Host DNA Degradation Research Articles

Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate

Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.

Effects of storage conditions and digestion time on DNA amplification of biting midge (Culicoides) blood meals

BackgroundMolecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium.MethodWe characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a − 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA.ResultsWe found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature.ConclusionsThese findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera.Graphical

Degradation of host translational machinery drives tRNA acquisition in viruses.

Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p= 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.

Host DNA integrity within blood meals of hematophagous larval gnathiid isopods (Crustacea, Isopoda, Gnathiidae)

BackgroundJuvenile gnathiid isopods are common ectoparasites of marine fishes. Each of the three juvenile stages briefly attach to a host to obtain a blood meal but spend most of their time living in the substrate, thus making it difficult to determine patterns of host exploitation. Sequencing of host blood meals from wild-caught specimens is a promising tool to determine host identity. Although established protocols for this approach exist, certain challenges must be overcome when samples are subjected to typical field conditions that may contribute to DNA degradation. The goal of this study was to address a key methodological issue associated with molecular-based host identification from free-living, blood-engorged gnathiid isopods—the degradation of host DNA within blood meals. Here we have assessed the length of time host DNA within gnathiid blood meals can remain viable for positive host identification.MethodsJuvenile gnathiids were allowed to feed on fish of known species and subsets were preserved at 4-h intervals over 24 h and then every 24 h up to 5 days post-feeding. Host DNA extracted from gnathiid blood meals was sequenced to validate the integrity of host DNA at each time interval. DNA was also extracted from blood meals of wild-fed gnathiids for comparison. Attempts were also made to extract host DNA from metamorphosed juveniles.ResultsUsing a cox1 universal fish primer set, known fish host DNA sequences were successfully identified for nearly 100% of third-stage juvenile gnathiid blood meals, digested for up to 5 days post-feeding. For second-stage juveniles, host identification was 100% successful when gnathiids were preserved within 24 h of collection. Fish hosts were positively identified for 69% of sequences from wild-fed gnathiid isopods. Of the 31% of sequences not receiving a ≥ 98 % match to a sequence in GenBank, 25 sequences were of possible invertebrate origin.ConclusionsTo our knowledge, this is the first study to examine the degradation rate of gnathiid isopod blood meals. Determining the rate at which gnathiids digest their blood meal is an important step in ensuring the successful host identification by DNA-based methods in large field studies.

Phage T4‐induced DNA breaks activate a tRNA repair‐defying anticodon nuclease

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.

Comparative metagenomic analyses reveal viral-induced shifts of host metabolism towards nucleotide biosynthesis

BackgroundViral genomes often contain metabolic genes that were acquired from host genomes (auxiliary genes). It is assumed that these genes are fixed in viral genomes as a result of a selective force, favoring viruses that acquire specific metabolic functions. While many individual auxiliary genes were observed in viral genomes and metagenomes, there is great importance in investigating the abundance of auxiliary genes and metabolic functions in the marine environment towards a better understanding of their role in promoting viral reproduction.ResultsIn this study, we searched for enriched viral auxiliary genes and mapped them to metabolic pathways. To initially identify enriched auxiliary genes, we analyzed metagenomic microbial reads from the Global Ocean Survey (GOS) dataset that were characterized as viral, as well as marine virome and microbiome datasets from the Line Islands. Viral-enriched genes were mapped to a “global metabolism network” that comprises all KEGG metabolic pathways. Our analysis of the viral-enriched pathways revealed that purine and pyrimidine metabolism pathways are among the most enriched pathways. Moreover, many other viral-enriched metabolic pathways were found to be closely associated with the purine and pyrimidine metabolism pathways. Furthermore, we observed that sequential reactions are promoted in pathways having a high proportion of enriched genes. In addition, these enriched genes were found to be of modular nature, participating in several pathways.ConclusionsOur naïve metagenomic analyses strongly support the well-established notion that viral auxiliary genes promote viral replication via both degradation of host DNA and RNA as well as a shift of the host metabolism towards nucleotide biosynthesis, clearly indicating that comparative metagenomics can be used to understand different environments and systems without prior knowledge of pathways involved.

Genome of low-temperature T4-related bacteriophage vB_EcoM-VR7

The complete genome sequence of the T4-related low-temperature Escherichia coli bacteriophage vB_EcoM-VR7 was determined. The genome sequence is 169,285bp long, with a G+C content of 40.3%. Overall, 95% of the phage genome is coding. It encodes 293 putative protein-encoding open reading frames (ORFs) and tRNA(Met). More than half (59%) of the genomic DNA lacks significant homology with the DNA of T4, but once translated, 72% of the vB_EcoM-VR7 genome (211 ORFs) encodes protein homologues of T4 genes. Overall, 46 vB_EcoM-VR7 ORFs have no homologues in T4 but are derived from other T4-related phages, nine ORFs show similarities to bacterial or non-T4-related phage genes, and 27 ORFs are unique to vB_EcoM-VR7. This phage lacks several T4 enzymes involved in host DNA degradation; however, there is extensive representation of the DNA replication, recombination and repair enzymes as well as the viral capsid and tail structural genes.

A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi

A reliable method for measuring disease progression is important when evaluating susceptibility in host—pathogen interactions. We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis. Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves the accuracy of pathogen measurement in both resistant (non-host A. thaliana–P. cinnamomi) and susceptible (host Lupinus angustifolius–P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis.

Structure of Bacteriophage T4 Endonuclease II Mutant E118A, a Tetrameric GIY-YIG Enzyme

Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P2 1 with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.

An E. coli B mutation, rpoB5081, that prevents growth of phage T4 strains defective in host DNA degradation.

An E. coli B Tab strain, EM121, was isolated that restricts T4 denA (DNA endonuclease II) mutants at 37 degrees C and above, but is permissive for wild-type T4 at all temperatures examined. At 42 degrees C, other mutants affected in nucleic acid metabolism (T4 dexA, regA and uvsW strains) are also restricted. Genetic analysis revealed that one mutation (rpoB5081) in the RNA polymerase beta subunit gene is sufficient for restricting all denA mutants. rpoB5081, together with a second linked mutation, is also required for restricting the other T4 mutants, rpoB5081 (P806S), previously shown to increase transcription termination in E. coli K-12, causes delayed synthesis of T4 late proteins and reduced DNA synthesis in denA infections. Thus, T4 DNA synthesis and gene expression are impaired by the rpoB5081 beta subunit when degradation of host DNA is reduced. Because the restricted T4 mutants are not readily distinguished from wild-type phage under typical plating conditions, EM121 is an important host for screening and mapping T4 denA mutations.

The genome and proteome of coliphage T1

The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation.

An E. coli B mutation, rpoB5081, that prevents growth of phage T4 strains defective in host DNA degradation

An E. coli B Tab strain, EM121, was isolated that restricts T4 denA (DNA endonuclease II) mutants at 37°C and above, but is permissive for wild-type T4 at all temperatures examined. At 42°C, other mutants affected in nucleic acid metabolism (T4 dexA, regA and uvsW strains) are also restricted. Genetic analysis revealed that one mutation ( rpoB5081) in the RNA polymerase β subunit gene is sufficient for restricting all denA mutants. rpoB5081, together with a second linked mutation, is also required for restricting the other T4 mutants. rpoB5081 (P806S), previously shown to increase transcription termination in E. coli K-12, causes delayed synthesis of T4 late proteins and reduced DNA synthesis in denA infections. Thus, T4 DNA synthesis and gene expression are impaired by the rpoB5081 β subunit when degradation of host DNA is reduced. Because the restricted T4 mutants are not readily distinguished from wild-type phage under typical plating conditions, EM121 is an important host for screening and mapping T4 denA mutations.

A hydrophobic region within the adenovirus E1B 19 kDa protein is necessary for the transient inhibition of NF-kappaB activated by different stimuli.

The early transcribed adenovirus proteins E1A and E1B display a variety of functions in the transformation of primary rodent cells and the regulation of apoptosis and transcription. We have recently shown recently that the E1B 19 kDa protein from Adenovirus 5 (Ad 5) can functionally antagonize the stimulatory effect of E1A 13S on the human transcription factor NF-kappaB. Here we show that expression of E1B 19 kDa negatively interfered with the activation of NF-kappaB by different stimuli, such as the E1A 13S protein, and treatment with phorbol ester and tumor necrosis factor alpha. This suggests that E1B 19 kDa acts on a common upstream signaling event. Band shift experiments showed that expression of E1B 19 kDa impaired the generation of the nuclear, DNA-binding form of NF-kappaB. Domain mapping experiments employing various E1B 19 kDa mutants revealed the necessity of a hydrophobic Bcl-2 homology region between amino acids 90 and 96 for NF-kappaB inhibition. Co-transfection experiments showed that the inhibitory effect of E1B 19 kDa on E1A 13S-activated NF-kappaB transcription was gradually lost in the course of time. Thus the continuous stimulatory action of E1A 13S can finally override the antagonistic effects of E1B 19 kDa on NF-kappaB activity. In contrast to E1B 19 kDa, expression of the E1B 55 kDa protein did not result in a de novo activation of NF-kappaB, but co-stimulated the transcriptional potential of activated NF-kappaB.

Isolation of a Mutant Bacteriophage T7 Deleted in Nonessential Genetic Elements, Gene19.5andm

Half of the 55 potential genes of bacteriophage T7 appear to be dispensable. One of the major obstacles in the study of these nonessential genes is the difficulty in obtaining mutants. During a study of genes involved in the packaging of bacteriophage T7, we hypothesized that some nonessential genes may be required for optimal growth. Mutant phages lacking such nonessential genes may form plaques but grow slowly. One gene located at the extreme right end of the linear T7 genome, gene19.5with no known mutants, and a genetic elementmresponsible for a unique hairpin end, were studied. Mutant T7 phages deleted in gene19.5andm(T7Δ19.5-M) were generatedin vivoby homologous recombination with a recombinant plasmid. This phage produces small plaques and the production of progeny phage particles per infected cell was reduced fourfold. Investigation of the intracellular DNA after infection with T7Δ19.5-Mshowed the persistence ofEscherichia coliDNA as well as delayed conversion of concatemers to unit-length T7 DNA. The inefficiency of concatemer processing confirmed the proposed function of the M-hairpin in duplication of the concatemer junction. Since it is not likely that the M-hairpin influences the degradation of host DNA, we propose that the gene19.5product is partly responsible for the degradation ofE. colichromosomal DNA.

Phage φ92: A New Choleraphage Infecting <i>Vibrio cholerae</i> Cells Belonging to Serovars O1 and Non-O1

A new choleraphage, psi 92, with an icosahedral head and no tail structure has been described. This phage has a wide host range. It infects and lyses strains belonging to different serovars and biotypes of Vibrio cholerae. The phage utilises host DNA degradation products for its own DNA synthesis. psi 92 contains a linear double-stranded DNA of 56 kbp. A physical map of the phage genome has been constructed using the restriction enzyme KpnI. Intracellular development of phage psi 92 following infection was examined by thin-section electron microscopy.

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.

5-Azacytidine-resistant mutants of Chlorella virus IL-3A

Many dsDNA-containing viruses which infect the unicellular, eukaryotic Chlorella-like green alga strain NC64A encode for DNA methyltransferases and DNA site-specific (restriction) endonucleases. We have hypothesized that these endonucleases help degrade host DNA permitting deoxynucleotides to recycle into virus DNA. This hypothesis was tested by isolating deletion mutants of Chlorella virus IL-3A lacking functional genes for the cytosine methyltransferase M. CviJI and the cognate site-specific endonuclease CviJI. The growth and burst sizes of the mutants and parent virus were identical. Also host nuclear and chloroplast DNAs disappeared from infected cells at the same rates. Thus M. CviJI and CviJI activities are not required for IL-3A replication and CviJI activity is not essential for host DNA degradation.

The virulent bacteriophage IRA of Salmonella typhimurium: Cloning of phage genes which are potentially lethal for the host cell

Morphological and biological properties of Salmonella typhimurium phage IRA were studied. The phage is a member of the Styloviridae family and exhibits a very wide spectrum of lytic activity. Molecular cloning of phage genes whose expression is lethal for the host cell has been performed and consequences of gene expression have been investigated. Expression of recombinant plasmid pKI71 causes structural changes of the cell wall and also degradation of host DNA while plasmid DNA remains intact. Expression of pKI72 blocks normal cell division. The possibility of applying such recombinant clones in marking pathways of microbial contamination in water areas is proposed.

Characterization of the phage P1 dam gene

The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation.