Structure, biogenesis and evolution of thylakoid membranes.

Cyanobacteria and chloroplasts of algae and plants harbor specialized thylakoid membranes that convert sunlight into chemical energy. These membranes house photosystems II and I, the vital protein-pigment complexes that drive oxygenic photosynthesis. In the course of their evolution, thylakoid membranes have diversified in structure. However, the core machinery for photosynthetic electron transport remained largely unchanged, with adaptations occurring primarily in the light-harvesting antenna systems. Whereas thylakoid membranes in cyanobacteria are relatively simple they become more complex in algae and plants. The chloroplasts of vascular plants contain intricate networks of stacked grana and unstacked stroma thylakoids. This review provides an in-depth view of thylakoid membrane architectures in phototrophs, and the determinants that shape their forms, as well as presenting recent insights into the spatial organization of their biogenesis and maintenance. Its overall goal is to define the underlying principles that have guided the evolution of these bioenergetic membranes.

Biocompatibility of Synechococcus sp. PCC 7002 with Human Dermal Cells In Vitro.

Being the green gold of the future, cyanobacteria have recently attracted considerable interest worldwide. This study investigates the adaptability and biocompatibility of the cyanobacterial strain Synechococcus sp. PCC 7002 with human dermal cells, focusing on its potential application in biomedical contexts. First, we investigated the adaptability of Synechococcus PCC 7002 bacteria to human cell culture conditions. Next, we evaluated the biocompatibility of cyanobacteria with common dermal cells, like 3T3 fibroblasts and HaCaT keratinocytes. Therefore, cells were directly and indirectly cocultured with the corresponding cells, and we measured metabolic activity (AlamarBlue assay) and proliferation (cell count and PicoGreen assay). The lactate dehydrogenase (LDH) assay was performed to determine the cytotoxic effect of cyanobacteria and their nutrition medium on human dermal cells. The cyanobacteria exhibited exponential growth under conventional human cell culture conditions, with the temperature and medium composition not affecting their viability. In addition, the effect of illumination on the proliferation capacity was investigated, showing a significant impact of light exposure on bacterial growth. The measured oxygen production under hypoxic conditions demonstrated a sufficient oxygen supply for further tissue engineering approaches depending on the number of bacteria. There were no significant adverse effects on human cell viability and growth under coculture conditions, whereas the LDH assay assessed signs of cytotoxicity regarding 3T3 fibroblasts after 2 days of coculturing. These negative effects were dismissed after 4 days. The findings highlight the potential of Synechococcus sp. PCC 7002 for integration into biomedical approaches. We found no cytotoxicity of cyanobacteria on 3T3 fibroblasts and HaCaT keratinocytes, thus paving the way for further in vivo studies to assess long-term effects and systemic reactions.

NTRC regulates CP12 to activate Calvin-Benson cycle during cold acclimation.

NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin-Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.

Plant organellar RNA maturation.

Plant organellar RNA metabolism is run by a multitude of nucleus-encoded RNA-binding proteins (RBPs) that control RNA stability, processing, and degradation. In chloroplasts and mitochondria, these post-transcriptional processes are vital for the production of a small number of essential components of the photosynthetic and respiratory machinery-and consequently for organellar biogenesis and plant survival. Many organellar RBPs have been functionally assigned to individual steps in RNA maturation, often specific to selected transcripts. While the catalog of factors identified is ever-growing, our knowledge of how they achieve their functions mechanistically is far from complete. This review summarizes the current knowledge of plant organellar RNA metabolism taking an RBP-centric approach and focusing on mechanistic aspects of RBP functions and the kinetics of the processes they are involved in.

Lysine acetylation regulates moonlighting activity of the E2 subunit of the chloroplast pyruvate dehydrogenase complex in Chlamydomonas.

The dihydrolipoamide acetyltransferase subunit DLA2 of the chloroplast pyruvate dehydrogenase complex (cpPDC) in the green alga Chlamydomonas reinhardtii has previously been shown to possess moonlighting activity in chloroplast gene expression. Under mixotrophic growth conditions, DLA2 forms part of a ribonucleoprotein particle (RNP) with the psbA mRNA that encodes the D1 protein of the photosystem II (PSII) reaction center. Here, we report on the characterization of the molecular switch that regulates shuttling of DLA2 between its functions in carbon metabolism and D1 synthesis. Determination of RNA-binding affinities by microscale thermophoresis demonstrated that the E3-binding domain (E3BD) of DLA2 mediates psbA-specific RNA recognition. Analyses of cpPDC formation and activity, as well as RNP complex formation, showed that acetylation of a single lysine residue (K197) in E3BD induces the release of DLA2 from the cpPDC, and its functional shift towards RNA binding. Moreover, Förster resonance energy transfer microscopy revealed that psbA mRNA/DLA2 complexes localize around the chloroplast's pyrenoid. Pulse labeling and D1 re-accumulation after induced PSII degradation strongly suggest that DLA2 is important for D1 synthesis during de novo PSII biogenesis.

Transcardial injection and vascular distribution of microalgae in Xenopus laevis as means to supply the brain with photosynthetic oxygen.

Oxygen in vertebrates is generally provided through respiratory organs and blood vessels. This protocol describes transcardial injection, vascular distribution, and accumulation of phototrophic microalgae in the brain of Xenopus laevis tadpoles. Following tissue isolation, oxygen dynamics and neuronal activity are recorded in semi-intact whole-head preparations. Illumination of such microalgae-filled preparations triggers the photosynthetic production of oxygen in the brain that, under hypoxic conditions, rescues neuronal activity. This technology is potentially able to ameliorate consequences of hypoxia under pathological conditions. For complete details on the use and execution of this protocol, please refer to Özugur et al. (2021).

Thylakoid attachment to the plasma membrane in Synechocystis sp. PCC 6803 requires the AncM protein.

Thylakoids are the highly specialized internal membrane systems that harbor the photosynthetic electron transport machinery in cyanobacteria and in chloroplasts. In Synechocystis sp. PCC 6803, thylakoid membranes (TMs) are arranged in peripheral sheets that occasionally converge on the plasma membrane (PM) to form thylakoid convergence membranes (TCMs). TCMs connect several thylakoid sheets and form local contact sites called thylapses between the two membrane systems, at which the early steps of photosystem II (PSII) assembly occur. The protein CurT is one of the main drivers of TCM formation known so far. Here, we identify, by whole-genome sequencing of a curT- suppressor strain, the protein anchor of convergence membranes (AncM) as a factor required for the attachment of thylakoids to the PM at thylapses. An ancM- mutant is shown to have a photosynthetic phenotype characterized by reductions in oxygen-evolution rate, PSII accumulation, and PS assembly. Moreover, the ancM- strain exhibits an altered thylakoid ultrastructure with additional sheets and TCMs detached from the PM. By combining biochemical studies with fluorescence and correlative light-electron microscopy-based approaches, we show that AncM is an integral membrane protein located in biogenic TCMs that form thylapses. These data suggest an antagonistic function of AncM and CurT in shaping TM ultrastructure.

Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

Green oxygen power plants in the brain rescue neuronal activity.

Neuronal activity in the brain depends on mostly aerobic generation of energy equivalents and thus on a constant O2 supply. Oxygenation of the vertebrate brain has been optimized during evolution by species-specific uptake and transport of O2 that originally derives from the phototrophic activity of prokaryotic and eukaryotic organisms in the environment. Here, we employed a concept that exploits transcardial injection and vascular distribution of unicellular green algae or cyanobacteria in the brain of Xenopus laevis tadpoles. Using oxygen measurements in the brain ventricle, we found that these microorganisms robustly produce sizable amounts of O2 upon illumination. In a severe hypoxic environment, when neuronal activity has completely ceased, the photosynthetic O2 reliably provoked a restart and rescue of neuronal activity. In the future, phototrophic microorganisms might provide a novel means to directly increase oxygen levels in the brain in a controlled manner under particular eco-physiological conditions or following pathological impairments.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

Use of photosynthetic transgenic cyanobacteria to promote lymphangiogenesis in scaffolds for dermal regeneration.

Impaired wound healing represents an unsolved medical need with a high impact on patients´ quality of life and global health care. Even though its causes are diverse, ischemic-hypoxic conditions and exacerbated inflammation are shared pathological features responsible for obstructing tissue restoration. In line with this, it has been suggested that promoting a normoxic pro-regenerative environment and accelerating inflammation resolution, by reinstating the lymphatic fluid transport, could allow the wound healing process to be resumed. Our group was first to demonstrate the functional use of scaffolds seeded with photosynthetic microorganisms to supply tissues with oxygen. Moreover, we previously proposed a photosynthetic gene therapy strategy to create scaffolds that deliver other therapeutic molecules, such as recombinant human growth factors into the wound area. In the present work, we introduce the use of transgenic Synechococcus sp. PCC 7002 cyanobacteria (SynHA), which can produce oxygen and lymphangiogenic hyaluronic acid, in photosynthetic biomaterials. We show that the co-culture of lymphatic endothelial cells with SynHA promotes their survival and proliferation under hypoxic conditions. Also, hyaluronic acid secreted by the cyanobacteria enhanced their lymphangiogenic potential as shown by changes to their gene expression profile, the presence of lymphangiogenic protein markers and their capacity to build lymph vessel tubes. Finally, by seeding SynHA into collagen-based dermal regeneration materials, we developed a viable photosynthetic scaffold that promotes lymphangiogenesis in vitro under hypoxic conditions. The results obtained in this study lay the groundwork for future tissue engineering applications using transgenic cyanobacteria that could become a therapeutic alternative for chronic wound treatment. STATEMENT OF SIGNIFICANCE: In this study, we introduce the use of transgenic Synechococcus sp. PCC 7002 (SynHA) cyanobacteria, which were genetically engineered to produce hyaluronic acid, to create lymphangiogenic photosynthetic scaffolds for dermal regeneration. Our results confirmed that SynHA cyanobacteria maintain their photosynthetic capacity under standard human cell culture conditions and efficiently proliferate when seeded inside fibrin-collagen scaffolds. Moreover, we show that SynHA supported the viability of co-cultured lymphatic endothelial cells (LECs) under hypoxic conditions by providing them with photosynthetic-derived oxygen, while cyanobacteria-derived hyaluronic acid stimulated the lymphangiogenic capacity of LECs. Since tissue hypoxia and impaired lymphatic drainage are two key factors that directly affect wound healing, our results suggest that lymphangiogenic photosynthetic biomaterials could become a treatment option for chronic wound management.

The versatile interactome of chloroplast ribosomes revealed by affinity purification mass spectrometry.

In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.

Acclimation in plants - the Green Hub consortium.

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.

Photosymbiosis for Biomedical Applications.

Without the sustained provision of adequate levels of oxygen by the cardiovascular system, the tissues of higher animals are incapable of maintaining normal metabolic activity, and hence cannot survive. The consequence of this evolutionarily suboptimal design is that humans are dependent on cardiovascular perfusion, and therefore highly susceptible to alterations in its normal function. However, hope may be at hand. "Photosynthetic strategies," based on the recognition that photosynthesis is the source of all oxygen, offer a revolutionary and promising solution to pathologies related to tissue hypoxia. These approaches, which have been under development over the past 20 years, seek to harness photosynthetic microorganisms as a local and controllable source of oxygen to circumvent the need for blood perfusion to sustain tissue survival. To date, their applications extend from the in vitro creation of artificial human tissues to the photosynthetic maintenance of oxygen-deprived organs both in vivo and ex vivo, while their potential use in other medical approaches has just begun to be explored. This review provides an overview of the state of the art of photosynthetic technologies and its innovative applications, as well as an expert assessment of the major challenges and how they can be addressed.

The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA.

Chloroplast RNAs are stabilized and processed by a multitude of nuclear-encoded RNA-binding proteins, often in response to external stimuli like light and temperature. A particularly interesting RNA-based regulation occurs with the psbA mRNA, which shows light-dependent translation. Recently, the chloroplast ribonucleoprotein CP33B was identified as a ligand of the psbA mRNA. We here characterized the interaction of CP33B with chloroplast RNAs in greater detail using a combination of RIP-chip, quantitative dot-blot, and RNA-Bind-n-Seq experiments. We demonstrate that CP33B prefers psbA over all other chloroplast RNAs and associates with the vast majority of the psbA transcript pool. The RNA sequence target motif, determined in vitro, does not fully explain CP33B's preference for psbA, suggesting that there are other determinants of specificity in vivo.

Towards a biotechnological platform for the production of human pro-angiogenic growth factors in the green alga Chlamydomonas reinhardtii.

The recent use of photosynthetic organisms such as Chlamydomonas reinhardtii in biomedical applications has demonstrated their potential for the treatment of acute and chronic tissue hypoxia. Moreover, transgenic microalgae have been suggested as an alternative in situ drug delivery system. In this study, we set out to identify the best available combination of strains and expression vectors to establish a robust platform for the expression of human pro-angiogenic growth factors, i.e., hVEGF-165, hPDGF-B, and hSDF-1, in biomedical settings. As a case study, combinations of two expression vectors (pOpt and pBC1) and two C. reinhardtii strains (UVM4 and UVM11) were compared with respect to hVEGF-165 transgene expression by determination of steady-state levels of transgenic transcripts and immunological detection of recombinant proteins produced and secreted by the generated strains. The results revealed the combination of the UVM11 strain with the pBC1 vector to be the most efficient one for high-level hVEGF-165 production. To assess the robustness of this finding, the selected combination was used to create hPDGF-B and hSDF-1 transgenic strains for optimized recombinant protein expression. Furthermore, biological activity and functionality of algal-produced recombinant pro-angiogenic growth factors were assessed by receptor phosphorylation and in vitro angiogenesis assays. The results obtained revealed a potentiating effect in the combinatorial application of transgenic strains expressing either of the three growth factors on endothelial cell tube formation ability, and thus support the idea of using transgenic algae expressing pro-angiogenic growth factors in wound healing approaches.

Plastoglobular protein 18 is involved in chloroplast function and thylakoid formation.

Plastoglobules are lipoprotein particles that are found in different types of plastids. They contain a very specific and specialized set of lipids and proteins. Plastoglobules are highly dynamic in size and shape, and are therefore thought to participate in adaptation processes during either abiotic or biotic stresses or transitions between developmental stages. They are suggested to function in thylakoid biogenesis, isoprenoid metabolism, and chlorophyll degradation. While several plastoglobular proteins contain identifiable domains, others provide no structural clues to their function. In this study, we investigate the role of plastoglobular protein 18 (PG18), which is conserved from cyanobacteria to higher plants. Analysis of a PG18 loss-of-function mutant in Arabidopsis thaliana demonstrated that PG18 plays an important role in thylakoid formation; the loss of PG18 results in impaired accumulation, assembly, and function of thylakoid membrane complexes. Interestingly, the mutant accumulated less chlorophyll and carotenoids, whereas xanthophyll cycle pigments were increased. Accumulation of photosynthetic complexes is similarly affected in both a Synechocystis and an Arabidopsis PG18 mutant. However, the ultrastructure of cyanobacterial thylakoids is not compromised by the lack of PG18, probably due to its less complex architecture.

Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane.

Little is known about how the photosynthetic machinery is arranged in time and space during the biogenesis of thylakoid membranes. Using in situ cryo-electron tomography to image the three-dimensional architecture of the cyanobacterium Synechocystis, we observed that the tips of multiple thylakoids merge to form a substructure called the 'convergence membrane'. This high-curvature membrane comes into close contact with the plasma membrane at discrete sites. We generated subtomogram averages of 70S ribosomes and array-forming phycobilisomes, then mapped these structures onto the native membrane architecture as markers for protein synthesis and photosynthesis, respectively. This molecular localization identified two distinct biogenic regions in the thylakoid network: thylakoids facing the cytosolic interior of the cell that were associated with both marker complexes, and convergence membranes that were decorated by ribosomes but not phycobilisomes. We propose that the convergence membranes perform a specialized biogenic function, coupling the synthesis of thylakoid proteins with the integration of cofactors from the plasma membrane and the periplasmic space.

Development of photosynthetic sutures for the local delivery of oxygen and recombinant growth factors in wounds.

Surgical sutures represent the gold standard for wound closure, however, their main purpose is still limited to a mechanical function rather than playing a bioactive role. Since oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors (VEGF, PDGF-BB, or SDF-1α) at the wound site. Here, photosynthetic genetically modified microalgae were seeded in commercially available sutures and their distribution and proliferation capacity was evaluated. Additionally, the mechanical properties of seeded sutures were compared to unseeded controls that showed no significant differences. Oxygen production, as well as recombinant growth factor release was quantified in vitro over time, and confirmed that photosynthetic sutures are indeed a feasible approach for the local delivery of bioactive molecules. Finally, photosynthetic sutures were tested in order to evaluate their resistance to mechanical stress and freezing. Significant stability was observed in both conditions, and the feasibility of their use in the clinical practice was therefore confirmed. Our results suggest that photosynthetic gene therapy could be used to produce a new generation of bioactive sutures with improved healing capacities. STATEMENT OF SIGNIFICANCE: Disruption of the vascular network is intrinsic to trauma and surgery, and consequently, wound healing is characterized by diminished levels of blood perfusion. Among all the blood components, oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process. Therefore, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors at the wound site. This novel concept has never been explored before for this type of material and represents the first attempt to create a new generation of bioactive sutures with improved regenerative capabilities.

Quantitative LC-MS Provides No Evidence for m6 dA or m4 dC in the Genome of Mouse Embryonic Stem Cells and Tissues.

Until recently, it was believed that the genomes of higher organisms contain, in addition to the four canonical DNA bases, only 5-methyl-dC (m5 dC) as a modified base to control epigenetic processes. In recent years, this view has changed dramatically with the discovery of 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNA from stem cells and brain tissue. N6 -methyldeoxyadenosine (m6 dA) is the most recent base reported to be present in the genome of various eukaryotic organisms. This base, together with N4 -methyldeoxycytidine (m4 dC), was first reported to be a component of bacterial genomes. In this work, we investigated the levels and distribution of these potentially epigenetically relevant DNA bases by using a novel ultrasensitive UHPLC-MS method. We further report quantitative data for m5 dC, hmdC, fdC, and cadC, but we were unable to detect either m4 dC or m6 dA in DNA isolated from mouse embryonic stem cells or brain and liver tissue, which calls into question their epigenetic relevance.