Protein Ocr Research Articles

Architecture and activation mechanism of the bacterial PARIS defence system.

Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race1-5. While the mechanisms of many bacterial antiphage defence systems are known1, how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

Ocr-mediated suppression of BrxX unveils a phage counter-defense mechanism.

The burgeoning crisis of antibiotic resistance has directed attention to bacteriophages as natural antibacterial agents capable of circumventing bacterial defenses. Central to this are the bacterial defense mechanisms, such as the BREX system, which utilizes the methyltransferase BrxX to protect against phage infection. This study presents the first in vitro characterization of BrxX from Escherichiacoli, revealing its substrate-specific recognition and catalytic activity. We demonstrate that BrxX exhibits nonspecific DNA binding but selectively methylates adenine within specific motifs. Kinetic analysis indicates a potential regulation of BrxX by the concentration of its co-substrate, S-adenosylmethionine, and suggests a role for other BREX components in modulating BrxX activity. Furthermore, we elucidate the molecular mechanism by which the T7 phage protein Ocr (Overcoming classical restriction) inhibits BrxX. Despite low sequence homology between BrxX from different bacterial species, Ocr effectively suppresses BrxX's enzymatic activity through high-affinity binding. Cryo-electron microscopy and biophysical analyses reveal that Ocr, a DNA mimic, forms a stable complex with BrxX, highlighting a conserved interaction interface across diverse BrxX variants. Our findings provide insights into the strategic counteraction by phages against bacterial defense systems and offer a foundational understanding of the complex interplay between phages and their bacterial hosts, with implications for the development of phage therapy to combat antibiotic resistance.

Generation of tools for expression and purification of the phage-encoded Type I restriction enzyme inhibitor, Ocr.

DNA manipulation is an essential tool in molecular microbiology research that is dependent on the ability of bacteria to take up and preserve foreign DNA by horizontal gene transfer. This process can be significantly impaired by the activity of bacterial restriction modification systems; bacterial operons comprising paired enzymatic activities that protectively methylate host DNA, while cleaving incoming unmodified foreign DNA. Ocr is a phage-encoded protein that inhibits Type I restriction modification systems, the addition of which significantly improves bacterial transformation efficiency. We recently established an improved and highly efficient transformation protocol for the important human pathogen group A Streptococcus using commercially available recombinant Ocr protein, manufacture of which has since been discontinued. In order to ensure the continued availability of Ocr protein within the research community, we have generated tools and methods for in-house Ocr production and validated the activity of the purified recombinant protein.

Methylome-dependent transformation of emm1 group A streptococci.

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A Streptococcus (GAS), a lethal human pathogen currently associated with an unprecedented surge of infections worldwide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this "restriction barrier" thus presents a major technical challenge. Here, we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Furthermore, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRDAG, encoded by all sequenced strains of the dominant and upsurge-associated emm1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol, whereby the restriction barrier is overcome by the addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRDAG strains including clinical isolates representing all emm1 lineages and will expedite critical research interrogating the genetics of emm1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation and the importance of defining lineage-specific mechanisms of genetic recalcitrance. IMPORTANCE Understanding the mechanisms by which bacterial pathogens are able to cause disease is essential to enable the targeted development of novel therapeutics. A key experimental approach to facilitate this research is the generation of bacterial mutants, through either specific gene deletions or sequence manipulation. This process relies on the ability to transform bacteria with exogenous DNA designed to generate the desired sequence changes. Bacteria have naturally developed protective mechanisms to detect and destroy invading DNA, and these systems severely impede the genetic manipulation of many important pathogens, including the lethal human pathogen group A Streptococcus (GAS). Many GAS lineages exist, of which emm1 is dominant among clinical isolates. Based on new experimental evidence, we identify the mechanism by which transformation is impaired in the emm1 lineage and establish an improved and highly efficient transformation protocol to expedite the generation of mutants.

Structural features of a minimal intact methyltransferase of a type I restriction-modification system

Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2–4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M2S1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S1/2-domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M1S1/2)2 structure reveals a symmetric (S1/2)2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M1S1/2 MTase dimer indicates a particular state resembling the structure of M2S1 MTases.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence.

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction–modification systems, is also active against BREX. In contrast to the wild–type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R–M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7.

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.

ArdB Protective Activity for Unmodified λ Phage Against EcoKI Restriction Decreases in UV-Treated Escherichia coli.

Anti-restriction proteins ArdB/KlcA specifically inhibit restriction (endonuclease) activity of restriction-modification (RM) type I systems. Molecular mechanisms of ArdB/KlcA-based anti-restriction remain unknown. In this study, we quantitate effects of ArdB on protection of unmodified λ phage DNA from EcoKI restriction. After UV irradiations, which produce significant amounts of unmodified chromosomal DNA in Escherichia coli K12 cells, the protective activity of ArdB decreases. Unlike ArdB, DNA-mimicking protein Ocr retains its ability to protect the unmodified λ phage regardless of UV dose. We hypothesize that the observed decrease in ArdB protective activity in UV-treated cells is due to its binding to unmodified chromosomal DNA, which decreases effective concentrations of free ArdB molecules available for λ phage protection against type I restriction enzymes.

The evolutionary pathway from a biologically inactive polypeptide sequence to a folded, active structural mimic of DNA.

The protein Ocr (overcome classical restriction) from bacteriophage T7 acts as a mimic of DNA and inhibits all Type I restriction/modification (RM) enzymes. Ocr is a homodimer of 116 amino acids and adopts an elongated structure that resembles the shape of a bent 24 bp DNA molecule. Each monomer includes 34 acidic residues and only six basic residues. We have delineated the mimicry of Ocr by focusing on the electrostatic contribution of its negatively charged amino acids using directed evolution of a synthetic form of Ocr, termed pocr, in which all of the 34 acidic residues were substituted for a neutral amino acid. In vivo analyses confirmed that pocr did not display any antirestriction activity. Here, we have subjected the gene encoding pocr to several rounds of directed evolution in which codons for the corresponding acidic residues found in Ocr were specifically re-introduced. An in vivo selection assay was used to detect antirestriction activity after each round of mutation. Our results demonstrate the variation in importance of the acidic residues in regions of Ocr corresponding to different parts of the DNA target which it is mimicking and for the avoidance of deleterious effects on the growth of the host.

Exploring the DNA mimicry of the Ocr protein of phage T7.

DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.

Antimodification activity of the ArdA and Ocr proteins

The ArdA and Ocr antirestriction proteins, whose genes are in transmissible plasmids (ardA) and bacteriophage genomes (0.3 (ocr)), specifically inhibit type I restriction-modification enzymes. The Ocr protein (T7 bacteriophage) was shown to inhibit both restriction (endonuclease) and modification (methylase) activities of the EcoKI enzyme in a broad range of intracellular concentrations (starting from 10-20 molecules per cell). In contrast to Ocr, the ArdA protein (ColIb-P9 transmissible plasmid) inhibited both of the EcoKI activities only at high intracellular concentrations (30000-40000 molecules per cell). When the ArdA concentration was several fold lower, only endonuclease activity of EcoKI was inhibited. It was assumed that a poorer ArdA ability to inhibit EcoKI modification activity is related to the substantial difference in life cycle between transmissible plasmids (symbiosis with the bacterial cell) and bacteriophages (infection and lysis of bacteria). The Ocr and ArdA mutants that inhibited exclusively endonuclease activity of EcoKI were obtained. Antirestriction proteins incapable of homodimerization were assumed to inhibit only endonuclease activity of type I restriction-modification enzymes.

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Förster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR+ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10−10 M versus 10−7 M).

A Dna Mimic Caught In The Act: 3D Electron Microscopy Shows Ecoki Methyltransferase In Complex With The T7 Antirestriction Protein Ocr.

Type I DNA restriction-modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of the DNA target or translocation, followed by cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein Ocr. Ocr mimics DNA with a pseudo-helical arrangement of charges, and is bent at a similar angle to that predicted for target DNA. We present a 3D density map generated by negative stain electron microscopy of the central core of the restriction complex, M.EcoKI M2S1 methyltransferase, bound to dimeric Ocr. Single particle analysis was carried out in IMAGIC and EMAN and resulted in a 3D reconstruction at ∼18 A resolution. An atomic model of all 5 subunits was generated by automated docking and homology modelling. This was computationally fitted into the EM density, giving excellent agreement. Ocr binds through the center of the M.EcoKI complex, spanning the two DNA recognition sites and matching the path predicted for its substrate DNA. We also present a complete atomic model of M.EcoKI in complex with its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of published experimental data on EcoKI spanning 40 years.

The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein.

Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.

A mutational analysis of DNA mimicry by ocr, the gene 0.3 antirestriction protein of bacteriophage T7

The ocr protein of bacteriophage T7 is a structural and electrostatic mimic of approximately 24 base pairs of double-stranded B-form DNA. As such, it inhibits all Type I restriction and modification (R/M) enzymes by blocking their DNA binding grooves and inactivates them. This allows the infection of the bacterial cell by T7 to proceed unhindered by the action of the R/M defence system. We have mutated aspartate and glutamate residues on the surface of ocr to investigate their contribution to the tight binding between the EcoKI Type I R/M enzyme and ocr. Contrary to expectations, all of the single and double site mutations of ocr constructed were active as anti-R/M proteins in vivo and in vitro indicating that the mimicry of DNA by ocr is very resistant to change.

Mocr: A novel fusion tag for enhancing solubility that is compatible with structural biology applications

A persistent problem in heterologous protein production is insolubility of the target protein when expressed to high level in the host cell. A widely employed strategy for overcoming this problem is the use of fusion tags. The best fusion tags promote solubility, may function as purification handles and either do not interfere with downstream applications or may be removed from the passenger protein preparation. A novel fusion tag is identified that meets these criteria. This fusion tag is a monomeric mutant of the Ocr protein (0.3 gene product) of bacteriophage T7. This fusion tag displays solubilizing activity with a variety of different passenger proteins. We show that it may be used as a purification handle similar to other fusion tags. Its small size and compact structure are compatible with its use in downstream applications of the passenger protein or it may be removed and purified away from the passenger protein. The use of monomeric Ocr (Mocr) as a complement to other fusion tags such as maltose-binding protein will provide greater flexibility in protein production and processing for a wide variety of protein applications.

Plasmid-encoded Antirestriction Protein ArdA Can Discriminate between Type I Methyltransferase and Complete Restriction–Modification System

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.

Interaction of the ocr gene 0.3 protein of bacteriophage T7 with EcoKI restriction/modification enzyme.

The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA. In total it mimics 22 phosphate groups over approximately 24 bp of DNA. This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes. We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI. One dimer binds to the core methyltransferase and two to the complete bifunctional restriction and modification enzyme. Ocr can also bind to the component subunits of EcoKI. Binding affinity to the methyltransferase core is extremely strong with a large favourable enthalpy change and an unfavourable entropy change. This strong interaction prevents the dissociation of the methyltransferase which occurs upon dilution of the enzyme. This stabilisation arises because the interaction appears to involve virtually the entire surface area of ocr and leads to the enzyme completely wrapping around ocr.