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Nature Communications Jan 2023The alternative sigma factor RpoS plays a central role in the critical host-adaptive response of the Lyme disease spirochete, Borrelia burgdorferi. We previously...
The alternative sigma factor RpoS plays a central role in the critical host-adaptive response of the Lyme disease spirochete, Borrelia burgdorferi. We previously identified bbd18 as a negative regulator of RpoS but could not inactivate bbd18 in wild-type spirochetes. In the current study we employed an inducible bbd18 gene to demonstrate the essential nature of BBD18 for viability of wild-type spirochetes in vitro and at a unique point in vivo. Transcriptomic analyses of BBD18-depleted cells demonstrated global induction of RpoS-dependent genes prior to lysis, with the absolute requirement for BBD18, both in vitro and in vivo, circumvented by deletion of rpoS. The increased expression of plasmid prophage genes and the presence of phage particles in the supernatants of lysing cultures indicate that RpoS regulates phage lysis-lysogeny decisions. Through this work we identify a mechanistic link between endogenous prophages and the RpoS-dependent adaptive response of the Lyme disease spirochete.
Topics: Animals; Bacterial Proteins; Borrelia burgdorferi; Gene Expression Regulation, Bacterial; Prophages; Sigma Factor; Ticks; Virulence Factors; Host-Pathogen Interactions
PubMed: 36639656
DOI: 10.1038/s41467-023-35897-3 -
The ISME Journal Dec 2023Environmental bacteria host an enormous number of prophages, but their diversity and natural functions remain largely elusive. Here, we investigate prophage activity and...
Environmental bacteria host an enormous number of prophages, but their diversity and natural functions remain largely elusive. Here, we investigate prophage activity and diversity in 63 Erwinia and Pseudomonas strains isolated from flag leaves of wheat grown in a single field. Introducing and validating Virion Induction Profiling Sequencing (VIP-Seq), we identify and quantify the activity of 120 spontaneously induced prophages, discovering that some phyllosphere bacteria produce more than 10 virions/mL in overnight cultures, with significant induction also observed in planta. Sequence analyses and plaque assays reveal E. aphidicola prophages contribute a majority of intraspecies genetic diversity and divide their bacterial hosts into antagonistic factions engaged in widespread microbial warfare, revealing the importance of prophage-mediated microdiversity. When comparing spontaneously active prophages with predicted prophages we also find insertion sequences are strongly correlated with non-active prophages. In conclusion, we discover widespread and largely unknown prophage diversity and function in phyllosphere bacteria.
Topics: Prophages; Triticum; Bacteria
PubMed: 37919394
DOI: 10.1038/s41396-023-01547-1 -
Applied and Environmental Microbiology Sep 2019The fast-growing marine bacterium represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that...
The fast-growing marine bacterium represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that the first chromosome of ATCC 14048 contains two prophage regions (VNP1 and VNP2) that are both inducible by the DNA-damaging agent mitomycin C and exhibit spontaneous activation under standard cultivation conditions. Their activation was also confirmed by live cell imaging of an mCherry fusion to the major capsid proteins of VNP1 and VNP2. Transmission electron microscopy visualized the release of phage particles belonging to the family into the culture supernatant. Freeing from its proviral load, followed by phenotypic characterization, revealed an improved robustness of the prophage-free variant toward DNA-damaging conditions, reduced cell lysis under hypo-osmotic conditions, and an increased pyruvate production compared to wild-type levels. Remarkably, the prophage-free strain outcompeted the wild type in a competitive growth experiment, emphasizing that this strain is a promising platform for future metabolic engineering approaches. The fast-growing marine bacterium represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. Genome analysis revealed the presence of two prophage regions in the genome that were shown to undergo spontaneous induction under standard cultivation conditions. In this study, we generated a prophage-free variant of Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypo-osmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive growth experiment. In conclusion, our study presents the prophage-free variant of as a promising platform strain for future biotechnological applications.
Topics: DNA Damage; Osmotic Pressure; Prophages; Vibrio
PubMed: 31253674
DOI: 10.1128/AEM.00853-19 -
Viruses Nov 2021Prophage 919TP is widely distributed among and is induced to produce free φ919TP phage particles. However, the interactions between prophage φ919TP, the induced phage...
Prophage 919TP is widely distributed among and is induced to produce free φ919TP phage particles. However, the interactions between prophage φ919TP, the induced phage particle, and its host remain unknown. In particular, phage resistance mechanisms and potential fitness trade-offs, resulting from phage resistance, are unresolved. In this study, we examined a prophage 919TP-deleted variant of and its interaction with a modified lytic variant of the induced prophage (φ919TP ). Specifically, the phage-resistant mutant was isolated by challenging a prophage-deleted variant with lytic phage φ919TP . Further, the comparative genomic analysis of wild-type and φ919TP -resistant mutant predicted that phage φ919TP selects for phage-resistant mutants harboring a mutation in key steps of lipopolysaccharide (LPS) O-antigen biosynthesis, causing a single-base-pair deletion in gene . Our study showed that the -mediated O-antigen defect can cause pleiotropic phenotypes, e.g., cell autoaggregation and reduced swarming motility, emphasizing the role of phage-driven diversification in . The developed approach assists in the identification of genetic determinants of host specificity and is used to explore the molecular mechanism underlying phage-host interactions. Our findings contribute to the understanding of prophage-facilitated horizontal gene transfer and emphasize the potential for developing new strategies to optimize the use of phages in bacterial pathogen control.
Topics: Bacterial Proteins; Cholera; Host Microbial Interactions; Host Specificity; Lysogeny; O Antigens; Prophages; Vibrio cholerae O1; Virus Activation
PubMed: 34960610
DOI: 10.3390/v13122342 -
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 -
The ISME Journal Jan 2024Filamentous prophages are widespread among bacteria and play crucial functions in virulence, antibiotic resistance, and biofilm structures. The filamentous Pf4...
Filamentous prophages are widespread among bacteria and play crucial functions in virulence, antibiotic resistance, and biofilm structures. The filamentous Pf4 particles, extruded by an important pathogen Pseudomonas aeruginosa, can protect producing cells from adverse conditions. Contrary to the conventional belief that the Pf4-encoding cells resist reinfection, we herein report that the Pf4 prophage is reciprocally and commonly exchanged within P. aeruginosa colonies, which can repair defective Pf4 within the community. By labeling the Pf4 locus with antibiotic resistance and fluorescence markers, we demonstrate that the Pf4 locus is frequently exchanged within colony biofilms, in artificial sputum media, and in infected mouse lungs. We further show that Pf4 trafficking is a rapid process and capable of rescuing Pf4-defective mutants. The Pf4 phage is highly adaptable and can package additional DNA doubling its genome size. We also report that two clinical P. aeruginosa isolates are susceptible to the Pf4-mediated exchange, and the Pf5 prophage can be exchanged between cells as well. These findings suggest that the genetic exchanging interactions by filamentous prophages may facilitate defect rescue and the sharing of prophage-dependent benefits and costs within the P. aeruginosa community.
Topics: Animals; Mice; Prophages; Pseudomonas aeruginosa; Bacteriophages; Pseudomonas Infections; Virulence; Biofilms
PubMed: 38365255
DOI: 10.1093/ismejo/wrad025 -
Microbiology Spectrum Feb 2015The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid... (Review)
Review
The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.
Topics: Centromere; Coliphages; DNA Replication; DNA, Circular; Gammaproteobacteria; Genes, Viral; Plasmids; Prophages; Replication Origin; Synteny
PubMed: 26104561
DOI: 10.1128/microbiolspec.PLAS-0032-2014 -
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 -
Microbiology (Reading, England) Mar 2022subspecies serovar Typhimurium (. Typhimurium) definitive phage type 104 (DT104), . Worthington, and produce ArtAB toxin, which catalyses ADP-ribosylation of...
subspecies serovar Typhimurium (. Typhimurium) definitive phage type 104 (DT104), . Worthington, and produce ArtAB toxin, which catalyses ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene () is encoded on a prophage in , and prophage induction by SOS-inducing agents is associated with increases in ArtAB production . However, little is known about the expression of . Here, we showed a significant increase in transcription of DT104 within macrophage-like RAW264.7 cells. Intracellular expression of ArtAB was also observed by immunofluorescence staining. The induced expression of in DT104 and was enhanced by treatment of RAW264.7 cells with phorbol 12-myristate 13-acetate (PMA), which stimulates the production of reactive oxygen species (ROS); however, such induction was not observed in . Worthington. Upregulation of , a major regulator of oxidative stress, and a repressor of prophage induction, was observed in . Worthington within RAW264.7 cells treated with PMA but not in the DT104 strain. Although the expression of was increased, was upregulated in which lacks the gene in the incomplete -encoded prophage. Taken together, oxidative stress plays a role in the production of toxins in macrophages, and high expression levels of and are responsible for the low expression of . Therefore, strain variation in the level of expression within macrophages could be explained by differences in the oxidative stress response of bacteria and might be reflected in its virulence.
Topics: Macrophages; Prophages; Salmonella typhimurium; Virulence
PubMed: 35333707
DOI: 10.1099/mic.0.001152 -
The Journal of General and Applied... Sep 2022Site-specific recombination (SSR) systems are employed in many genetic mobile elements, including temperate phages, for their integration and excision. Recently, they...
Site-specific recombination (SSR) systems are employed in many genetic mobile elements, including temperate phages, for their integration and excision. Recently, they have also been used as tools for applications in fields ranging from basic to synthetic biology. SPβ is a temperate phage of the Siphoviridae family found in the laboratory standard Bacillus subtilis strain 168. SPβ encodes a serine-type recombinase, SprA, and recombination directionality factor (RDF), SprB. SprA catalyzes recombination between the attachment site of the phage, attP, and that of the host, attB, to integrate phage genome into the attB site of the host genome and generate attL and attR at both ends of the prophage genome. SprB works in conjunction with SprA and switches from attB/attP to attL/R recombination, which leads to excision of the prophage. In the present study, we took advantage of this highly efficient recombination system to develop a site-specific integration and excision plasmid vector, named pSSβ. It was constructed using pUC plasmid and the SSR system components, attP, sprA and sprB of SPβ. pSSβ was integrated into the attB site with a significantly high efficiency, and the resulting pSSβ integrated strain also easily eliminated pSSβ itself from the host genome by the induction of SprB expression with xylose. This report presents two applications using pSSβ that are particularly suitable for gene complementation experiments and for a curing system of SPβ prophage, that may serve as a model system for the removal of prophages in other bacteria.
Topics: Bacillus subtilis; Bacteriophages; DNA; Integrases; Prophages; Recombination, Genetic
PubMed: 35387911
DOI: 10.2323/jgam.2021.10.004