ABSTRACT
Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.
Subject(s)
Bacteriophages , Guanine , Bacteria/genetics , Bacteriophages/genetics , DNA/genetics , Guanine/analogs & derivativesABSTRACT
The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.
Subject(s)
Bacteriophages , Lyases , Amino Acids/metabolism , Bacteriophages/genetics , DNA/metabolism , Thymidine/metabolismABSTRACT
TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis. Both enzymes have roles in epigenetic regulation. It has been hypothesized that all TET/JBPs have their ancestral origins in bacteriophages, but only eukaryotic orthologs have been described. Here we demonstrate the 5mC-dioxygenase activity of several phage TETs encoded within viral metagenomes. The clustering of these TETs in a phylogenetic tree correlates with the sequence specificity of their genomically cooccurring cytosine C5-methyltransferases, which install the methyl groups upon which TETs operate. The phage TETs favor Gp5mC dinucleotides over the 5mCpG sites targeted by the eukaryotic TETs and are found within gene clusters specifying complex cytosine modifications that may be important for DNA packaging and evasion of host restriction.
Subject(s)
5-Methylcytosine/metabolism , Bacteriophages/metabolism , DNA/metabolism , Amino Acid Sequence , DNA Methylation , Dioxygenases , Hydroxylation , Metagenomics , Nucleotide Motifs/genetics , Oxidation-Reduction , PhylogenyABSTRACT
Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.
Subject(s)
Bacteriophages , Bacteriophages/physiology , Phylogeny , Biological Evolution , Bacteria , EnglandABSTRACT
Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the Delftia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635-7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.
Subject(s)
Bacteria/metabolism , Bacterial Infections/microbiology , Bacterial Proteins/metabolism , Bacteriophages/genetics , DNA, Viral/biosynthesis , Thymidine/chemistry , Uridine/chemistry , Bacteriophages/growth & development , Bacteriophages/metabolism , Genome, ViralABSTRACT
Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation, suggesting that these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: the genera Firehammervirus and Fletchervirus Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2'-deoxyguanosine (dG) residues are replaced by modified bases. Fletcherviruses replace dG with 2'-deoxyinosine, while the firehammerviruses replace dG with 2'-deoxy-7-amido-7-deazaguanosine (dADG), noncanonical nucleotides previously described, but a 100% base substitution has never been observed to have been made in a virus. We analyzed the genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these noncanonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA.IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts' defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain noncanonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been underreported. To date, two genomic guanosine modifications have been observed in phage genomes, and both are low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which replace all guanosine bases in the genome in a genus-specific manner.
Subject(s)
Bacteriophages/genetics , Campylobacter jejuni/virology , Deoxyguanosine/genetics , Inosine/genetics , Biosynthetic Pathways/genetics , DNA, Viral/genetics , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Escherichia coli/metabolism , Escherichia coli/virology , Genome, Viral , Inosine/analogs & derivatives , Inosine/metabolism , Myoviridae/genetics , PhylogenyABSTRACT
Using amber suppression in coordination with a mutant pyrrolysyl-tRNA synthetase-tRNAPyl pair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site-specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su-H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post-translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.
Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Lysine/analysis , Protein Processing, Post-Translational , Acylation , Amino Acyl-tRNA Synthetases/genetics , Azides , Histones/genetics , Lysine/genetics , Norleucine/analogs & derivatives , Norleucine/geneticsABSTRACT
Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.
Subject(s)
Bacterial Proteins/chemistry , Phenylalanine/analogs & derivatives , Phenylalanine/chemistry , Phosphoinositide Phospholipase C/chemistry , Tyrosine/analogs & derivatives , Amino Acid Sequence , Cations , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Membrane Lipids/chemistry , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Transport , Staphylococcus aureus/enzymology , Tyrosine/chemistryABSTRACT
A new type of click reaction between an alkyl phosphine and acrylamide was developed and applied for site-specific protein labeling in vitro and in live cells. Acrylamide is a small electrophilic olefin that readily undergoes phospha-Michael addition with an alkyl phosphine. Our kinetic study indicated a second-order rate constant of 0.07 m(-1) s(-1) for the reaction between tris(2-carboxyethyl)phosphine and acrylamide at pHâ 7.4. To demonstrate its application in protein functionalization, we used a dansyl-phosphine conjugate to successfully label proteins that were site-specifically installed with N(É) -acryloyl-l-lysine and employed a biotin-phosphine conjugate to selectively probe human proteins that were metabolically labeled with N-acryloyl-galactosamine.
Subject(s)
Click Chemistry , Proteins/chemistry , Acrylamide/chemistry , Kinetics , Spectrometry, Mass, Electrospray IonizationABSTRACT
Will Ub my partner? The ubiquitin (Ub)-activating enzyme (E1) was used to catalyze an amidation reaction to functionalize the C terminus of Ub with unique functional groups, such as thiol, azide, alkyne, and alkene groups, with high efficiency and yield (>90 %). These groups were then applied for the facile synthesis of fluorophore-conjugated ubiquitin and specifically conjugated diubiquitin substrates for deubiquitinases.
Subject(s)
Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Specific Proteases/metabolism , Ubiquitin/metabolism , Alkenes/chemistry , Alkenes/metabolism , Alkynes/chemistry , Alkynes/metabolism , Animals , Azides/chemistry , Azides/metabolism , Biocatalysis , Kinetics , Protein Structure, Tertiary , Substrate Specificity , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolism , Ubiquitin/chemistry , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Specific Proteases/chemistry , UbiquitinationABSTRACT
Detailed kinetic analyses of inverse electron-demand DielsAlder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these "click" reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.
Subject(s)
Alkenes/chemistry , Alkynes/chemistry , Click Chemistry , Proteins/chemistry , Staining and Labeling/methods , Catalysis , Cyclooctanes/chemistry , Escherichia coli Proteins/chemistry , Kinetics , Models, Molecular , Protein Conformation , Tyrosine/chemistryABSTRACT
The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.
Subject(s)
Bacteriophages , Enterococcus faecalis , Viral Proteins , Enterococcus faecalis/virology , Enterococcus faecalis/genetics , Bacteriophages/genetics , Bacteriophages/physiology , Viral Proteins/metabolism , Viral Proteins/genetics , DNA Restriction Enzymes/metabolism , DNA Restriction Enzymes/genetics , Drug Resistance, Multiple, Bacterial/genetics , Plasmids/genetics , Vancomycin-Resistant Enterococci/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Bacterial Proteins/geneticsABSTRACT
Golden Gate Assembly is a flexible method of DNA assembly and cloning that permits the joining of multiple fragments in a single reaction through predefined connections. The method depends on cutting DNA using a Type IIS restriction enzyme, which cuts outside its recognition site and therefore can generate overhangs of any sequence while separating the recognition site from the generated fragment. By choosing compatible fusion sites, Golden Gate permits the joining of multiple DNA fragments in a defined order in a single reaction. Conventionally, this method has been used to join five to eight fragments in a single assembly round, with yield and accuracy dropping off rapidly for more complex assemblies. Recently, we demonstrated the application of comprehensive measurements of ligation fidelity and bias data using data-optimized assembly design (DAD) to enable a high degree of assembly accuracy for very complex assemblies with the simultaneous joining of as many as 52 fragments in one reaction. Here, we describe methods for applying DAD principles and online tools to evaluate the fidelity of existing fusion site sets and assembly standards, selecting new optimal sets, and adding fusion sites to existing assemblies. We further describe the application of DAD to divide known sequences at optimal points, including designing one-pot assemblies of small genomes. Using the T7 bacteriophage genome as an example, we present a protocol that includes removal of native Type IIS sites (domestication) simultaneously with parts generation by PCR. Finally, we present recommended cycling protocols for assemblies of medium to high complexity (12-36 fragments), methods for producing high-quality parts, examples highlighting the importance of DNA purity and fragment stoichiometric balance for optimal assembly outcomes, and methods for assessing assembly success. © 2023 New England Biolabs, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Assessing the fidelity of an overhang set using the NEBridge Ligase Fidelity Viewer Basic Protocol 2: Generating a high-fidelity overhang set using the NEBridge GetSet Tool Alternate Protocol 1: Expanding an existing overhang set using the NEBridge GetSet Tool Basic Protocol 3: Dividing a genomic sequence with optimal fusion sites using the NEBridge SplitSet Tool Basic Protocol 4: One-pot Golden Gate Assembly of 12 fragments into a destination plasmid Alternate Protocol 2: One-pot Golden Gate Assembly of 24+ fragments into a destination plasmid Basic Protocol 5: One-pot Golden Gate Assembly of the T7 bacteriophage genome from 12+ parts Support Protocol 1: Generation of high-purity amplicons for assembly Support Protocol 2: Cloning assembly parts into a holding vector Support Protocol 3: Quantifying DNA concentration using a Qubit 4 fluorometer Support Protocol 4: Visualizing large assemblies via TapeStation Support Protocol 5: Validating phage genome assemblies via ONT long-read sequencing.
Subject(s)
Bacteriophage T7 , Bacteriophages , Bicycling , DNA Restriction Enzymes , DomesticationABSTRACT
Nucleic acids in living organisms are more complex than the simple combinations of the four canonical nucleotides. Recent advances in biomedical research have led to the discovery of numerous naturally occurring nucleotide modifications and enzymes responsible for the synthesis of such modifications. In turn, these enzymes can be leveraged towards toolkits for DNA and RNA manipulation for epigenetic sequencing or other biotechnological applications. Here, we present the protocol to obtain purified 5-hydroxymethylcytosine carbamoyltransferase enzymes and the associated assays to convert 5-hydroxymethylcytosine to 5-carbamoyloxymethylcytosine in vitro . We include detailed assays using DNA, RNA, and single nucleotide/deoxynucleotide as substrates. These assays can be combined with downstream applications for genetic/epigenetic regulatory mechanism studies and next-generation sequencing purposes.
ABSTRACT
The antibiotic neocarzinostatin comprises a carrier protein with a well-defined cavity for accommodating an active enediyne chromophore. The protein has two disulfides, one (Cys(37)-Cys(47)) lies on the cavity bottom and the other (Cys(88)-Cys(93)) in a constrained short loop. When the chromophore is not bound to the protein, a thiol-induced cycloaromatization of the enediyne into a tetrahydroindacene derivative is responsible for the potent antitumor activity. When it is protein-bound, the protein diverts the cycloaromatization pathway to form a distinct hydroxyisochromene-type product. How the protein directs the enediyne chemistry is an interesting puzzle, and various suggestions have been proposed in the past. We screened more than fifty thiols and manipulated conditions to locate reaction features and search for factors that could influence the protein directing strength. Thiol- and oxygen-concentration-dependence studies suggested that disulfides, which maintain the steric rigidity of the protein, could play a key role in diverting the cycloaromatization pathway. For direct proofs, we made mutations at each of the two disulfides by replacing sulfur atoms with oxygen. Circular dichroism and two-dimensional NMR spectroscopy studies suggested that the mutations changed neither the protein conformation nor the ligand interactions. Analyses of the thiol-induced cycloaromatization revealed that rupture of Cys(37)-Cys(47) made the protein almost completely lose its chemical directing ability, whereas rupture of Cys(88)-Cys(93) had only a minor influence. The results demonstrated that the steric rigidity of the binding cavity, but not necessary the whole protein, played an important role in the protein-directed mechanism.
Subject(s)
Carrier Proteins/chemistry , Cysteine/chemistry , Enediynes/chemistry , Zinostatin/chemistry , Antibiotics, Antineoplastic/chemistry , Carrier Proteins/metabolism , Cysteine/metabolism , Ligands , Magnetic Resonance Spectroscopy , Molecular Structure , Protein Binding , Protein Conformation , Sulfhydryl Compounds/chemistryABSTRACT
Collectively, the dsDNA tailed bacteriophages (Caudovirales) contain the largest chemical diversity of naturally occurring deoxynucleotides in DNA observed to date. The continuing discovery of new modifications in phages suggest many more are waiting to be found. Thus, methods for the observation and characterization of noncanonical nucleosides are timely. We present here protocols for extraction of genomic DNA from bacteriophage particles, enzymatic hydrolysis of DNA to free nucleosides, and examination of nucleoside composition by HPLC and mass spectrometry.
Subject(s)
Bacteriophages/genetics , DNA, Viral , Epigenesis, Genetic , Epigenomics , Genome, Viral , Chromatography, High Pressure Liquid , DNA Methylation , Epigenomics/methods , Hydrolysis , Mass SpectrometryABSTRACT
The DNA in bacterial viruses collectively contains a rich, yet relatively underexplored, chemical diversity of nucleobases beyond the canonical adenine, guanine, cytosine, and thymine. Herein, we review what is known about the genetic and biochemical basis for the biosynthesis of complex DNA modifications, also called DNA hypermodifications, in the DNA of tailed bacteriophages infecting Escherichia coli and Salmonella enterica. These modifications, and their diversification, likely arose out of the evolutionary arms race between bacteriophages and their cellular hosts. Despite their apparent diversity in chemical structure, the syntheses of various hypermodified bases share some common themes. Hypermodifications form through virus-directed synthesis of noncanonical deoxyribonucleotide triphosphates, direct modification DNA, or a combination of both. Hypermodification enzymes are often encoded in modular operons reminiscent of biosynthetic gene clusters observed in natural product biosynthesis. The study of phage-hypermodified DNA provides an exciting opportunity to expand what is known about the enzyme-catalyzed chemistry of nucleic acids and will yield new tools for the manipulation and interrogation of DNA.
Subject(s)
Bacteriophages , Salmonella enterica , Bacteriophages/genetics , DNA , Escherichia coli/genetics , ThymineABSTRACT
Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1-3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria4,5 that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6-10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11-13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.14 In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family ("Naomiviridae"). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment.
Subject(s)
Bacteriophages , DNA, Viral/chemistry , Genome, Viral , Roseobacter , Bacteriophages/classification , Deoxyuridine/chemistry , Ecosystem , Phylogeny , Roseobacter/virology , Thymidine/chemistryABSTRACT
Shotgun metagenomic sequencing is a powerful approach to study microbiomes in an unbiased manner and of increasing relevance for identifying novel enzymatic functions. However, the potential of metagenomics to relate from microbiome composition to function has thus far been underutilized. Here, we introduce the Metagenomics Genome-Phenome Association (MetaGPA) study framework, which allows linking genetic information in metagenomes with a dedicated functional phenotype. We applied MetaGPA to identify enzymes associated with cytosine modifications in environmental samples. From the 2365 genes that met our significance criteria, we confirm known pathways for cytosine modifications and proposed novel cytosine-modifying mechanisms. Specifically, we characterized and identified a novel nucleic acid-modifying enzyme, 5-hydroxymethylcytosine carbamoyltransferase, that catalyzes the formation of a previously unknown cytosine modification, 5-carbamoyloxymethylcytosine, in DNA and RNA. Our work introduces MetaGPA as a novel and versatile tool for advancing functional metagenomics.
Many industrial processes, such as starch processing and oil refinement, use chemicals that cause harm to the environment. These can often be switched to more sustainable biological processes that are powered by proteins called enzymes. Enzymes are micro-factories that speed up biochemical reactions in most living things. Communities of microorganisms (also known as microbiomes) are an amazing but often untapped resource for discovering enzymes that can be harnessed for industrial purposes. To gain a better picture of the microbes present within a population, researchers often extract and sequence the genetic material of all microorganisms in an environmental sample, also known as the metagenome. While current methods for analyzing the metagenome are good at identifying new species, they often provide limited information about the microorganism's functional role within the community. This makes it difficult to find new enzymes that may be useful for industry. Here, Yang, Lin et al. have developed a new technique called Metagenomics Genome-Phenome Association, or MetaGPA for short. The method works in a similar way to genome-wide association studies (GWAS) which are used to identify genes involved in human disease. However, instead of disease associated genes in humans, MetaGPA finds microbial genes that are associated with a biological process useful for biotechnology. Like GWAS, the new approach created by Yang, Lin et al. compares two groups: the first contains microorganisms that carry out a specific process, and the second contains all organisms in the microbiome. The metagenome of each group is extracted and a computational pipeline is then applied to identify genes, including those coding for enzymes, that are found more often in the group performing the desired task. To test the technique, Yang, Lin et al. used MetGPA to find new enzymes involved in DNA modification. Microbiome samples were collected from coastal water and sewage, and the computational pipeline was applied to discover genes that are associated with this process. Further analysis revealed that one of the identified genes codes for an enzyme that introduces a previously unknown change to DNA. MetaGPA could be applied to other processes and microbiomes, and, if successful, may help researchers to identify more diverse enzymes than is currently available. This could scale up the discovery of new enzymes that can be used to power industrial reactions.
Subject(s)
Cytosine/metabolism , DNA, Bacterial/metabolism , Escherichia coli K12/genetics , Genome, Bacterial , Microbiota/genetics , RNA, Bacterial/metabolismABSTRACT
To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5-NedU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among >200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC (TS, S=C, or G), implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine.