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Anaerobe Apr 2023We set out to identify and characterize prophages within genomes of published Fusobacterium strains, and to develop qPCR-based methods to characterize intra- and...
OBJECTIVES
We set out to identify and characterize prophages within genomes of published Fusobacterium strains, and to develop qPCR-based methods to characterize intra- and extra-cellular induction of prophage replication in a variety of environmental contexts.
METHODS
Various in silico tools were used to predict prophage presence across 105 Fusobacterium spp. Genomes. Using the example of the model pathogen, Fusobacterium nucleatum subsp. animalis strain 7-1, qPCR was used with DNase I treatment to determine induction of its 3 predicted prophages ɸFunu1, ɸFunu2, and ɸFunu3, across several conditions.
RESULTS
116 predicted prophage sequences were found and analyzed. An emerging association between the phylogenetic history of a Fusobacterium prophage and that of its host was detected, as was the presence of genes encoding putative host fitness factors (e.g. ADP-ribosyltransferases) in distinct subclusters of prophage genomes. For strain 7-1, a pattern of expression for ɸFunu1, ɸFunu2, and ɸFunu3 was established indicating that ɸFunu1 and ɸFunu2 are capable of spontaneous induction. I Salt and mitomycin C exposure were able to promote induction of ɸFunu2. A range of other biologically relevant stressors, including exposure to pH, mucin and human cytokines showed no or minimal induction of these same prophages. ɸFunu3 induction was not detected under tested conditions.
CONCLUSION
The heterogeneity of Fusobacterium strains is matched by their prophages. While the role of Fusobacterium prophages in host pathogenicity remains unclear, this work provides the first overview of clustered prophage distribution among this enigmatic genus and describes an effective assay for quantifying mixed samples of prophages that cannot be detected by plaque assay.
Topics: Humans; Prophages; Phylogeny; Fusobacterium
PubMed: 36801248
DOI: 10.1016/j.anaerobe.2023.102718 -
International Journal of Molecular... Dec 2022The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, K-12. We show here that the CRISPR-Cas system is...
The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, K-12. We show here that the CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including ), Rac, Qin (including ), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.
Topics: Prophages; Escherichia coli; CRISPR-Cas Systems; Escherichia coli K12; Bacteria
PubMed: 36555835
DOI: 10.3390/ijms232416195 -
MSphere Feb 2022Modern sequencing technologies have provided insight into the genetic diversity of numerous species, including the human pathogen Pseudomonas aeruginosa. Bacterial...
Modern sequencing technologies have provided insight into the genetic diversity of numerous species, including the human pathogen Pseudomonas aeruginosa. Bacterial genomes often harbor bacteriophage genomes (prophages), which can account for upwards of 20% of the genome. Prior studies have found P. aeruginosa prophages that contribute to their host's pathogenicity and fitness. These advantages come in many different forms, including the production of toxins, promotion of biofilm formation, and displacement of other P. aeruginosa strains. While several different genera and species of P. aeruginosa prophages have been studied, there has not been a comprehensive study of the overall diversity of P. aeruginosa-infecting prophages. Here, we present the results of just such an analysis. A total of 6,852 high-confidence prophages were identified from 5,383 P. aeruginosa genomes from strains isolated from the human body and other environments. In total, 3,201 unique prophage sequences were identified. While 53.1% of these prophage sequences displayed sequence similarity to publicly available phage genomes, novel and highly mosaic prophages were discovered. Among these prophages, there is extensive diversity, including diversity within the functionally conserved integrase and C repressor coding regions, two genes responsible for prophage entering and persisting through the lysogenic life cycle. Analysis of integrase, C repressor, and terminase coding regions revealed extensive reassortment among P. aeruginosa prophages. This catalog of P. aeruginosa prophages provides a resource for future studies into the evolution of the species. Prophages play a critical role in the evolution of their host species and can also contribute to the virulence and fitness of pathogenic species. Here, we conducted a comprehensive investigation of prophage sequences from 5,383 publicly available Pseudomonas aeruginosa genomes from human as well as environmental isolates. We identified a diverse population of prophages, including tailed phages, inoviruses, and microviruses; 46.9% of the prophage sequences found share no significant sequence similarity with characterized phages, representing a vast array of novel P. aeruginosa-infecting phages. Our investigation into these prophages found substantial evidence of reassortment. In producing this, the first catalog of P. aeruginosa prophages, we uncovered both novel prophages as well as genetic content that have yet to be explored.
Topics: Bacteriophages; Integrases; Lysogeny; Prophages; Pseudomonas aeruginosa
PubMed: 35196122
DOI: 10.1128/msphere.01015-21 -
MSystems Oct 2023species include several human pathogens and mycobacteriophages show potential for therapeutic use to control infections. However, phage infection profiles vary greatly...
species include several human pathogens and mycobacteriophages show potential for therapeutic use to control infections. However, phage infection profiles vary greatly among clinical isolates and phage therapies must be personalized for individual patients. phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced strains. The prophages are numerous and diverse, especially in genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages.
Topics: Humans; Prophages; Mycobacterium; Bacteriophages; Mycobacteriophages; Genome, Bacterial
PubMed: 37791767
DOI: 10.1128/msystems.00446-23 -
Bioinformatics (Oxford, England) Jun 2020Phigaro is a standalone command-line application that is able to detect prophage regions taking raw genome and metagenome assemblies as an input. It also produces...
SUMMARY
Phigaro is a standalone command-line application that is able to detect prophage regions taking raw genome and metagenome assemblies as an input. It also produces dynamic annotated 'prophage genome maps' and marks possible transposon insertion spots inside prophages. It is applicable for mining prophage regions from large metagenomic datasets.
AVAILABILITY AND IMPLEMENTATION
Source code for Phigaro is freely available for download at https://github.com/bobeobibo/phigaro along with test data. The code is written in Python.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: High-Throughput Nucleotide Sequencing; Metagenome; Metagenomics; Prophages; Software
PubMed: 32311023
DOI: 10.1093/bioinformatics/btaa250 -
Frontiers in Microbiology 2021Prophage activation in intestinal environments has been frequently reported to affect host adaptability, pathogen virulence, gut bacterial community composition, and... (Review)
Review
Prophage activation in intestinal environments has been frequently reported to affect host adaptability, pathogen virulence, gut bacterial community composition, and intestinal health. Prophage activation is mostly caused by various stimulators, such as diet, antibiotics, some bacterial metabolites, gastrointestinal transit, inflammatory environment, oxidative stress, and quorum sensing. Moreover, with advancements in biotechnology and the deepening cognition of prophages, prophage activation regulation therapy is currently applied to the treatment of some bacterial intestinal diseases such as Shiga toxin-producing infection. This review aims to make headway on prophage induction in the intestine, in order to make a better understanding of dynamic changes of prophages, effects of prophage activation on physiological characteristics of bacteria and intestinal health, and subsequently provide guidance on prophage activation regulation therapy.
PubMed: 34966370
DOI: 10.3389/fmicb.2021.785634 -
Nucleic Acids Research Jul 2023PHASTEST (PHAge Search Tool with Enhanced Sequence Translation) is the successor to the PHAST and PHASTER prophage finding web servers. PHASTEST is designed to support...
PHASTEST (PHAge Search Tool with Enhanced Sequence Translation) is the successor to the PHAST and PHASTER prophage finding web servers. PHASTEST is designed to support the rapid identification, annotation and visualization of prophage sequences within bacterial genomes and plasmids. PHASTEST also supports rapid annotation and interactive visualization of all other genes (protein coding regions, tRNA/tmRNA/rRNA sequences) in bacterial genomes. Given that bacterial genome sequencing has become so routine, the need for fast tools to comprehensively annotate bacterial genomes has become progressively more important. PHASTEST not only offers faster and more accurate prophage annotations than its predecessors, it also provides more complete whole genome annotations and much improved genome visualization capabilities. In standardized tests, we found that PHASTEST is 31% faster and 2-3% more accurate in prophage identification than PHASTER. Specifically, PHASTEST can process a typical bacterial genome in 3.2 min (raw sequence) or in 1.3 min when given a pre-annotated GenBank file. Improvements in PHASTEST's ability to annotate bacterial genomes now make it a particularly powerful tool for whole genome annotation. In addition, PHASTEST now offers a much more modern and responsive visualization interface that allows users to generate, edit, annotate and interactively visualize (via zooming, rotating, dragging, panning, resetting), colourful, publication quality genome maps. PHASTEST continues to offer popular options such as an API for programmatic queries, a Docker image for local installations, support for multiple (metagenomic) queries and the ability to perform automated look-ups against thousands of previously PHAST-annotated bacterial genomes. PHASTEST is available online at https://phastest.ca.
Topics: Databases, Nucleic Acid; Genome, Bacterial; Molecular Sequence Annotation; Plasmids; Prophages; Search Engine; Software
PubMed: 37194694
DOI: 10.1093/nar/gkad382 -
Journal of Bacteriology Aug 2023The operon of Qin cryptic prophage in Escherichia coli K-12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic...
The operon of Qin cryptic prophage in Escherichia coli K-12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the operon have remained unclear. Transcription from the major promoter of the operon, is repressed by DicA. In this study, we discovered that transcription of the operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the operon, and filamentation is mediated by DicF and DicB. Spontaneous derepression of occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of λ phage with DicA and DicC performing functions similar to those of CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the operon. Collectively, our results illustrate that toxic genes carried on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. Cryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host, or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examined the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.
Topics: Escherichia coli; Prophages; Escherichia coli K12; Bacteriophage lambda; Bacteria; RNA, Small Untranslated
PubMed: 37439671
DOI: 10.1128/jb.00129-23 -
Molecular Microbiology Nov 2022Compared to bacteria of the gut microbiota, bacteriophages are still poorly characterised, and their physiological importance is far less known. Temperate phages are... (Review)
Review
Compared to bacteria of the gut microbiota, bacteriophages are still poorly characterised, and their physiological importance is far less known. Temperate phages are probably a major actor in the gut, as it is estimated that 80% of intestinal bacteria are lysogens, meaning that they are carrying prophages. In addition, prophage induction rates are higher in the gut than in vitro. However, studies on the signals leading to prophage induction have essentially focused on genotoxic agents with poor relevance for this environment. In this review, we sum up recent findings about signals able to trigger prophage induction in the gut. Three categories of signals are at play: those originating from interactions between intestinal microbes, those from the human or animal host physiology and those from external intakes. These recent results highlight the diversity of factors influencing prophage induction in the gut, and start to unveil ways by which microbiota composition may be modulated.
Topics: Animals; Humans; Gastrointestinal Microbiome; Lysogeny; Virus Activation; Prophages; Bacteriophages
PubMed: 36164818
DOI: 10.1111/mmi.14983 -
MSphere Aug 2023Prophages are bacteriophages integrated into the bacterial host's chromosome. This research aims to analyze and characterize the existing prophages within a collection...
Prophages are bacteriophages integrated into the bacterial host's chromosome. This research aims to analyze and characterize the existing prophages within a collection of 53 strains from intensive care units (ICUs) in Portugal and Spain. A total of 113 prophages were localized in the collection, with 18 of them being present in more than one strain simultaneously. After annotation, five of them were discarded as incomplete, and the 13 remaining prophages were characterized. Of 13, 10 belonged to the siphovirus tail morphology group, 2 to the podovirus tail morphology group, and 1 to the myovirus tail morphology group. All prophages had a length ranging from 20,199 to 63,401 bp and a GC% between 56.2% and 63.6%. The number of open reading frames (ORFs) oscillated between 32 and 88, and in 3/13 prophages, more than 50% of the ORFs had an unknown function. With our findings, we show that prophages are present in the majority of the strains isolated from Portuguese and Spanish critically ill patients, many of them found in more than one circulating strain at the same time and following a similar clonal distribution pattern. Although a great sum of ORFs had an unknown function, number of proteins in relation to viral defense (anti-CRISPR proteins, toxin/antitoxin modules, proteins against restriction-modification systems) as well as to prophage interference into their host's quorum sensing system and regulatory cascades were found. This supports the idea that prophages have an influence in bacterial pathogenesis and anti-phage defense. IMPORTANCE Despite being known for decades, prophages remain understudied when compared to the lytic phages employed in phage therapy. This research aims to shed some light into the nature, composition, and role of prophages found within a set of circulating strains of , with special attention to high-risk clones. Given the fact that prophages can effectively influence bacterial pathogenesis, prophage basic research constitutes a topic of growing interest. Furthermore, the abundance of viral defense and regulatory proteins within prophage genomes detected in this study evidences the importance of characterizing the most frequent prophages in circulating clinical strains and in high-risk clones if phage therapy is to be used.
Topics: Humans; Prophages; Pseudomonas aeruginosa; Genome, Viral; Critical Care; Spain
PubMed: 37366636
DOI: 10.1128/msphere.00128-23