Diverse Durham collection phages demonstrate complex BREX defense responses.

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.

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.

Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor.

Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.

Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system.

Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.

The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses.

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.

Structural and functional diversity among Type III restriction-modification systems that confer host DNA protection via methylation of the N4 atom of cytosine.

We report a new subgroup of Type III Restriction-Modification systems that use m4C methylation for host protection. Recognition specificities for six such systems, each recognizing a novel motif, have been determined using single molecule real-time DNA sequencing. In contrast to all previously characterized Type III systems which modify adenine to m6A, protective methylation of the host genome in these new systems is achieved by the N4-methylation of a cytosine base in one strand of an asymmetric 4 to 6 base pair recognition motif. Type III systems are heterotrimeric enzyme complexes containing a single copy of an ATP-dependent restriction endonuclease-helicase (Res) and a dimeric DNA methyltransferase (Mod). The Type III Mods are beta-class amino-methyltransferases, examples of which form either N6-methyl adenine or N4-methyl cytosine in Type II RM systems. The Type III m4C Mod and Res proteins are diverged, suggesting ancient origin or that m4C modification has arisen from m6A MTases multiple times in diverged lineages. Two of the systems, from thermophilic organisms, required expression of both Mod and Res to efficiently methylate an E. coli host, unlike previous findings that Mod alone is proficient at modification, suggesting that the division of labor between protective methylation and restriction activities is atypical in these systems. Two of the characterized systems, and many homologous putative systems, appear to include a third protein; a conserved putative helicase/ATPase subunit of unknown function and located 5' of the mod gene. The function of this additional ATPase is not yet known, but close homologs co-localize with the typical Mod and Res genes in hundreds of putative Type III systems. Our findings demonstrate a rich diversity within Type III RM systems.

Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by CryoEM.

Restriction enzymes that combine methylation and cleavage into a single assemblage and modify one DNA strand are capable of efficient adaptation toward novel targets. However, they must reliably cleave invasive DNA and methylate newly replicated unmodified host sites. One possible solution is to enforce a competition between slow methylation at a single unmodified host target, versus faster cleavage that requires multiple unmodified target sites in foreign DNA to be brought together in a reaction synapse. To examine this model, we have determined the catalytic behavior of a bifunctional type IIL restriction-modification enzyme and determined its structure, via cryoelectron microscopy, at several different stages of assembly and coordination with bound DNA targets. The structures demonstrate a mechanism in which an initial dimer is formed between two DNA-bound enzyme molecules, positioning the endonuclease domain from each enzyme against the other's DNA and requiring further additional DNA-bound enzyme molecules to enable cleavage.

Complete Genome Sequence and Methylome Analysis of Deinococcus wulumuqiensis 479.

Deinococcus wulumuqiensis 479 (formerly known as Deinococcus radiodurans 479) is the original source strain for the restriction enzyme DrdI. Its complete sequence and full methylome were determined using Pacific Biosciences single-molecule real-time (SMRT) sequencing.

A model for the evolution of prokaryotic DNA restriction-modification systems based upon the structural malleability of Type I restriction-modification enzymes.

Restriction Modification (RM) systems prevent the invasion of foreign genetic material into bacterial cells by restriction and protect the host's genetic material by methylation. They are therefore important in maintaining the integrity of the host genome. RM systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Comparing the structures of the different types, similarities can be observed suggesting an evolutionary link between these different types. This work describes the 'deconstruction' of a large Type I RM enzyme into forms structurally similar to smaller Type II RM enzymes in an effort to elucidate the pathway taken by Nature to form these different RM enzymes. Based upon the ability to engineer new enzymes from the Type I 'scaffold', an evolutionary pathway and the evolutionary pressures required to move along the pathway from Type I RM systems to Type II RM systems are proposed. Experiments to test the evolutionary model are discussed.

The non-specific adenine DNA methyltransferase M.EcoGII.

We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo - using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using purified recombinant M.EcoGII enzyme, demonstrate that up to 99% of dA bases in duplex DNA substrates can be methylated thereby rendering them insensitive to cleavage by multiple restriction endonucleases. These properties suggest that the enzyme could also be used for high resolution mapping of protein binding sites in DNA and RNA substrates.

DNA target recognition domains in the Type I restriction and modification systems of Staphylococcus aureus.

Staphylococcus aureus displays a clonal population structure in which horizontal gene transfer between different lineages is extremely rare. This is due, in part, to the presence of a Type I DNA restriction-modification (RM) system given the generic name of Sau1, which maintains different patterns of methylation on specific target sequences on the genomes of different lineages. We have determined the target sequences recognized by the Sau1 Type I RM systems present in a wide range of the most prevalent S. aureus lineages and assigned the sequences recognized to particular target recognition domains within the RM enzymes. We used a range of biochemical assays on purified enzymes and single molecule real-time sequencing on genomic DNA to determine these target sequences and their patterns of methylation. Knowledge of the main target sequences for Sau1 will facilitate the synthesis of new vectors for transformation of the most prevalent lineages of this 'untransformable' bacterium.

Novel m4C modification in type I restriction-modification systems.

We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase subunits from these systems group into two families, one of which has NPPF in the highly conserved catalytic motif IV and modifies adenine to m6A, and one having an NPPY catalytic motif IV and modifying cytosine to m4C. The high degree of similarity among their cytosine-recognizing components (MTase and S) suggest they have recently evolved, most likely from the far more common m6A Type I systems. Type I enzymes that modify cytosine exclusively were formed by replacing the adenine target recognition domain (TRD) with a cytosine-recognizing TRD. These are the first examples of m4C modification in Type I RM systems.

Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities.

The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5'-TCCRAC-3'; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5'-TCCRAC-3'). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5'-TCCRAC-3'). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N(6)-adenine DNA methyltransferases of Neisseria meningitidis.

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N(6)-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N(6)-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY M6A: G-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC M6A: CC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC M6A: GC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY M6A: G-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC M6A: GG-3' sites, to 100% at 5'-ACGT M6A: GG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity.

The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature.

The complete genome of Teredinibacter turnerae T7901: an intracellular endosymbiont of marine wood-boring bivalves (shipworms).

Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2-40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels.

The MmeI family: type II restriction-modification enzymes that employ single-strand modification for host protection.

The type II restriction endonucleases form one of the largest families of biochemically-characterized proteins. These endonucleases typically share little sequence similarity, except among isoschizomers that recognize the same sequence. MmeI is an unusual type II restriction endonuclease that combines endonuclease and methyltransferase activities in a single polypeptide. MmeI cuts DNA 20 bases from its recognition sequence and modifies just one DNA strand for host protection. Using MmeI as query we have identified numerous putative genes highly similar to MmeI in database sequences. We have cloned and characterized 20 of these MmeI homologs. Each cuts DNA at the same distance as MmeI and each modifies a conserved adenine on only one DNA strand for host protection. However each enzyme recognizes a unique DNA sequence, suggesting these enzymes are undergoing rapid evolution of DNA specificity. The MmeI family thus provides a rich source of novel endonucleases while affording an opportunity to observe the evolution of DNA specificity. Because the MmeI family enzymes employ modification of only one DNA strand for host protection, unlike previously described type II systems, we propose that such single-strand modification systems be classified as a new subgroup, the type IIL enzymes, for Lone strand DNA modification.

Quantitative imaging of nitrogen fixation by individual bacteria within animal cells.

Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonia for biosynthesis, is exclusively performed by a few bacteria and archaea. Despite the essential importance of biological nitrogen fixation, it has been impossible to quantify the incorporation of nitrogen by individual bacteria or to map the fate of fixed nitrogen in host cells. In this study, with multi-isotope imaging mass spectrometry we directly imaged and measured nitrogen fixation by individual bacteria within eukaryotic host cells and demonstrated that fixed nitrogen is used for host metabolism. This approach introduces a powerful way to study microbes and global nutrient cycles.

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.

BACKGROUND: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. RESULTS: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. CONCLUSION: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

Extensive variation in intracellular symbiont community composition among members of a single population of the wood-boring bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae).

Shipworms (wood-boring bivalves of the family Teredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species, Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the gamma subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiont Teredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.