Saccharibacteria (formerly TM7) are a group of environmentally diverse, ultrasmall bacteria with highly reduced genomes belonging to Patescibacteria (formerly Candidate Phyla Radiation), a newly identified bacterial lineage accounting for over a quarter of microbial diversity. Nanosynbacter lyticus strain TM7x was isolated from the human oral cavity and was the first culture representative of Saccharibacteria. It displays an obligate episymbiotic lifestyle where TM7x lives on the surface of its bacterial host Schaalia odontolytica strain XH001. Saccharibacteria rely on host bacteria for growth. TM7x multiplies through budding division, and daughter cells can disassociate from host bacteria during their horizontal transmission stage and establish symbiosis with new bacterial hosts. However, how these metabolically constrained symbionts maintain their viability and infectivity during their horizontal transmission phase, when they are disassociated from hosts, remains poorly understood. By applying targeted mutagenesis using recently developed genetic tools for Saccharibacteria, we demonstrate that the TM7x-encoded arginine deiminase system (ADS) plays a critical role in ATP production and impacts TM7x-host bacterium interaction. Furthermore, we present the first empirical evidence showing that TM7x can uptake and utilize glucose via the glycolysis pathway. Glycolysis is particularly important for episymbiont ATP production under anoxic conditions during horizontal transmission between hosts. Our study demonstrates that TM7x employs two ATP-generating metabolic pathways, ADS and glycolysis, to ensure its viability and infectivity under different microenvironments when disassociated from its hosts during horizontal transmission, a critical phase of its life cycle.

Saccharibacteria are ultrasmall episymbionts that require host-bacteria to grow. They are positively associated with inflammatory diseases within the human microbiome, yet their mechanisms for interacting with the human host and contributing to diseases remain unknown. This study investigated the tripartite interactions between Saccharibacteria (Nanosynbacter lyticus strain TM7x and other strains), their host/non-host-bacteria, and human oral gingival epithelial cells. Both host and non-host-bacteria strongly induce proinflammatory cytokines in epithelial cells, while Saccharibacteria alone elicits limited immune activation. Remarkably, Saccharibacteria dampened proinflammatory cytokine responses to host/non-host-bacteria during coinfection. Mechanistically, this effect results from Saccharibacteria-mediated clustering and endocytosis of surface TLR2 receptor, ultimately leading to reduce TLR2-mediated cytokine signalling. Sacchribacteria type IV pili appendages facilitate epithelial cell binding and subsequent immune dampening via direct interaction between pili adhesins and TLR2. High resolution imaging shows that Saccharibacteria are internalized by epithelial cells through caveolin-mediated endocytosis, subsequently colocalize with endosome markers, and eventually are trafficked to lysosomes for degradation. Moreover, a subset of the Saccharibacteria survives lysosomal degradation and retains the ability to reinfect host-bacteria, highlighting a mechanism for transient persistence in the oral microbiome and a vital role in human immune and microbiome modulation.

Patescibacteria are an elusive linage of "microbial dark matter" bacteria predicted to represent ~25% of total bacterial diversity. Despite this abundance and ubiquity, these organisms are challenging to cultivate, resulting from their specialized episymbiotic lifestyle. All cultivated representatives to date, predominantly composed of Saccharibacteria from the oral microbiome, depend on cognate prokaryotic hosts for growth and reproduction. Studying the growth dynamics of episymbiotic bacteria and their hosts in batch cultures has suggested that many episymbionts initially reduce host populations, and that hosts eventually adapt to episymbiont stress after serial passaging. However, discontinuous batch cultures do not reflect natural interactions between these organisms due to their drastically different growth rates. An episymbiont requires several (~2-4) serial passages alongside its host to reach the high cell densities needed to impact host growth, which complicates investigation of host inhibition and adaptation to episymbiont stress. To describe these dynamics accurately, we utilized continuous culture via small-scale Raspberry Pi powered bioreactors, called Pioreactors. Within a bioreactor, host bacteria can be cultivated at a consistent growth rate indefinitely, providing the perfect substrate for cultivation of model Saccharibacteria. Quantification of time until host crash, crash severity, time until recovery, and stable co-culture density provides mechanistic ways to describe episymbiont-host interactions. First, we used these techniques to compare episymbiont infection by three different episymbionts, revealing distinct infection patterns ranging from mild inhibition with rapid host adaptation, to rapid host collapse followed by "arms race" oscillation dynamics. Then, bioreactors were used to quantify the episymbiotic role played by a known host-binding type 4 pili (T4P-2), demonstrating that loss of long-distance host binding significantly delayed the host crash without altering general crash dynamics. These experiments reveal that episymbionts can have drastically different effects on bacterial communities and provide the tools necessary to describe strain/species differences and molecular interactions.

Abstract Introduction Saccharibacteria (TM7) are episymbiont, that grow on the surface of host bacteria such as Schaalia and Actinomyces (Actinobacteria). They are positively associated with inflammatory diseases within the human microbiome, yet their mechanisms for interacting with the human host and contributing to diseases remain unknown. This study investigated tripartite interactions between TM7, their host/non-host bacteria and their human host. Methods Numerous oral epithelial cell lines were infected with TM7 strains their host/non-host bacteria, and their co-culture, followed by analysis of pro-inflammatory cytokine responses and downstream interaction with epithelial cells. Results TM7 dampened innate immune responses induced by both their host and non-host Actinobacteria in oral epithelial cells. Follow up transcriptomic analysis revealed host bacteria activated TLR2 pathway, while TM7 inhibited it through close interaction between TM7 Type IV pili (T4P) and TLR2 receptor, leading to TLR2 clustering and endocytosis via caveolin, thereby inhibiting host and non-host Actinobacteria induced epithelial innate immune response. Endocytosed TM7 was processed to lysosome, however, a fraction of TM7 persisted and survived to reinfect its host-bacteria upon mechanical lysis. Conclusion This study highlights the role of TM7 in inflammatory diseases and offer novel mechanistic insights into how TM7 influence immune activation and inflammation, highlighting their potential role in shaping the microbiome and contributing to inflammatory disease dynamics. Funding Source National Institute of Dental and Craniofacial Research (NIDCR) under awards 1R01DE031274 (B.B.) and 1R01DE023810 (X.H., J.S.M.). Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

The Patescibacteriota, also known as the Candidate Phyla Radiation (CPR), represent a large lineage of ultrasmall bacteria with highly reduced genomes and obligate dependence on bacterial hosts. Although genomic analyses have revealed CRISPR-Cas and restriction-modification systems in many CPR genomes, no cognate bacteriophages (phages) have been isolated, leaving CPR-phage interactions unexplored. Nanosynbacter lyticus TM7x, the first cultivated CPR bacterium, grows episymbiotically on its host, Schaalia odontolytica XH001, in the human oral microbiome. Here, we identify Xhp1, an inducible prophage of XH001 that is preferentially activated during episymbiosis with TM7x. Released Xhp1 particles infect prophage-free XH001 via distinct strategies determined by host growth mode, establishing lysogeny under planktonic conditions but driving lytic infection during surface-associated growth. Xhp1 also binds efficiently to TM7x and exhibits limited infection under the conditions tested, indicating direct phage-CPR interactions. Importantly, TM7x modulates Xhp1 availability in a spatially dependent manner. In planktonic culture, free-floating TM7x reduces lysogenic conversion of XH001ΔXhp1, consistent with TM7x acting as a phage sink that lowers effective phage concentration. In contrast, during surface-associated growth, TM7x increases XH001ΔXhp1 susceptibility to lytic infection, likely by locally concentrating phage particles within a constrained niche. These results demonstrate that CPR bacteria can regulate viral encounter rates through spatial organization. In spatially structured environments such as oral biofilms, such modulation may shape infection dynamics and community structure. Together, this work characterizes the first CPR-targeting phage and reveals a an important role for phages in CPR-host bacteria interactions.

The recently validly published phylum Minisyncoccota (previously Ca. Patescibacteriota or candidate phyla radiation) had long been an enigma, with members having remained uncultivated for several decades since the first SSU ribosomal sequence was recovered. We reported on the first isolated strain of Minisyncoccota, Nanosynbacter lyticus type strain TM7xT (ATCC TSD-290T), in 2015, using directed cultivation from human oral samples. Strain TM7xT had an extremely reduced genome (705 CDS), ultra-small cell size (200-300 nm), and replicated as an obligate epibiont on the surface of another commonly found oral species: Pauljensenia odontolytica_C strain XH001 (host bacterium). TM7xT also exhibited what has been defined as a parasitic phase, where it disrupts the membrane of the bacterial host cell, resulting in host bacteria lysis. However, free-floating, host-free TM7xT cells remain viable and can re-infect fresh host bacterial cells when available. This discovery and coculture marked the first concrete evidence of how these ultrasmall organisms within the Minisyncoccota survive and persist despite their reduced genomes missing de novo biosynthesis of many essential compounds, including all amino acids and vitamins. This model dual species system has now allowed a deeper understanding of the TM7x-host bacterium interaction and dynamics, with over 15 publications reporting on various aspects of this strain to date. We propose the name Nanosynbacter lyticus gen. nov. sp. nov. for strain TM7xT. We also propose Nanosynbacteraceae fam. nov., Nanosynbacterales ord. nov. and Nanosynbacteria class nov. to accommodate the genus Nanosynbacter.

Implant-related infections demand advanced biomaterials capable of delivering localized, sustained antimicrobial activity while supporting tissue repair. Here, we present an engineered thermoresponsive hydrogel based on poly(N-vinylcaprolactam) (PNVCL), designed as a dual-function platform that enables temperature-triggered gelation, controlled tetracycline release, and osteogenic support. The PNVCL network was tailored to provide rapid sol-gel transition at physiological temperature, strong adhesion to moist surfaces, injectability into narrow implant geometries, high swelling capacity, slow degradation, and a stable drug-release profile-properties rarely combined within a single hydrogel system. To evaluate material performance, we employed a tiered biological framework comprising cytocompatibility assays, controlled physicochemical analyses, and antimicrobial testing against complex, polymicrobial biofilms, complemented by human in situ and in vivo multispecies infection models. The PNVCL-tetracycline hydrogel demonstrated potent antibacterial activity, preserved human gingival fibroblast viability, and sustained its structural integrity and release characteristics even under infection-associated inflammatory conditions. In vivo, the material simultaneously reduced bacterial burden, modulated pathogenic community structure, and promoted new bone formation with a higher degree of maturation, confirming its dual antimicrobial and osteogenic behavior. By integrating precisely engineered thermoresponsive behavior, controlled drug delivery, robust mechanical and interfacial properties, and validated biological functionality, this PNVCL-tetracycline hydrogel represents a material-driven, clinically relevant solution for treating implant-related infections.

Saccharibacteria are episymbionts that require host-bacteria to grow. They are positively associated with inflammatory diseases within the human microbiome, yet their mechanisms for interacting with the human host and contributing to diseases remain unknown. This study investigated interactions between a Saccharibacterium (Nanosynbacter lyticus), its host-bacteria (Schaalia odontolytica), and oral epithelial cells. The host-bacteria induced proinflammatory cytokines in epithelial cells, while Saccharibacteria were immune silent. Remarkably, Saccharibacteria dampened cytokine responses to host-bacteria during coinfection. This effect was driven by Saccharibacteria-induced clustering of TLR2 receptors, a process likely facilitated by type IV, ultimately leading to reduced TLR2-mediated cytokine signalling. High resolution imaging showed that Saccharibacteria were endocytosed by oral epithelial cells, and colocalized with endosome markers, eventually trafficking to lysosomes. Moreover, a subset of the Saccharibacteria survive endocytosis long-term, and retains their capability to reinfect host-bacteria, highlighting a mechanism for persistence in the oral microbiome and a vital role in mammalian immune system modulation.

All cultivated Patescibacteria, also known as the candidate phyla radiation, are obligate episymbionts residing on other microbes. Despite being ubiquitous in many diverse environments, including mammalian microbiomes, molecular mechanisms of host identification and binding amongst ultrasmall bacterial episymbionts remain largely unknown. Type 4 pili are well conserved in this group and could potentially facilitate these symbiotic interactions. To test this hypothesis, we genetically targeted pili genes in Saccharibacteria Nanosynbacter lyticus strain TM7x to assess their essentiality and roles in symbiosis. Our results revealed that N. lyticus assembles two distinct type 4 pili: a nonessential thin pilus that has the smallest diameter of any type 4 pili and contributes to host-binding and episymbiont growth; and an essential thick pilus involved in twitching motility. To understand the role of these pili in vivo we developed Saccharibacteria competition assays and species-specific Fluorescence in situ hybridization probes. Competition between different Saccharibacteria within mock communities demonstrated consistent competitive outcomes that were not driven by priority effects but were dependent on the thin pilus. Collectively, our findings demonstrate that Saccharibacteria encode unique extracellular pili that enable their underexplored episymbiotic lifestyle and competitive fitness within a community.

We are pleased to introduce a new series, "Innovations of Excellence, powered by ADA Forsyth," which provides a forum for brief introductions to new technologies, therapeutic approaches, and health care trends of interest to oral health clinicians and researchers-a "window" into the latest scientific discoveries.Inspired

The oral environment is composed of a diverse array of proteins, and any substrate inserted into this habitat promptly becomes subjected to protein adsorption and bacterial colonization. However, the predictive and modulatory nature of implant surfaces coated with salivary pellicle proteomes in microbial adhesion has not been explored using high-throughput techniques. Thus, using human saliva for salivary pellicle adsorption and microbial accumulation, we compared adsorption and community formation on titanium (Ti) biomaterials (implant devices) and dental surfaces (enamel and dentine). The proteomic profile was evaluated by liquid chromatography coupled with tandem mass spectrometry, and the microbiome was assessed using 16S RNA sequencing. Linear discriminant analysis (LDA) and canonical correlation analysis (CCA) were used to quantify variation in analyte amounts and identify likely biomarkers. Substrates were analyzed regarding their physical, chemical, and topographical properties. Our results showed that the salivary pellicle proteomes on Ti exhibited differences in composition and protein intensities compared with dental surfaces. These differences in proteomes affected the biological processes at the level of microbiome accumulation. Geometric analysis showed greater similarity between Ti and enamel proteomes, while dentine differed markedly. Ti harbors a microbiome community that differs from that of dental surfaces. Canonical correlation analysis (CCA) pinpointed proteins that promoted or inhibited the adherence of specific microbes. Apolipoprotein E showed a strong negative correlation (>0.8) with Streptococcus parasanguinis. Higher levels of the protein on dental surfaces were associated with reduced microbial adhesion, whereas its absence on Ti surfaces facilitated increased bacterial adhesion. These findings provide valuable insights into the initial biological responses after the insertion of implanted devices, which can be leveraged by biomedical engineering to develop biomaterials with enhanced outcomes and prevent microbial accumulation.

Saccharibacteria (formerly TM7) are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to Candidate Phyla Radiation, a large monophyletic lineage with poorly understood biology. Nanosynbacter lyticus type strain TM7x is the first Saccharibacteria member isolated from the human oral microbiome. With restrained metabolic capacities, TM7x lives on the surface of, and forms an obligate episymbiotic relationship with its bacterial host, Schaalia odontolytica strain XH001. The symbiosis allows TM7x to propagate but presents a burden to host bacteria by inducing stress response. Here, we employed super-resolution fluorescence imaging to investigate the physical association between TM7x and XH001. We showed that the binding with TM7x led to a substantial alteration in the membrane fluidity of XH001. We also revealed the formation of intracellular lipid droplets in XH001 when forming episymbiosis with TM7x, a feature that has not been reported in oral bacteria. The TM7x-induced lipid droplets accumulation in XH001 was confirmed by label-free Raman spectroscopy, which also unveiled additional phenotypical features when XH001 cells are physically associated with TM7x. Further exploration through culturing XH001 under various stress conditions showed that lipid droplets accumulation was a general response to stress. A survival assay demonstrated that the presence of lipid droplets plays a protective role in XH001, enhancing its survival under adverse conditions. In conclusion, our study sheds new light on the intricate interaction between Saccharibacteria and their host bacteria, highlighting the potential benefit conferred by TM7x to its host and further emphasizing the context-dependent nature of symbiotic relationships.

Bacteria of the candidate phyla radiation (CPR), constituting about 25

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long - term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface- associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA- Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down- regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x- associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont- mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source- sink" dynamics between phage and bacteria in biofilm, promoting their long - term coexistence within the human microbiome.

The human oral and nasal cavities can act as reservoirs for opportunistic pathogens capable of causing acute infection. These microbes asymptomatically colonize the human oral and nasal cavities which facilitates transmission within human populations via the environment, and they routinely possess clinically significant antibiotic resistance genes. Among these opportunistic pathogens, the Klebsiella genus stands out as a notable example, with its members frequently linked to nosocomial infections and multidrug resistance. As with many colonizing opportunistic pathogens, the essential transmission factors influencing the spread of Klebsiella species among both healthy and diseased individuals remain unclear. Here, we explored a possible explanation by investigating the ability of oral and nasal Klebsiella species to outcompete their native microbial community members under in vitro starvation conditions, which could be analogous to external hospital environments or the microenvironment of mechanical ventilators. When K. pneumoniae and K. aerogenes were present within a healthy human oral or nasal sample, the bacterial community composition shifted dramatically under starvation conditions and typically became enriched in Klebsiella species. Furthermore, introducing K. pneumoniae exogenously into a native microbial community lacking K. pneumoniae, even at low inoculum, led to repeated enrichment under starvation. Precise monitoring of K. pneumoniae within these communities undergoing starvation indicated rapid initial growth and prolonged viability compared to other members of the microbiome. K. pneumoniae strains isolated from healthy individuals’ oral and nasal cavities also exhibited resistance to multiple classes of antibiotics and were genetically similar to clinical and gut isolates. In addition, we found that in the absence of Klebsiella species, other understudied opportunistic pathogens, such as Peptostreptococcus, increased in relative abundance under starvation conditions. Our findings establish an environmental and microbiome community circumstance that allows for the enrichment of Klebsiella species and other opportunistic pathogens. Klebsiella’s enrichment may hinge on its ability to quickly outgrow other members of the microbiome. The ability to outcompete other commensal bacteria and to persist under harsh environmental conditions could be an important factor that contributes to enhanced transmission in both commensal and pathogenic contexts.

Saccharibacteria (formerly TM7) Nanosynbacter lyticus type strain TM7x exhibits a remarkably compact genome and an extraordinarily small cell size. This obligate epibiotic parasite forms a symbiotic relationship with its bacterial host, Schaalia odontolytica, strain XH001 (formerly Actinomyces odontolyticus strain XH001). Due to its limited genome size, TM7x possesses restrained metabolic capacities, predominantly living on the surface of its bacterial host to sustain this symbiotic lifestyle. To comprehend this intriguing, yet understudied interspecies interaction, a thorough understanding of the physical interaction between TM7x and XH001 is imperative. In this study, we employed super-resolution fluorescence imaging to investigate the physical association between TM7x and XH001. We found that the binding with TM7x led to a substantial alteration in the membrane fluidity of the host bacterium XH001. Unexpectedly, we revealed the formation of intracellular lipid droplets in XH001 when forming episymbiosis with TM7x, a feature not commonly observed in oral bacteria cells. The TM7x-induced LD accumulation in XH001 was further confirmed by label-free non-invasive Raman spectroscopy, which also unveiled additional phenotypical features when XH001 cells are physically associated with TM7x. Further exploration through culturing host bacterium XH001 alone under various stress conditions showed that LD accumulation was a general response to stress. Intriguingly, a survival assay demonstrated that the presence of LDs likely plays a protective role in XH001, enhancing its overall survival under adverse conditions. In conclusion, our study sheds new light on the intricate interaction between Saccharibacteria and its host bacterium, highlighting the potential benefit conferred by TM7x to its host, and further emphasizing the context-dependent nature of symbiotic relationships.

Background: Oral Saccharibacteria Nanosynbacter lyticus strain TM7x lives as an ultrasmall epibiont on the surface of its host, Schaalia odontolytica strain XH001. Establishing this interaction is a poorly understood multi-step process. The recovery phase marks a shift in the TM7x/host interaction, switching from the early killing phase, with extensive host cell death, to a stable symbiosis phase where the host and epibiont can grow together. Results: Transcriptomes of TM7x and host, XH001, were captured during the recovery phase and compared to uninfected host and the early host/epibiont interaction (initial encounter). XH001 showed increased expression for rhamnose cell wall components and for the precursor to peptidoglycan while TM7x showed increases in the peptidoglycan pathway. Transporter expression was generally increased for both organisms during recovery compared to the initial encounter, though, XH001 showed lower amino acid transporter expression. Consistent with host parasitism, XH001 showed increased expression of various stress-related genes during recovery while TM7x showed reduced stress. TM7x displayed higher expression of type IV pili, consistent with increased attachment to new hosts. Conclusion: As TM7x is a member of the broadly distributed Candidate Phyla Radiation with small genomes lacking numerous biosynthetic pathways, this study provides further insights into how these epibionts interact and modulate their host bacteria.

Decades of ongoing research has established that oral microbial communities play a role in oral diseases such as periodontitis and caries. Yet the detection of oral bacteria and the profiling of oral polymicrobial communities currently rely on methods that are costly, slow, and technically complex, such as qPCR or next-generation sequencing. For the widescale screening of oral microorganisms suitable for point-of-care settings, there exists the need for a low-cost, rapid detection technique. Here, we tailored the novel CRISPR-Cas-based assay SHERLOCK for the species-specific detection of oral bacteria. We developed a computational pipeline capable of generating constructs suitable for SHERLOCK and experimentally validated the detection of seven oral bacteria. We achieved detection within the single-molecule range that remained specific in the presence of off-target DNA found within saliva. Further, we adapted the assay for detecting target sequences directly from unprocessed saliva samples. The results of our detection, when tested on 30 healthy human saliva samples, fully aligned with 16S rRNA sequencing. Looking forward, this method of detecting oral bacteria is highly scalable and can be easily optimized for implementation at point-of-care settings.

Background Bacteria of the Candidate Phyla Radiation (CPR), constituting about 25% of the bacterial biodiversity, are characterized by small cell size and patchy genomes without complete key metabolic pathways, suggesting a symbiotic lifestyle. Gracilibacteria (BD1-5), which are part of the CPR branch, possess alternate coded genomes and have not yet been cultivated. However, besides genomic evidence, little is known about the lifestyle of Gracilibacteria, their temporal dynamics, and activity in natural ecosystems, particularly in groundwater, where they were initially been genomically resolved. Therefore, we here aimed to investigate Gracilibacteria activity in situ and to discern expressed genes involved in their lifestyle, using the metaproteogenome of Gracilibacteria as a function of time in the cold-water geyser Wallender Born in the Volcanic Eifel region in Germany.Results We coupled genome-resolved metagenomics and metaproteomics to investigate a cold-water geyser microbial community enriched in Gracilibacteria across a 12-day time-series. Groundwater was collected and sequentially filtered to fraction CPR and other bacteria. Based on 670 Gbps of metagenomic data, 1129 different ribosomal protein S3 marker genes and 751 high-quality genomes (123 population genomes after dereplication), we identified dominant bacteria belonging to Galionellales and Gracilibacteria along with keystone microbes, which were low in genomic abundance but substantially contributing to proteomic abundance. Seven high-quality Gracilibacteria genomes showed typical limitations, such as limited amino acid or nucleotide synthesis, in their central metabolism but no co-occurrence with potential hosts. The genomes of these Gracilibacteria encoded for a high number of proteins related to a symbiotic or even predatory lifestyle, e.g. , type IV and type II secretion system subunits and features related to cell-cell interactions and cell motility, which were also detected on protein level.Conclusions Coupling metagenomics to metaproteomics enabled us to identify microbial keystone taxa in a high-CO2 aquifer, and to reveal microbial dynamics of Gracilibacteria. We posit that Gracilibacteria might be successful microbial predators in this ecosystem, potentially aiding in population control of this highly perturbed microbial geyser community from the deep biosphere.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACTWithin human microbiomes, Gracilibacteria, Absconditabacteria, and Saccharibacteria, members of Candidate Phyla Radiation (CPR), are increasingly correlated with human oral health and disease. We profiled the diversity of CRISPR-Cas systems in the genomes of these bacteria and sought phages that are capable of infecting them by matching their spacer inventories to large phage sequence databases. Gracilibacteria and Absconditabacteria recode the typical TGA stop codon to glycine and are putatively infected by phages that share their host’s alternate genetic code. Unexpectedly, however, other predicted phages of Gracilibacteria and Absconditabacteria do not use an alternative genetic code. Some of these phages may infect both alternatively coded CPR bacteria and standard-coded bacteria. These phages typically rely on other stop codons besides TGA and thus should be capable of producing viable gene products in either bacterial host type. By avoiding the acquisition of in-frame stop codons, these phages may have a broadened host range. Interestingly, we additionally predict that some phages of Saccharibacteria are targeted by spacers encoded in Actinobacteria, a phylum that includes known hosts for episymbiotic Saccharibacteria.IMPORTANCEHere, we profiled putative phages of Saccharibacteria, which are of particular importance as Saccharibacteria influence some human oral diseases. We additionally profiled putative phages of Gracilibacteria and Absconditabacteria, two Candidate Phyla Radiation (CPR) lineages of interest given their use of an alternative genetic code. Among the phages identified in this study, some are targeted by spacers from both CPR and non-CPR bacteria and others by both bacteria that use the standard genetic code as well as bacteria that use an alternative genetic code. These findings represent new insights into possible phage replication strategies and have relevance for phage therapies that seek to manipulate microbiomes containing CPR bacteria.