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Nucleic Acids Research May 2020The increasing use of CRISPR-Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms...
The increasing use of CRISPR-Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.
Topics: Bacterial Proteins; CRISPR-Associated Protein 9; Machine Learning; Prophages; Proteome; Sequence Analysis, Protein; Streptococcus
PubMed: 32286628
DOI: 10.1093/nar/gkaa219 -
MBio Feb 2024Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. phage P22 has four such systems...
Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the (repressor), , , and genes. Here we report that the P22-encoded SieA protein is necessary and sufficient for exclusion by the SieA system and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants that overcome the SieA block were isolated, and they have amino acid changes in the C-terminal regions of the gene and encoded ejection proteins. Three different single-amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in phage target specificity. Our data strongly suggest a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.IMPORTANCEThe ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called "superinfection exclusion." A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection.
Topics: Humans; Bacteriophage P22; Superinfection; Bacteriophages; Prophages; Membrane Proteins; DNA; Amino Acids
PubMed: 38236051
DOI: 10.1128/mbio.02169-23 -
The CRISPR Journal Aug 2021Prophages are widely spread among bacterial genomes, and they can have positive or negative effects on their hosts. A key aspect in the study of prophages is the...
Prophages are widely spread among bacterial genomes, and they can have positive or negative effects on their hosts. A key aspect in the study of prophages is the discovery of their induction signals. Prophage induction can occur by inactivating a phage transcriptional repressor, which is responsible for maintaining the lysogenic state. This repressor can be inactivated through the bacterial SOS response. However, the induction signals for numerous prophages do not involve the SOS system, and therefore significant efforts are needed to identify these conditions. Similarly, curing bacterial strains of inducible prophages is a tedious process, requiring the screening of several colonies. Here, we investigated whether transcriptional silencing of a prophage repressor using CRISPR interference (CRISPRi) would lead to prophage induction. Using phages λ and P2 as models, we demonstrated the efficiency of CRISPRi for prophage induction and for curing lysogenic strains of their prophages.
Topics: Bacteriophage lambda; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Escherichia coli; Gene Editing; Gene Order; Gene Targeting; Genetic Engineering; Genome, Viral; Plasmids; Prophages; Virus Activation
PubMed: 34406037
DOI: 10.1089/crispr.2021.0026 -
Nucleic Acids Research Dec 2020CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing...
CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.
Topics: Autoimmunity; Bacteria; Base Sequence; CRISPR-Associated Protein 9; CRISPR-Associated Proteins; CRISPR-Cas Systems; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Genome, Bacterial; Prophages; Software
PubMed: 33219687
DOI: 10.1093/nar/gkaa1071 -
Nucleic Acids Research Sep 2023Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPβ prophage whose lysogenic...
Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPβ prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPβ c2 mutant, we demonstrate that the lytic cycle of SPβ c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPβ genome. MrpR functions as a master repressor of SPβ that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPβ c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPβ.
Topics: Bacillus Phages; Bacillus subtilis; Bacteriophages; Lysogeny; Prophages; Recombinases; Viral Proteins
PubMed: 37602373
DOI: 10.1093/nar/gkad675 -
Science (New York, N.Y.) Apr 2024Phage viruses shape the evolution and virulence of their bacterial hosts. The genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein,...
Phage viruses shape the evolution and virulence of their bacterial hosts. The genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNA. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.
Topics: Animals; Endodeoxyribonucleases; Oxidative Stress; Prophages; RNA; RNA, Transfer; Salmonella enterica; Salmonella Phages; Viral Proteins
PubMed: 38574144
DOI: 10.1126/science.adl3222 -
Science (New York, N.Y.) Mar 2024The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA)...
The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA) and B (CifB) proteins, encoded by prophage WO of the endosymbiont alter long noncoding RNA (lncRNA) and DNA during sperm development to establish a paternal-effect embryonic lethality known as cytoplasmic incompatibility (CI). CifA is a ribonuclease (RNase) that depletes a spermatocyte lncRNA important for the histone-to-protamine transition of spermiogenesis. Both CifA and CifB are deoxyribonucleases (DNases) that elevate DNA damage in late spermiogenesis. lncRNA knockdown enhances CI, and mutagenesis links lncRNA depletion and subsequent sperm chromatin integrity changes to embryonic DNA damage and CI. Hence, prophage proteins interact with eukaryotic macromolecules during gametogenesis to create a symbiosis that is fundamental to insect evolution and vector control.
Topics: Animals; Male; Cytoplasm; DNA; Prophages; RNA, Long Noncoding; Spermatozoa; Wolbachia; Paternal Inheritance; Viral Proteins; Drosophila melanogaster; Bacterial Proteins; Deoxyribonucleases
PubMed: 38452081
DOI: 10.1126/science.adk9469 -
Infection, Genetics and Evolution :... Sep 2023To systematically investigate the prophages carrying in Porphyromonas gingivalis (P. gingivalis) strains, analyze potential antibiotic resistance genes (ARGs) and...
To systematically investigate the prophages carrying in Porphyromonas gingivalis (P. gingivalis) strains, analyze potential antibiotic resistance genes (ARGs) and virulence genes in these prophages. We collected 90 whole genome sequences of P. gingivalis from NCBI and utilized the Prophage Hunter online software to predict prophages; Comprehensive antibiotic research database (CARD) and virulence factors database (VFDB) were adopted to analyze the ARGs and virulence factors (VFs) carried by the prophages. Sixty-nine prophages were identified among 24/90 P. gingivalis strains, including 17 active prophages (18.9%) and 52 ambiguous prophages (57.8%). The proportion of prophages carried by each P. gingivalis genome ranged from 0.5% to 6.7%. A total of 188 antibiotic resistance genes belonging to 25 phenotypes and 46 different families with six mechanisms of antibiotic resistance were identified in the 17 active prophages. Three active prophages encoded 4 virulence genes belonging to type III and type VI secretion systems. The potential hosts of these virulence genes included Escherichia coli, Shigella sonnei, Salmonella typhi, and Klebsiella pneumoniae. In conclusion, 26.7% P. gingivalis strains carry prophages, while the proportion of prophage genes in the P. gingivalis genome is relatively low. In addition, approximately 39.7% of the P. gingivalis prophage genes have ARGs identified, mainly against streptogramin, peptides, and aminoglycosides. Only a few prophages carry virulence genes. Prophages may play an important role in the acquisition, dissemination of antibiotic resistance genes, and pathogenicity evolution in P. gingivalis.
Topics: Prophages; Genome, Bacterial; Porphyromonas gingivalis; Virulence Factors; Virulence; Escherichia coli; Anti-Bacterial Agents
PubMed: 37572952
DOI: 10.1016/j.meegid.2023.105489 -
Current Microbiology Feb 2020Mobile genetic elements (MGE) play a large role in the plasticity of genomes, participating in several phenomena which involve genes acquisition. Pseudomonas stutzeri is...
Mobile genetic elements (MGE) play a large role in the plasticity of genomes, participating in several phenomena which involve genes acquisition. Pseudomonas stutzeri is an environmental widely distributed bacteria. This bacteria has a very large genomic plasticity, which would explain its occurrence in several different environments. NCBI data bank and online programs were used to build an inventory to investigate diversity and structure of MGE in Pseudomonas stutzeri, searching for insertion sequences (IS), integrases/transposases, plasmids and prophages. Five hundred and forty-eight ISs, 62 integrases, 166 transposases, five plasmids and eight complete prophages were found. MGE location and adjacent genes were investigated. Possible implications of the presence of these mobile elements explaining phenotypic diversity of Pseudomonas stutzeri were discussed. The study showed that MGEs might be good clues to understand the dynamics of genomes and their phenotypic plasticity, although they are not the only elements responsible for these characteristics.
Topics: Conjugation, Genetic; DNA Transposable Elements; Genetic Variation; Genome, Bacterial; Genomics; Phenotype; Plasmids; Prophages; Pseudomonas stutzeri
PubMed: 31754823
DOI: 10.1007/s00284-019-01812-7 -
Cell Host & Microbe Nov 2021Bacteria have evolved many immune systems to combat their viral parasites (i.e., phages). In this issue of Cell Host & Microbe, Owen et al. discover a mechanism of...
Bacteria have evolved many immune systems to combat their viral parasites (i.e., phages). In this issue of Cell Host & Microbe, Owen et al. discover a mechanism of anti-phage immunity that is mediated by a phage-encoded protein, and thus provide an example of how inter-phage conflict can promote survival of the bacterial population.
Topics: Bacteria; Bacteriophages; Prophages
PubMed: 34762825
DOI: 10.1016/j.chom.2021.10.004