Significance In animals, gut microbes are essential for digestion. Here, we show that bacteria outside the gut can also play a critical role in digestion. In shipworms, wood-eating marine bivalves, ...endosymbiotic bacteria are found within specialized cells in the gills. We show that these endosymbionts produce wood-degrading enzymes that are selectively transported to the shipworm’s bacteria-free gut, where wood digestion occurs. Because only selected wood-degrading enzymes are transported, the shipworm system naturally identifies those endosymbiont enzymes most relevant to lignocellulose deconstruction without interference from other microbial proteins. Thus, this work expands the known biological repertoire of bacterial endosymbionts to include digestion of food and identifies previously undescribed enzymes and enzyme combinations of potential value to biomass-based industries, such as cellulosic biofuel production.
Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea , wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.
Modified DNA bases in mammalian genomes, such as 5-methylcytosine ((5m)C) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic ...conversion of (5m)C to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like (5m)C oxygenase (NgTET1) from Naegleria gruberi, a single-celled protist evolutionarily distant from vertebrates. Here we show that NgTET1 is a 5-methylpyrimidine oxygenase, with activity on both (5m)C (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine ((5f)U) and 5-carboxyuridine ((5ca)U) in vitro. Mutagenesis studies reveal a delicate balance between choice of (5m)C or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to (5m)CpG and TpG dinucleotide sites in DNA. Intriguingly, NgTET1 displays higher T-oxidation activity in vitro than mammalian TET1, supporting a closer evolutionary relationship between NgTET1 and the base J-binding proteins from trypanosomes. Finally, we demonstrate that NgTET1 can be readily used as a tool in (5m)C sequencing technologies such as single molecule, real-time sequencing to map (5m)C in bacterial genomes at base resolution.
Modified DNA bases in mammalian genomes, such as 5-methylcytosine (5mC) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic ...conversion of5mC to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like5mC oxygenase (NgTET1) fromNaegleria gruberi, a single-celled protist evolutionarily distant from vertebrates. Here we show that NgTET1 is a 5-methylpyrimidine oxygenase, with activity on both5mC (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine (5fU) and 5-carboxyuridine (5caU) in vitro. Mutagenesis studies reveal a delicate balance between choice of5mC or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to5mCpG and TpG dinucleotide sites in DNA. Intriguingly, NgTET1 displays higher T-oxidation activity in vitro than mammalian TET1, supporting a closer evolutionary relationship between NgTET1 and the base J-binding proteins from trypanosomes. Finally, we demonstrate that NgTET1 can be readily used as a tool in5mC sequencing technologies such as single molecule, realtime sequencing to map5mC in bacterial genomes at base resolution.
Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the ...pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX (BacteRiophage EXclusion) systems. These RM-like activities employ host protection by DNA modification, but immediate replication arrest occurs without evident of nuclease action on unmodified phage DNA. Here we show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a variant BREX system. A laboratory strain disabled for both the restriction and methylation activity of StySA nevertheless has wild type sequence in pglX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (μ) in their C-terminal domains. We further investigate this system in situ, replacing the mutated pglZμ and brxCμ genes with the WT counterpart. PglZ-WT supports methylation in the presence of either BrxCμ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. These results suggests that while BrxC, PglZ and PglX are principal components of the BREX modification activity, BrxL is required for restriction only. Furthermore, we show that a partial disruption of brxL disrupts transcription globally.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to ...prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT®) DNA sequencing that is capable of ...direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.
Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as ...straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that "write" these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A rising incidence of meningococcal serogroup W disease has been evident in many countries worldwide. Serogroup W isolates belonging to the sequence type (ST)-11 clonal complex have been associated ...with atypical symptoms and increased case fatality rates. The continued expansion of this clonal complex in the later part of the 2010s has been largely due to a shift from the so-called original UK strain to the 2013 strain. Here we used single-molecule real-time (SMRT) sequencing to determine the methylomes of the two major serogroup W strains belonging to ST-11 clonal complex. Five methylated motifs were identified in this study, and three of the motifs, namely 5'-GATC-3', 5'-GAAGG-3', 5'-GCGCGC-3', were found in all 13 isolates investigated. The results showed no strain-specific motifs or difference in active restriction modification systems between the two strains. Two phase variable methylases were identified and the enrichment or depletion of the methylation motifs generated by these methylases varied between the two strains. Results from this work give further insight into the low diversity of methylomes in highly related strains and encourage further research to decipher the role of regions with under- or overrepresented methylation motifs.
Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as ...opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genomes and their exact roles in the cell remain to be defined. In this study, we developed a simple and convenient technique called EcoWI-seq based on a modification-dependent restriction endonuclease to identify genomic positions of PT modifications. EcoWI-seq shows similar performance than other PT modification detection techniques and additionally, is easily scalable while requiring little starting material. As a proof of principle, we applied EcoWI-seq to map the PT modifications at base resolution in the genomes of both the Salmonella enterica cerro 87 and E. coli expressing the dnd+ gene cluster. Specifically, we address whether the partial establishment of modified PT positions is a stochastic or deterministic process. EcoWI-seq reveals a systematic usage of the same subset of target sites in clones for which the PT modification has been independently established.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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