Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity.

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3'-5' exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3'-5' exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme's activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme's moderate thermal stability.

CPR-C4 is a highly conserved novel protease from the Candidate Phyla Radiation with remote structural homology to human vasohibins.

The Candidate Phyla Radiation is a recently uncovered and vast expansion of the bacterial domain of life, made up of largely uncharacterized phyla that lack isolated representatives. This unexplored territory of genetic diversity presents an abundance of novel proteins with potential applications in the life-science sectors. Here, we present the structural and functional elucidation of CPR-C4, a hypothetical protein from the genome of a thermophilic Candidate Phyla Radiation organism, identified through metagenomic sequencing. Our analyses revealed that CPR-C4 is a member of a family of highly conserved proteins within the Candidate Phyla Radiation. The function of CPR-C4 as a cysteine protease was predicted through remote structural similarity to the Homo sapiens vasohibins and subsequently confirmed experimentally with fluorescence-based activity assays. Furthermore, detailed structural and sequence alignment analysis enabled identification of a noncanonical cysteine-histidine-leucine(carbonyl) catalytic triad. The unexpected structural and functional similarities between CPR-C4 and the human vasohibins suggest an evolutionary relationship undetectable at the sequence level alone.

Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6.

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Šresolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.

Going to extremes - a metagenomic journey into the dark matter of life.

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.

Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.

As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPß were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel ß-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each ß-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.

Modification of linear (ß1→3)-linked gluco-oligosaccharides with a novel recombinant ß-glucosyltransferase (trans-ß-glucosidase) enzyme from Bradyrhizobium diazoefficiens.

Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (ß1→3) linkages preferably and to a lesser extent (ß1→6) or (ß1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (ß1→3)(ß1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic ß-glucans in B. diazoefficiens.

Isolation, growth and genome of the Rhodothermus RM378 thermophilic bacteriophage.

Several bacteriophages that infect different strains of the thermophilic bacterium Rhodothermus marinus were isolated and their infection pattern was studied. One phage, named RM378 was cultivated and characterized. The RM378 genome was also sequenced and analyzed. The phage was grouped as a member of the Myoviridae family with A2 morphology. It had a moderately elongated head, with dimensions of 85 and 95 nm between opposite apices and a 150 nm long tail, attached with a connector to the head. RM378 showed a virulent behavior that followed a lytic cycle of infection. It routinely gave lysates with 10(11) pfu/ml, and sometimes reached titers as high as 10(13) pfu/ml. The titer remained stable up to 65 °C but the phage lost viability when incubated at higher temperatures. Heating for 30 min at 96 °C lowered the titer by 10(4). The RM378 genome consisted of ds DNA of 129.908 bp with a GC ratio of 42.0% and contained about 120 ORFs. A few structural proteins, such as the major head protein corresponding to the gp23 in T4, could be identified. Only 29 gene products as probable homologs to other proteins of known function could be predicted, with most showing only low similarity to known proteins in other bacteriophages. These and other studies based on sequence analysis of a large number of phage genomes showed RM378 to be distantly related to all other known T4-like phages.

Toxoid construction of AsaP1, a lethal toxic aspzincin metalloendopeptidase of Aeromonas salmonicida subsp. achromogenes, and studies of its activity and processing.

AsaP1 is a toxic aspzincin metalloendopeptidase secreted by the fish pathogen Aeromonas salmonicida subsp. achromogenes. The protease is highly immunogenic and antibodies against AsaP1 evoke a passive protection against infection with A. salmonicida subsp. achromogenes. The protease is expressed as 37 kDa pre-pro-protein and processed to an active enzyme of 19kDa in A. salmonicida subsp. achromogenes. Recombinant expression of AsaP1(rec) in E. coli results in a protease of 22 kDa that is not secreted. AsaP1(rec) induces comparable pathological changes in Atlantic salmon (Salmo salar L.) to native AsaP1(wt). The aim of the study was to construct AsaP1 toxoids by exchanging catalytically important amino acids in the active site region of the protease. Four different AsaP1 mutants (AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A), and AsaP1(Y309F)) were successfully constructed by one step site directed mutagenesis, expressed in E. coli BL21 C43 as pre-pro-proteins and purified by His-tag affinity chromatography and gel filtration. Three of the resulting mutants (AsaP1(E294A), AsaP1(E294Q), and AsaP1(Y309A)) were not caseinolytic active and are detected as unprocessed pre-pro-proteins of 37 kDa. Caseinolytic active AsaP1(rec) and a mutant with reduced activity, AsaP1(Y309F), were processed to a size of 22 kDa. Furthermore, AsaP1(rec) is able to process the inactive mutants to the mature size of 22 kDa, allowing the conclusion that AsaP1 is autocatalytically processed. All four mutants AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A) and AsaP1(Y309F) are non-toxic in fish but induce a specific anti-AsaP1 antibody response in Arctic charr (Salvelinus alpinus L.) and are therefore true toxoids and possible vaccine additives.

Exploring novel non-Leloir ß-glucosyltransferases from proteobacteria for modifying linear (ß1->3)-linked gluco-oligosaccharide chains.

Over the years several ß-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding ß-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-ß-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a ß-glucan donor to a ß-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (ß1 â†’ 3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (ß1 â†’ 3)-elongation activity, (ß1 â†’ 4)- or (ß1 â†’ 6)-elongation, or (ß1 â†’ 6)-branching activities were also detected.

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

Characterization of a 5'-polynucleotide kinase/3'-phosphatase from bacteriophage RM378.

A polynucleotide kinase from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus was identified, expressed, and purified. This polynucleotide kinase was demonstrated to have a 5'-kinase domain as well as a 3'-phosphohydrolase domain. The RM378 polynucleotide kinase had limited sequence similarity to the 5'-kinase domain of the T4 bacteriophage polynucleotide kinase, but apparent homology was not evident within the 3'-phosphohydrolase domain. The domain order of RM378 polynucleotide kinase was reversed relative to that of the T4 polynucleotide kinase. The RM378 phosphohydrolase domain displayed some sequence similarity with the bacterial poly(A) polymerase family, including an HD motif characteristic of the diverse superfamily of metal-dependent HD phosphohydrolases. The RM378 polynucleotide kinase was biochemically characterized and shown to possess 5'-kinase activity on RNA and single- and double-stranded DNA at elevated temperatures. It also showed phosphohydrolase activity on 2':3'-cyclic adenosine monophosphate. This description of the RM378 polynucleotide kinase, along with the recently described RM378 RNA ligase, suggests that the RM378 bacteriophage has to counter a similar anti-phage mechanism in R. marinus as the one that the T4 phage has to counter in Escherichia coli.

Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.