) including glycoside hydrolases (GH), polysaccharide lyases (PL), and carboxyl esterases (CE) following the classification scheme of the Carbohydrate active enzyme (CAZy) database [37].

Results
Phylogenetic affiliation and general genomic features of "Latescibacteria" SAGs All four SAGS were affiliated with the candidate order PBS_III_9 based on phylogenetic analysis of the candidate phylum "Latescibacteria" using 1198 near-full length 16S rRNA gene sequences (Fig 1, Table A in S1 File, and Supplementary Text in S1 File). Sakinaw lake SAGs belonged to family I, while Etoliko lagoon SAG E-K07 belonged to family VI within this order ( Fig 1B). General genomic features for each SAG are shown in Table 1.

Metabolic potential of Sakinaw Lake SAGs
Anabolic pathways identified in S-E07 and S-B13 include machinery for the production of amino acids, cofactors, fatty acids, purines and pyrimidines, terpenoid unit backbone, and glycerophospholipids. In addition, the SAGs encode near-complete replication, transcriptional, and translational machineries. The presence of genes for lipopolysaccharide (LPS) biosynthesis and pathway for LPS insertion in the outer membrane suggests a Gram-negative cell wall (Supplementary Text).
Catabolical pathways identified in S-E07 and S-B13 indicate a heterotrophic lifestyle. Moreover, the apparent absence of a respiratory chain suggests sole dependence on fermentative pathways and substrate level phosphorylation for coupled energy release and conservation. Both S-E07 and S-B13 encode a diverse array of carbohydrate active enzymes (CAZymes), with a conspicuous enrichment (Genes/Mbp), and diversity (number of different families) of polysaccharide lyases (PLs) (Fig 2, Figure A in S1 File, Table B in S1 File). In contrast, the SAGs are relatively depauperate in genes encoding glycoside hydrolases (GHs) including enzymes involved in the degradation of cellulose (1 putative endoglucanase (GH5), 3 putative β-glucosidases (GH3, GH116, and GH9), and no putative cellobiohydrolase), and enzymes involved in the degradation of xylans (xylanases, andβ-xylosidases).  Interestingly, many of the polymers that S-E07 and S-B13 are predicted to degrade are integral components of cell walls of the green algal phyla Charophyta (most commonly encountered in freshwater habitats), and Chlorophyta (widely distributed in freshwater, marine, and terrestrial habitats), as well as the brown algal Class Phaeophyceae. Green and brown algal cell walls are complex, with a diverse array of structural fibrillar polymers enmeshed in complex matrices with crystalline polymer components (Fig 3). Both S-E07 and S-B13 encode genes necessary for the conversion of these cell wall components, including pectin, alginate, ulvans, fucans, hydroxyproline-rich glycoproteins (HRGP), e.g. arabinogalactan proteins (AGP) and extensins, and xyloglucan (Table 2, Figure B in S1 File, Supplementary Text in S1 File). Moreover, the SAGs also encode pathways mediating the conversion of soluble organic compounds commonly utilized for storage in algae (e.g. starch and trehalose). A more in depth description of these capabilities follows.
Algal cell wall degradation potential 1. Pectins. Pectins are components of the amorphous matrix and outer lattice of Charophyta cell wall (Fig 3A) [38]. Both S-E07 and S-B13 encode machinery for depolymerizing the pectic polysaccharide homogalacturonan (HG) ( Table 2). They encode carboxyl esterases (CE8 and CE12) for the removal of accessory acetyl and methyl groups attached to the backbone, pectin lyase and pectate lyase (PL1, and PL10) to breakdown the backbone to oligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at their non-reducing ends, exopolygalacturonate lyase (PL9) to cleave digalacturonate unit, and oligogalacturonide lyase (PL22) to degrade the digalacturonate units to 5-dehydro-4-deoxy-D-glucuronate and galacturonic acid as the final end products of HG degradation [39,40]. In addition to HG, S-E07 and S-B13 encode all  [43]. Within the Charophyta (A), the wall is formed of an inner fibrillar layer made of cellulose microfibrils. The fibrillar layer is enmeshed in and surrounded by a middle amorphous matrix of pectin (homogalacturonan, HG, and rhamnogalacturonan I, RGI) that anchors the inner fibrillar cellulose layer to an outer lattice of homogalacturonan. Extracellular polymeric substances or mucilages are also present outside the outer lattice [38,43,101]. Similarly, cell walls of Chlorophyta (B) contain skeletal polysaccharides enmeshed in a matrix. However, the skeletal polysaccharides in Chlorophyta cell walls form double fibrillar layers (inner layer and outer layer) with an amorphous matrix in between. The fibrillar layers vary in composition between cellulose, β-1,3-xylans or β-1,4-mannans or complex heteropolymers, and are rich in hydroproline-rich glycoprotein such as extensins and AGPs. The amorphous matrix polysaccharides are generally in the form of ulvans (e.g. in Ulva species). Brown algal cell walls (C) consist of a fibrillar framework of cellulose microfibrils present in layers parallel to the cell surface but with no clear orientation within each layer. Two such layers are depicted in the figure. All cellulose layers are enmeshed in acidic polysaccharides, e.g. alginates. The interfibrillar matrices are composed of alginates and fucans [41,43].   Single Cell Genomics of the "Latescibacteria" the necessary machinery to degrade rhamnogalacturonan I (RGI) ( Table 2). These include carboxyl esterases (CE8 and CE12), rhamnogalacturonan endolyase (PL11) that attack the backbone to produce oligosaccharides with L-rhamnopyranose at the reducing end and 4-deoxy-4,5-unsaturated D-galactopyranosyl uronic acid at the non-reducing end, rhamnogalacturonan exolyase (PL11) that attacks those oligosaccharides to release the disaccharide 2-O-(4-deoxybeta-L-threo-hex-4-enopyranuronosyl)-alpha-L-rhamnopyranose from the reducing end, and d-4,5-unsaturated β-glucuronyl hydrolase (GH88) that degrades those disaccharides to rhamnose and 5-dehydro-4-deoxy-D-glucuronate. The SAGs also encode β-galactosidase (GH42) for removal of galactosyl sugar substitutions [39,40].
4. Ulvans. Ulvans are present in the amorphous interfibrillar matrix of Chlorophyta cell walls (Fig 3B) [43][44][45]. Ulvan backbones are made of a few repeating disaccharides (Supplementary Text). However, the exact composition of ulvans is largely unknown. One important characteristic of ulvans is the presence of unusual sugars, e.g. iduronic acid, in its backbone [44]. Iduronic acid is also an important constituent of mammalian glycosaminoglycans (GAGs), e.g. heparan sulfate, dermatan sulfate, heparin [46]. "Latescibacteria" SAGs harbor several PLs annotated as heparin and heparan lyase (PL12 and PL21). Structural similarity in sugar composition between ulvans and mammalian GAGs such as heparin suggest that those polysaccharide lyases (annotated as PL12 and PL21 with heparinase activity) might be potential ulvan lyases responsible for ulvan backbone cleavage to produce di-and tetrasaccharides with an unsaturated β-glucuronyl residue located at the non-reducing end [47]. SAGs also harbor several copies of unsaturated glucuronyl hydrolases (GH88) that could potentially act on the oligosaccharides produced and release 5-dehydro-4-deoxy-D-glucuronate and other sugar residues, e.g. rhamnose, and xylose, as end products.
8. Sulfatase activity on sulfated polysaccharide. Both S-E07 and S-B13 encode multiple sulfatases (n = 14 in S-E07 and n = 3 in S-B13) belonging to the family of arylsulfatases (pfam 00884). Many of the polymers in marine algal cell walls are sulfated, e.g. ulvans, homofucans, sulfated-xylofucoglucan, and sulfated-xylofucoglucoronan [58]. Removal of the sulfate groups from such polysaccharides prior to their degradation facilitates access of GHs and PLs to side chains and backbones [59]. The SAGs also harbor the essential anaerobic sulfatase maturation enzyme-coding gene [60] for post-translational modification of a critical Cys or Ser in the active site to a C-α-formylglycine [61].

Degradation of algal storage compounds and additional polymers of non-algal origin
In addition to algal cell wall components, both S-E07 and S-B13 encode GHs that could potentially target algal intracellular carbon storage compounds, or secreted polysaccharides sourced from other organisms. The SAGs encode GHs specific for starch (α-amylase belonging to GH119, GH57, GH13, and α-glucosidase belonging to GH97), as well as for trehalose (trehalase/maltase belonging to GH65) degradation. Starch is recognized as an important intracellular storage compound in green algae and green plants [62], while trehalose is an intracellular storage compound in brown algae [41]. In addition, S-E07 and S-B13 encode β-fructofuranosidase (GH32) specific for sucrose, and endo1,4-poly-D-galactosaminidase (GH114) specific for poly-D-galactosamine (Table 2). Extracellular polymeric substance (EPS) as additional potential source of energy for the "Latescibacteria" EPS forms extensive mucilaginous sheath outside the algal cell wall and function in adhesion, gliding motility, biofilm formation, and protection. Although the exact chemistry of EPS is not entirely known, EPS was shown to be composed mainly of polysaccharides (up to 75%), with minor protein content (2-10%). The polysaccharide fraction is rich in uronic acids, as well as monosaccharides, mainly glucose, galactose, mannose, xylose, arabinose, fucose, and rhamnose [63,64]. As mentioned above, "Latescibacteria" SAGs harbor genes involved in the uptake and catabolism of all such components.
"Latescibacteria" SAGs harbor extensive transport systems for sugars, and amino acids/oligopeptides uptake Both S-E07 and S-B13 encode several non-specific porins for transport of substrates across the outer membrane, coupled to specialized transporters in the inner membrane, including multiple secondary (symport), ABC (ATP-binding cassette), and phosphotransferase system (PTS) transporters for the uptake of a wide array of monomers, e.g. those putatively produced from the degradation of all polymers described above (Fig 4, Table 2). Uronic acids and uronic acid derivatives are potentially imported using a single common transporter (a sugar phosphate permease transporter of the major facilitator superfamily similar to ExuT transporter of Ralstonia solanacearum [65]). Fucose, rhamnose, as well as xylose are potentially imported via dedicated proton symporters, while glucose and galactose are potentially imported via dedicated sodium symporters. Moreover, the SAGs encode components of dedicated ABC transporters for arabinose, ribose, and oligopeptides and dipeptides as well as components of the PTS specific for N-acetylgalactosamine, fructose, and mannose import. The SAGs also encode a complete two-component signal transduction system for sensing di/tricarboxylates, e.g. malate, citrate, (DctBD), as well as a tripartite ATP-independent di/tricarboxylate transport system (TRAP) (DctPQM) [66].

Catabolism of imported sugars
Both S-E07 and S-B13 encode extensive pathways for the catabolism of a wide array of sugars, sugar acids, amino sugars, amino acids, as well as citrate and malate. Monomer degradation pathways in the SAGs are predicted to converge on one of three central metabolic routes, (i) feeding into the EMP pathway (for glucose, galactose, mannose, fructose, sugar acids, amino sugars, aspartate, and citrate and malate), (ii) feeding into PPP (for xylose, ribose, and arabinose), or (iii) the special fucose and rhamnose degradation pathways to propionate and propanol.
Monomer catabolism is detailed in the supplementary text and Fig 5. Briefly, the genomes encode a complete glycolytic pathway for metabolism of various C6 sugars to pyruvate, including glucose, galactose, mannose, and fructose and the amino sugars N-acetylgalactosamine, Nacetylglucosamine, and D-galactosamine. The genomes also encode the necessary enzymes for channeling the C6 sugar acids galacturonic acid, glucuronic acid, and 5-dehydro-4-deoxy-Dglucuronate to the central metabolite 2-dehydro-3-deoxy-D-gluconate (KDG), which is subsequently converted to pyruvate and glyceraldehyde-3-phosphate (GAP), that feed into the EMP. In addition, the amino acid aspartate, as well as dicarboxylates (malate) and tricarboxylates (citrate) that could potentially serve as C and energy source are catabolized via conversion to oxaloacetate and subsequently to phosphoenolpyruvate (PEP). On the other hand, the C5 sugars xylose, ribose, and arabinose are metabolized via the non-oxidative branch of the pentose phosphate pathway by first conversion to xylulose-5-P. Collectively, the metabolism of these compunds via the EMP or the PPP results in the production of pyruvate. Pyruvate could potentially be converted to acetyl-CoA via the action of pyruvate:ferredoxin oxidoreductase. Indeed, as indicated previously, the SAGs encode the machinery necessary for substrate-level phosphorylation including acetyl CoA synthase, as well as propanediol transacetylase and acetate kinase, both of which convert acetyl-CoA to acetate with concomitant ATP production (Fig 5).
Fucose and rhamnose metabolism requires a different catabolic pathway and partially occurs in an intracellular bacterial microcompartment (BMC) to protect against cellular damage  Table 2, results in the production of monomers that could potentially be transported across the outer membrane (OM) of "Latescibacteria" cell wall through non-specific outer membrane porins (OMP). In the periplasm, those monomers are then transported across the inner membrane (IM) via dedicated transporters including (1) Secondary transporters: glucosamine (GluA), galactosamine (GalA), and 5-dehydro-4-deoxy-glucosamine (5-dehydro-4-deoxy-GluA) are potentially imported using a single common transporter ExuT. Fucose (Fuc), rhamnose (Rha), and xylose (Xyl) are imported via dedicated proton symporters, while glucose (Glu), and galactose (Gal) are imported via dedicated sodium symporters. (2) ATP-binding cassette (ABC) transporters: ribose (Rib) and arabinose (Ara) sugars, as well as oligopeptides and dipeptides have dedicated ABC transporters with specific periplasmic substrate binding protein (SBP), two membrane permeases (P), and an ATPase. And (3) Phosphotransferase system (PTS) transporters: mannose (Man), fructose (Fru), galactosamine (GalN), and N-acetyl galactosamine (N-Ac-GalN) are imported via dedicated PTS transporters with cytoplasmic enzyme-I component (E-I) and membrane associated enzyme II components (IIA, IIB, and IIC). Sugars are phosphorylated during this kind of transport. The SAGs also encode a dedicated signal transduction system, and a tripartite ATP-independnent transporter (TRAP) for sensing, and importing, respectively, dicarboxylates, e.g. malate, and tricarboxylates, e.g. citrate, across the inner membrane. The signal transduction system is composed of the sensor histidine kinase DctB, and the cytoplasmic response regulator DctD, while the TRAP transporter is composed of the periplasmic solute receptor (DctP), the membrane small permease component (DctQ), and the membrane large permease component (DctM). TonB-dependent import of vitamin B12 and iron complexes is also predicted from the SAGs. Several proteins with Plug domains could potentially act as the outer membrane receptor protein for vitamin B12 and iron complexes. Binding of the ligand to the receptor activates TonB-dependent import across the outer membrane via three proteins TonB, ExbB, and ExbD, that couple proton motive force to ligand transport across the outer membrane. In the periplasm, vitamin B12 or iron complexes are then transported across the inner membrane via a dedicated ABC transporter. doi:10.1371/journal.pone.0127499.g004 Single Cell Genomics of the "Latescibacteria" by containing the reactive intermediate propionaldehyde [67,68]. Both S-E07 and S-B13 encode a dedicated pathway for the degradation of fucose and rhamnose to lactaldehyde and dihydroxyacetone-phosphate. Several genes encoding for BMC structural shell proteins with BMC domains (pfam 00936, as well as pfam 03319) were identified in the SAGs consistent with a recent observation by Axen and colleagues exploring the taxonomic distribution of BMCs across bacterial phyla [69]. Inside the BMC, lactaldehyde is converted to 1,2-propanediol (1, 2-PD). Although homologues for 1,2-PD dehydratase, the enzyme responsible for conversion of 1,2-PD to propionaldehyde, were not identified in S-E07 and S-B13, both SAGs harbor NAD-dependent aldehyde dehydrogenase, and NADH-dependent alcohol dehydrogenase for conversion of propionaldehyde to propionyl-CoA, and propanol, respectively. Propionyl-CoA can then be converted to propionate with the concomitant production of 1 mole of ATP per propionate produced. Single Cell Genomics of the "Latescibacteria" Additional genomic features Both S-E07 and S-B13 encode machinery for pili and flagella production, enabling potential attachment to surfaces [70], as well as gas vesicles production for maintaining a position in the water column with the most favorable growth conditions [71] (Supplementary Text). In addition, the SAGs encode multiple oxidative stress enzymes that counter harmful effects of changing oxygen tension caused by vertical migration in the stratified water column while in pursuit of decaying algal cells or other food particles. These include rubrerythrin, rubredoxin, rubredoxin oxidoreductase, superoxide reductase (desulfoferredoxin), ferritin-like protein, NADPH-dependent alkyl hydroperoxide reductase, and glutathione peroxidase [72], as well as machinery for bacillithiol biosynthesis, a thiol implicated in peroxide sensing [72][73][74][75].

Discussion
Our analysis of four "Latescibacteria" SAGs obtained from the anaerobic monimolimnion water column of Sakinaw Lake, and the anaerobic sediments of Etoliko lagoon revealed extensive saccharolytic and proteolytic capabilities, with preference for specific polysaccharides and glycoproteins such as pectins, alginates, fucans, ulvans, xyloglucans, starch, extensins, and arabinogalactan protein originating from algal cell walls and EPS. While the degradation of some of these polymers (e.g. pectins and alginates) have been fairly well characterized at the genomic, enzymatic, and organismal levels [39,40,42], limited information is available regarding the pathways, genes, and microorganisms mediating the degradation of others (e.g. fucans, ulvans, extensins and arabinogalactan proteins) [44,46,47,53,54,80]. More importantly, our knowledge of the degradation of many of these compounds is based on the study of model aerobic organisms with little knowledge of such pathways in anaerobes.
We argue that the observed patterns of polymer degradation, and monomer/oligomer transport and catabolism reflect niche specialization within "Latescibacteria" for survival and substrate acquisition in aquatic ecosystems. Specifically, we hypothesize that "Latescibacteria" SAGs analyzed in Sakinaw lake and Etoliko lagoon are involved in the degradation of a considerable fraction of algal cell wall polysaccharides and glycoprotein, algal EPS, and algal storage molecules within the detritus of green and brown algae originating at the oxic and photic zones and sinking to the anoxic and aphotic zones through sedimentation. Primary productivity is an important source for organic matter deposited in lakes [81,82]. Algal cells represent up to 90% of such sinking organic matters, especially in stratified lakes like Sakinaw. Prior studies have demonstrated that CO 2 fixation by algae represents the major source of organic carbon input in Sakinaw Lake, with the water column being the main site for the degradation of fixed organic carbon [83]. The stratified nature and lack of upwelling within meromictic lakes results in greater accumulation of organic matter into the lake's deeper anoxic layers [84]. The overall contribution of algal detritus to lacustrine sediments is often enhanced by the frequent occurrence of algal blooms, an ecological phenomenon predicted to increase due to global warming trends, and the progressive increase in fertilizers usage [85]. This has been reported in the lagoon systems of Western Greece, where the occurrence of algal blooms and subsequent sedimentation of organic matter represent one of the driving forces for the observed progressive eutrophication and anoxia within this ecosystem [86,87].
It should also be noted that, in addition to polymers putatively degraded "Latescibacteria", algal cells are known to produce considerable quantities of oils (up to 60% of their weight), especially under unfavorable conditions (e.g. N and P starvation, temperature, salinity, or pH shifts, or heavy metal accumulation) [88,89]. Interestingly, the analyzed SAGs lack all enzymes of the fatty acid degradation pathway to acetyl CoA. Similarly, cellulose represents an important constituent of green and brown algal cell wall [43], but the analyzed "Latescibacteria" SAGs display an extremely sparse cellulose degradation capacity (Figure A in S1 File). We reason that readily degradable components within algal detritus, e.g. cellular lipids and fatty acids, free proteins, and cellulose, are promptly utilized by microorganisms in the algal phycosphere [90][91][92], as well as by aerobic and anaerobic copiotrophs in the surrounding water column during the sedimentation process. Thus "Latescibacteria" residing in the deeper anaerobic layers of Sakinaw lake and Etoliko lagoon sediments have evolved to specialize in the degradation of the more recalcitrant substrates that accumulate as algal detritus descends to deeper anoxic layers in stratified aquatic ecosystems. Indeed, studies in meromictic lakes have demonstrated that degradation of algal blooms occurs during sedimentation leading to biomass loss and chemical structure alteration of the algal blooms with depth [81,82].
The proposed ecological role for members of the "Latescibacteria" strongly suggests cellular attachment to sinking algal detritus. "Latescibacteria" SAGs encode genes for flagella and pili production, and formation of gas vesicles; traits that could enhance cellular capacity for tracking and attachment to particulate organic matter. A recent survey of microbial communities in the oxygen starved Black Sea with considerable primary productivity within the upper oxic zone, shows higher relative abundance of "Latescibacteria" in particulate-associated samples derived from the deep anoxic zone when compared to water samples from the same location [24].
In addition to the major contribution to sinking organic matter in water bodies, algal biomass degradation under anaerobic conditions has recently received additional attention due to its potential use for biogas production [93][94][95][96][97][98][99]. Surprisingly, little is currently known regarding the microbial community involved in algal biomass degradation under anaerobic conditions [93]. Thus analysis of "Latescibacteria" SAGs directly contributes to our understanding of potential bacterial lineages involved in the anaerobic turnover of algal cell components.
Finally, the "Latescibacteria" SAGs encode numerous biosynthetic capabilities and a rich repertoire of catabolic enzymes and transporters with the potential to promote growth on a large number of substrates. Such capabilities are in contrast to multiple recently obtained genomes of several uncultured bacterial and archaeal CP, where sparse anabolic capabilities, small genome size, and apparent dependence on syntrophic interactions for growth were observed [9,13]. As such, the reported physiological properties (anaerobic nature and predicted slow growth rate due to possession of a relatively large genome size and a single rRNA operon), metabolic capabilities (distinct preference to specific polymers and sugars/sugar acids, auxotrophy to specific amino acids), and ecological distribution (preference to anaerobic and eutrophic habitats) should be considered when designing strategies for the isolation of members of the "Latescibacteria".
Supporting Information S1 File. Supporting Information document containing supplementary text, Tables A-C, and Figures A-B, accompany this manuscript. Table A. Genbank accession numbers, candidate order, and study site of all near-full-length 16S rRNA gene sequences affiliated with "Latescibacteria" that were used to construct phylogenetic trees shown in Fig 1. Table B. Total number of glycosyl hydrolases (GHs), polysaccharide lyases (PLs), and carbohydrate esterases (CEs) in the two most complete "Latescibacteria" SAGs compared to other lignocellulolytic and alginolytic organisms. Table C. Number of peptidases belonging to various Merops peptidase families identified in "Latescibacteria" genomes and their possible physiological roles. Figure A. Total number of "Latescibacteria" genes belonging to the different families of glycosyl hydrolases (GHs) and polysaccharide lyases (PLs) shown on the X-axis for SAGs S-E07 and S-B13. Figure B. Schematic representation of polymers shown in Table 2. (DOCX)

Author Contributions
Conceived and designed the experiments: NY CR TW ME. Performed the experiments: NY IF CR. Analyzed the data: NY IF CR SJH TW ME. Contributed reagents/materials/analysis tools: SJH. Wrote the paper: NY IF ME.