Functionally comparable but evolutionarily distinct nucleotide-targeting effectors help identify conserved paradigms across diverse immune systems.

While nucleic acid-targeting effectors are known to be central to biological conflicts and anti-selfish element immunity, recent findings have revealed immune effectors that target their building blocks and the cellular energy currency-free nucleotides. Through comparative genomics and sequence-structure analysis, we identified several distinct effector domains, which we named Calcineurin-CE, HD-CE, and PRTase-CE. These domains, along with specific versions of the ParB and MazG domains, are widely present in diverse prokaryotic immune systems and are predicted to degrade nucleotides by targeting phosphate or glycosidic linkages. Our findings unveil multiple potential immune systems associated with at least 17 different functional themes featuring these effectors. Some of these systems sense modified DNA/nucleotides from phages or operate downstream of novel enzymes generating signaling nucleotides. We also uncovered a class of systems utilizing HSP90- and HSP70-related modules as analogs of STAND and GTPase domains that are coupled to these nucleotide-targeting- or proteolysis-induced complex-forming effectors. While widespread in bacteria, only a limited subset of nucleotide-targeting effectors was integrated into eukaryotic immune systems, suggesting barriers to interoperability across subcellular contexts. This work establishes nucleotide-degrading effectors as an emerging immune paradigm and traces their origins back to homologous domains in housekeeping systems.

Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.

One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homolog of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.

Comprehensive classification of ABC ATPases and their functional radiation in nucleoprotein dynamics and biological conflict systems.

ABC ATPases form one of the largest clades of P-loop NTPase fold enzymes that catalyze ATP-hydrolysis and utilize its free energy for a staggering range of functions from transport to nucleoprotein dynamics. Using sensitive sequence and structure analysis with comparative genomics, for the first time we provide a comprehensive classification of the ABC ATPase superfamily. ABC ATPases developed structural hallmarks that unambiguously distinguish them from other P-loop NTPases such as an alternative to arginine-finger-based catalysis. At least five and up to eight distinct clades of ABC ATPases are reconstructed as being present in the last universal common ancestor. They underwent distinct phases of structural innovation with the emergence of inserts constituting conserved binding interfaces for proteins or nucleic acids and the adoption of a unique dimeric toroidal configuration for DNA-threading. Specifically, several clades have also extensively radiated in counter-invader conflict systems where they serve as nodal nucleotide-dependent sensory and energetic components regulating a diversity of effectors (including some previously unrecognized) acting independently or together with restriction-modification systems. We present a unified mechanism for ABC ATPase function across disparate systems like RNA editing, translation, metabolism, DNA repair, and biological conflicts, and some unexpected recruitments, such as MutS ATPases in secondary metabolism.

The catalytic core of DEMETER guides active DNA demethylation in Arabidopsis.

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.

Diversification of AID/APOBEC-like deaminases in metazoa: multiplicity of clades and widespread roles in immunity.

AID/APOBEC deaminases (AADs) convert cytidine to uridine in single-stranded nucleic acids. They are involved in numerous mutagenic processes, including those underpinning vertebrate innate and adaptive immunity. Using a multipronged sequence analysis strategy, we uncover several AADs across metazoa, dictyosteliida, and algae, including multiple previously unreported vertebrate clades, and versions from urochordates, nematodes, echinoderms, arthropods, lophotrochozoans, cnidarians, and porifera. Evolutionary analysis suggests a fundamental division of AADs early in metazoan evolution into secreted deaminases (SNADs) and classical AADs, followed by diversification into several clades driven by rapid-sequence evolution, gene loss, lineage-specific expansions, and lateral transfer to various algae. Most vertebrate AADs, including AID and APOBECs1-3, diversified in the vertebrates, whereas the APOBEC4-like clade has a deeper origin in metazoa. Positional entropy analysis suggests that several AAD clades are diversifying rapidly, especially in the positions predicted to interact with the nucleic acid target motif, and with potential viral inhibitors. Further, several AADs have evolved neomorphic metal-binding inserts, especially within loops predicted to interact with the target nucleic acid. We also observe polymorphisms, driven by alternative splicing, gene loss, and possibly intergenic recombination between paralogs. We propose that biological conflicts of AADs with viruses and genomic retroelements are drivers of rapid AAD evolution, suggesting a widespread presence of mutagenesis-based immune-defense systems. Deaminases like AID represent versions "institutionalized" from the broader array of AADs pitted in such arms races for mutagenesis of self-DNA, and similar recruitment might have independently occurred elsewhere in metazoa.

Expansions, diversification, and interindividual copy number variations of AID/APOBEC family cytidine deaminase genes in lampreys.

Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.

A Nonhemolytic Group B Streptococcus Strain Exhibits Hypervirulence.

Group B streptococci (GBS) are Gram-positive bacteria that are a leading cause of neonatal infections. Most invasive isolates are ß-hemolytic, and hemolytic activity is critical for GBS virulence. Although nonhemolytic GBS strains are occasionally isolated, they are often thought to be virulence attenuated. In this study, we show that a nonhemolytic GBS strain (GB37) isolated from a septic neonate exhibits hypervirulence. Substitution of tryptophan to leucine (W297L) in the sensor histidine kinase CovS results in constitutive kinase signaling, leading to decreased hemolysis and increased activity of the GBS hyaluronidase, HylB. These results describe how nonpigmented and nonhemolytic GBS strains can exhibit hypervirulence.

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification.

While N(6) -methyladenosine (m(6) A) is a well-known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well-studied 5-methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction-modification and counter-restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m(6) A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m(6) A-binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m(6) A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m(6) A demethylases, this proposal needs more careful evaluation. Also watch the Video Abstract.

Transposons to toxins: the provenance, architecture and diversification of a widespread class of eukaryotic effectors.

Enzymatic effectors targeting nucleic acids, proteins and other cellular components are the mainstay of conflicts across life forms. Using comparative genomics we identify a large class of eukaryotic proteins, which include effectors from oomycetes, fungi and other parasites. The majority of these proteins have a characteristic domain architecture with one of several N-terminal 'Header' domains, which are predicted to play a role in trafficking of these effectors, including a novel version of the Ubiquitin fold. The Headers are followed by one or more diverse C-terminal domains, such as restriction endonuclease (REase), protein kinase, HNH endonuclease, LK-nuclease (a RNase) and multiple distinct peptidase domains, which are predicted to carry their toxicity determinants. The most common types of these proteins appear to have originated from prokaryotic transposases (e.g. TN7 and Mu) and combine a CDC6/ORC1-STAND clade NTPase domain with a C-terminal REase domain. Other than the so-called Crinkler effectors of oomycetes and fungi, these effectors are encoded by other eukaryotic parasites such as trypanosomatids (the RHS proteins) and the rhizarian Plasmodiophora, and symbionts like Capsaspora Remarkably, we also find these proteins in free-living eukaryotes, including several viridiplantae, fungi, amoebozoans and animals. These versions might either still be transposons or function in other poorly understood eukaryote-specific inter-organismal and inter-genomic conflicts. These include the Medea1 selfish element of Tribolium that spreads via post-zygotic killing. We present a unified mechanism for the recombination-dependent diversification and action of this widespread class of molecular weaponry deployed across diverse conflicts ranging from parasitic to free-living forms.

Polyvalent Proteins, a Pervasive Theme in the Intergenomic Biological Conflicts of Bacteriophages and Conjugative Elements.

Intense biological conflicts between prokaryotic genomes and their genomic parasites have resulted in an arms race in terms of the molecular "weaponry" deployed on both sides. Using a recursive computational approach, we uncovered a remarkable class of multidomain proteins with 2 to 15 domains in the same polypeptide deployed by viruses and plasmids in such conflicts. Domain architectures and genomic contexts indicate that they are part of a widespread conflict strategy involving proteins injected into the host cell along with parasite DNA during the earliest phase of infection. Their unique feature is the combination of domains with highly disparate biochemical activities in the same polypeptide; accordingly, we term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large fraction are enzymatic domains predicted to modify proteins, target nucleic acids, alter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal components. They further contain nucleic acid-binding domains, virion structural domains, and 40 novel uncharacterized domains. Analysis of their architectural network reveals both pervasive common themes and specialized strategies for conjugative elements and plasmids or (pro)phages. The themes include likely processing of multidomain polypeptides by zincin-like metallopeptidases and mechanisms to counter restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA-binding domains acquired by eukaryotes from such systems have been reused in XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kinetoplastids. Characterization of the novel domains discovered here, such as RNases and peptidases, are likely to aid in the development of new reagents and elucidation of the spread of antibiotic resistance.IMPORTANCE This is the first report of the widespread presence of large proteins, termed polyvalent proteins, predicted to be transmitted by genomic parasites such as conjugative elements, plasmids, and phages during the initial phase of infection along with their DNA. They are typified by the presence of multiple domains with disparate activities combined in the same protein. While some of these domains are predicted to assist the invasive element in replication, transcription, or protection of their DNA, several are likely to target various host defense systems or modify the host to favor the parasite's life cycle. Notably, DNA-binding domains from these systems have been transferred to eukaryotes, where they have been incorporated into DNA repair and mitochondrial genome replication systems.

Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling.

Cyclic di- and linear oligo-nucleotide signals activate defenses against invasive nucleic acids in animal immunity; however, their evolutionary antecedents are poorly understood. Using comparative genomics, sequence and structure analysis, we uncovered a vast network of systems defined by conserved prokaryotic gene-neighborhoods, which encode enzymes generating such nucleotides or alternatively processing them to yield potential signaling molecules. The nucleotide-generating enzymes include several clades of the DNA-polymerase ß-like superfamily (including Vibrio cholerae DncV), a minimal version of the CRISPR polymerase and DisA-like cyclic-di-AMP synthetases. Nucleotide-binding/processing domains include TIR domains and members of a superfamily prototyped by Smf/DprA proteins and base (cytokinin)-releasing LOG enzymes. They are combined in conserved gene-neighborhoods with genes for a plethora of protein superfamilies, which we predict to function as nucleotide-sensors and effectors targeting nucleic acids, proteins or membranes (pore-forming agents). These systems are sometimes combined with other biological conflict-systems such as restriction-modification and CRISPR/Cas. Interestingly, several are coupled in mutually exclusive neighborhoods with either a prokaryotic ubiquitin-system or a HORMA domain-PCH2-like AAA+ ATPase dyad. The latter are potential precursors of equivalent proteins in eukaryotic chromosome dynamics. Further, components from these nucleotide-centric systems have been utilized in several other systems including a novel diversity-generating system with a reverse transcriptase. We also found the Smf/DprA/LOG domain from these systems to be recruited as a predicted nucleotide-binding domain in eukaryotic TRPM channels. These findings point to evolutionary and mechanistic links, which bring together CRISPR/Cas, animal interferon-induced immunity, and several other systems that combine nucleic-acid-sensing and nucleotide-dependent signaling.

Lineage-specific expansions of TET/JBP genes and a new class of DNA transposons shape fungal genomic and epigenetic landscapes.

TET/JBP dioxygenases oxidize methylpyrimidines in nucleic acids and are implicated in generation of epigenetic marks and potential intermediates for DNA demethylation. We show that TET/JBP genes are lineage-specifically expanded in all major clades of basidiomycete fungi, with the majority of copies predicted to encode catalytically active proteins. This pattern differs starkly from the situation in most other organisms that possess just a single or a few copies of the TET/JBP family. In most basidiomycetes, TET/JBP genes are frequently linked to a unique class of transposons, KDZ (Kyakuja, Dileera, and Zisupton) and appear to have dispersed across chromosomes along with them. Several of these elements typically encode additional proteins, including a divergent version of the HMG domain. Analysis of their transposases shows that they contain a previously uncharacterized version of the RNase H fold with multiple distinctive Zn-chelating motifs and a unique insert, which are predicted to play roles in structural stabilization and target sequence recognition, respectively. We reconstruct the complex evolutionary history of TET/JBPs and associated transposons as involving multiple rounds of expansion with concomitant lineage sorting and loss, along with several capture events of TET/JBP genes by different transposon clades. On a few occasions, these TET/JBP genes were also laterally transferred to certain Ascomycota, Glomeromycota, Viridiplantae, and Amoebozoa. One such is an inactive version, calnexin-independence factor 1 (Cif1), from Schizosaccharomyces pombe, which has been implicated in inducing an epigenetically transmitted prion state. We argue that this unique transposon-TET/JBP association is likely to play important roles in speciation during evolution and epigenetic regulation.

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA(+) and VLRC(+) lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αß and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.

Selection of the lamprey VLRC antigen receptor repertoire.

The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC(+) peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line-encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA(+) cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC(+) lymphocyte lineage.

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea.

TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.

Gene networks and chromatin and transcriptional regulation of the phaseolin promoter in Arabidopsis.

The complete lack of seed storage protein expression in vegetative tissues and robust expression during embryogenesis makes seed development an ideal system to study tissue-specific expression of genes. The promoter for the Phaseolin (phas) gene, which encodes the major seed storage protein in bean (Phaseolus vulgaris), is activated in two sequential steps: Phaseolus vulgaris ABI3-like factor (Pv-ALF)-dependent potentiation and abscisic acid-mediated activation. In this study, a heterologous in vivo Pv-ALF/phas-GUS (for ß-glucuronidase) expression system in transgenic Arabidopsis thaliana leaves was used in conjunction with the powerful RNA-Seq approach to capture transcriptional landscapes of phas promoter expression. Remarkably, expression of over 1300 genes from 11 functional categories coincided with changes in the transcriptional status of the phas promoter. Gene network analysis of induced genes and artificial microRNA-mediated loss-of-function genetic assays identified transcriptional regulators RINGLET 2 (RLT2) and AINTEGUMENTA-LIKE 5 (AIL5) as being essential for phas transcription. Pv-ALF binding to the RLT2 and AIL5 promoter regions was confirmed by electrophoretic mobility shift assay. RLT2 and AIL5 knockdown lines displayed reduced expression of several endogenous seed genes, suggesting that these factors are involved in activation of endogenous Arabidopsis seed storage gene expression. Overall, the identification of these key factors involved in phas activation provides important insight into the two-step transcriptional regulation of seed-specific gene expression.

The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.

Catalysis of NAD(+)-dependent ADP-ribosylation of proteins, nucleic acids, or small molecules has evolved in at least three structurally unrelated superfamilies of enzymes, namely ADP-ribosyltransferase (ART), the Sirtuins, and probably TM1506. Of these, the ART superfamily is the most diverse in terms of structure, active site residues, and targets that they modify. The primary diversification of the ART superfamily occurred in the context of diverse bacterial conflict systems, wherein ARTs play both offensive and defensive roles. These include toxin-antitoxin systems, virus-host interactions, intraspecific antagonism (polymorphic toxins), symbiont/parasite effectors/toxins, resistance to antibiotics, and repair of RNAs cleaved in conflicts. ARTs evolving in these systems have been repeatedly acquired by lateral transfer throughout eukaryotic evolution, starting from the PARP family, which was acquired prior to the last eukaryotic common ancestor. They were incorporated into eukaryotic regulatory/epigenetic control systems (e.g., PARP family and NEURL4), and also used as defensive (e.g., pierisin and CARP-1 families) or immunity-related proteins (e.g., Gig2-like ARTs). The ADP-ribosylation system also includes other domains, such as the Macro, ADP-ribosyl glycohydrolase, NADAR, and ADP-ribosyl cyclase, which appear to have initially diversified in bacterial conflict-related systems. Unlike ARTs, sirtuins appear to have a much smaller presence in conflict-related systems.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA.

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology.

A TET homologue protein from Coprinopsis cinerea (CcTET) that biochemically converts 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine.

DNA methylation (5-methylcytosine, 5mC) plays critical biological functions in mammals and plants as a vital epigenetic marker. The Ten-Eleven translocation dioxygenases (TET1, 2, and 3) have been found to oxidize 5mC to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in mammalian cells. We report herein three mushroom TET homologues from Coprinopsis cinerea that can mediate 5mC oxidation. Specifically, one homologue (CC1G_05589, CcTET) shows similar activity to its mammalian TET homologues. Biochemically, CcTET actively converts 5mC to 5hmC, 5fC, and 5caC under natural conditions (pH 7.0). Interestingly, CcTET also converts the majority of 5mC to 5fC under slightly acidic (pH 5.8) and neutral conditions. Kinetics analyses of the oxidation by CcTET under neutral conditions indicate that conversion of 5mC to 5hmC and 5hmC to 5fC are faster than that of 5fC to 5caC, respectively. Our results provide an example of a TET homologue in a non-mammalian organism that exhibits full 5mC-to-5caC oxidation activity and a slight preference to producing 5fC. The preferential accumulation of 5fC in the in vitro oxidation reactions under both neutral and acidic conditions may have biological implications for 5mC oxidation in fungi species.

Identification of a proteolysis regulator for an essential enzyme in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis proteins that are post-translationally modified with Pup, a prokaryotic ubiquitin-like protein, can be degraded by proteasomes. While pupylation is reversible, mechanisms regulating substrate specificity have not been identified. Here, we identify the first depupylation regulators: CoaX, a pseudokinase, and pantothenate, an essential, central metabolite. In a Δ coaX mutant, pantothenate synthesis enzymes were more abundant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome-dependent manner. In vitro , CoaX accelerated depupylation of Pup∼PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pup∼PanB in response to pantothenate levels. One Sentence Summary: A pseudo-pantothenate kinase regulates proteasomal degradation of a pantothenate synthesis enzyme in M. tuberculosis .