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1.
ACS Synth Biol ; 12(3): 709-721, 2023 03 17.
Article in English | MEDLINE | ID: mdl-36802585

ABSTRACT

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.


Subject(s)
Anti-Infective Agents , CRISPR-Cas Systems , CRISPR-Cas Systems/genetics , Gene Editing , Bacteriophage P1/genetics , Zebrafish/genetics , Shigella flexneri/genetics , Animals
2.
J Mol Biol ; 434(21): 167829, 2022 11 15.
Article in English | MEDLINE | ID: mdl-36116540

ABSTRACT

Enterobacteria phage P1 expresses two types of tail fibre, S and S'. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S' tail fibre was never determined. Here, a ΔS-cin Δpac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S' tail fibre. P1(S') could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S') transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S') transduction, indicating that this E. coli strain could be susceptible to P1(S')-mediated transduction. Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S') transduction efficiency. A higher transduction efficiency of P1(S') improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the bacterial strains which are susceptible to P1(S')-mediated transduction, as well as demonstrating its potential for delivering a DNA sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.


Subject(s)
Bacteriophage P1 , Escherichia coli , O Antigens , Shigella flexneri , Transduction, Genetic , Viral Tail Proteins , Escherichia coli/genetics , Escherichia coli/virology , O Antigens/genetics , O Antigens/physiology , Shigella flexneri/genetics , Shigella flexneri/virology , Bacteriophage P1/genetics , Bacteriophage P1/physiology , Viral Tail Proteins/genetics
3.
Virus Genes ; 58(6): 570-583, 2022 Dec.
Article in English | MEDLINE | ID: mdl-35986820

ABSTRACT

Phage P1 was isolated from the abnormal fermented liquid using Lactobacillus plantarum (L. plantarum) IMAU10120. To date, genetic knowledge regarding L. plantarum phage diversity is still limited, and further in-depth sequencing analysis of isolated L. plantarum phages can fill this gap. Here, we investigated the whole genome sequence of L. plantarum phage P1, sequenced by Illumina HiSeq platform, to decipher its genomic characteristics and putative DNA packaging mechanism. It was revealed that phage P1 was 73,787 bp in length, which was composed of linear double-stranded DNA (dsDNA), and the GC content was 39.17%. Its genome contained 86 coding sequences for various functions, such as adsorption, injection, replication, assembly, and release. Moreover, it was observed that L. plantarum phage P1 utilized the 'cohesive ends' DNA packaging mechanism. This study furthered the genomic knowledge of L. plantarum phages and provided some basis for the control of L. plantarum phages in the dairy fermentation industry.


Subject(s)
Bacteriophages , Lactobacillus plantarum , Lactobacillus plantarum/genetics , Bacteriophage P1/genetics , Bacteriophages/genetics , DNA Packaging , DNA , Sequence Analysis , Genome, Viral
4.
Int J Mol Sci ; 23(8)2022 Apr 11.
Article in English | MEDLINE | ID: mdl-35457047

ABSTRACT

P1 is a model temperate myovirus. It infects different Enterobacteriaceae and can develop lytically or form lysogens. Only some P1 adaptation strategies to propagate in different hosts are known. An atypical feature of P1 is the number and organization of cell lysis-associated genes. In addition to SAR-endolysin Lyz, holin LydA, and antiholin LydB, P1 encodes other predicted holins, LydC and LydD. LydD is encoded by the same operon as Lyz, LydA and LydB are encoded by an unlinked operon, and LydC is encoded by an operon preceding the lydA gene. By analyzing the phenotypes of P1 mutants in known or predicted holin genes, we show that all the products of these genes cooperate with the P1 SAR-endolysin in cell lysis and that LydD is a pinholin. The contributions of holins/pinholins to cell lysis by P1 appear to vary depending on the host of P1 and the bacterial growth conditions. The pattern of morphological transitions characteristic of SAR-endolysin-pinholin action dominates during lysis by wild-type P1, but in the case of lydC lydD mutant it changes to that characteristic of classical endolysin-pinholin action. We postulate that the complex lytic system facilitates P1 adaptation to various hosts and their growth conditions.


Subject(s)
Bacteriophage P1 , Viral Proteins , Bacteriophage P1/genetics , Bacteriophage P1/metabolism , Biological Transport , Endopeptidases/metabolism , Operon , Viral Proteins/genetics , Viral Proteins/metabolism
5.
Viruses ; 14(4)2022 03 25.
Article in English | MEDLINE | ID: mdl-35458408

ABSTRACT

Bacteriophage P1 is the premier transducing phage of E. coli. Despite its prominence in advancing E. coli genetics, modern molecular techniques have not been applied to thoroughly understand P1 structure. Here, we report the proteome of the P1 virion as determined by liquid chromatography tandem mass-spectrometry. Additionally, a library of single-gene knockouts identified the following five previously unknown essential genes: pmgA, pmgB, pmgC, pmgG, and pmgR. In addition, proteolytic processing of the major capsid protein is a known feature of P1 morphogenesis, and we identified the processing site by N-terminal sequencing to be between E120 and S121, producing a 448-residue, 49.3 kDa mature peptide. Furthermore, the P1 defense against restriction (Dar) system consists of six known proteins that are incorporated into the virion during morphogenesis. The largest of these, DarB, is a 250 kDa protein that is believed to translocate into the cell during infection. DarB deletions indicated the presence of an N-terminal packaging signal, and the N-terminal 30 residues of DarB are shown to be sufficient for directing a heterologous reporter protein to the capsid. Taken together, the data expand on essential structural P1 proteins as well as introduces P1 as a nanomachine for cellular delivery.


Subject(s)
Bacteriophage P1 , Escherichia coli , Bacteriophage P1/genetics , Bacteriophage P1/metabolism , Capsid/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , DNA, Viral/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
6.
Microb Genom ; 8(12)2022 12.
Article in English | MEDLINE | ID: mdl-36748517

ABSTRACT

Antimicrobial-resistance (AMR) genes can be transferred between microbial cells via horizontal gene transfer (HGT), which involves mobile and integrative elements such as plasmids, bacteriophages, transposons, integrons and pathogenicity islands. Bacteriophages are found in abundance in the microbial world, but their role in virulence and AMR has not fully been elucidated in the Enterobacterales. With short-read sequencing paving the way to systematic high-throughput AMR gene detection, long-read sequencing technologies now enable us to establish how such genes are structurally connected into meaningful genomic units, raising questions about how they might cooperate to achieve their biological function. Here, we describe a novel ~98 kbp circular P1-bacteriophage-like plasmid termed ph681355 isolated from a clinical Salmonella enterica serovar Typhi isolate. It carries bla CTX-M-15, an IncY plasmid replicon (repY gene) and the ISEcP1 mobile element and is, to our knowledge, the first reported P1-bacteriophage-like plasmid (phage-plasmid) in S. enterica Typhi. We compared ph681355 to two previously described phage-plasmids, pSJ46 from S. enterica serovar Indiana and pMCR-1-P3 from Escherichia coli, and found high nucleotide similarity across the backbone. However, we saw low ph681355 backbone similarity to plasmid p60006 associated with the extensively drug-resistant S. enterica Typhi outbreak isolate in Pakistan, providing evidence of an alternative route for bla CTX-M-15 transmission. Our discovery highlights the importance of utilizing long-read sequencing in interrogating bacterial genomic architecture to fully understand AMR mechanisms and their clinical relevance. It also raises questions regarding how widespread bacteriophage-mediated HGT might be, suggesting that the resulting genomic plasticity might be higher than previously thought.


Subject(s)
Bacteriophages , Salmonella typhi , Salmonella typhi/genetics , Bacteriophages/genetics , Bacteriophage P1/genetics , Drug Resistance, Multiple, Bacterial/genetics , Plasmids/genetics , beta-Lactamases/genetics
7.
mBio ; 12(5): e0101321, 2021 10 26.
Article in English | MEDLINE | ID: mdl-34517752

ABSTRACT

Phage P1 is a temperate phage which makes the lytic or lysogenic decision upon infecting bacteria. During the lytic cycle, progeny phages are produced and the cell lyses, and in the lysogenic cycle, P1 DNA exists as a low-copy-number plasmid and replicates autonomously. Previous studies at the bulk level showed that P1 lysogenization was independent of multiplicity of infection (MOI; the number of phages infecting a cell), whereas lysogenization probability of the paradigmatic phage λ increases with MOI. However, the mechanism underlying the P1 behavior is unclear. In this work, using a fluorescent reporter system, we demonstrated this P1 MOI-independent lysogenic response at the single-cell level. We further observed that the activity of the major repressor of lytic functions (C1) is a determining factor for the final cell fate. Specifically, the repression activity of P1, which arises from a combination of C1, the anti-repressor Coi, and the corepressor Lxc, remains constant for different MOI, which results in the MOI-independent lysogenic response. Additionally, by increasing the distance between phages that infect a single cell, we were able to engineer a λ-like, MOI-dependent lysogenization upon P1 infection. This suggests that the large separation of coinfecting phages attenuates the effective communication between them, allowing them to make decisions independently of each other. Our work establishes a highly quantitative framework to describe P1 lysogeny establishment. This system plays an important role in disseminating antibiotic resistance by P1-like plasmids and provides an alternative to the lifestyle of phage λ. IMPORTANCE Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI. In this work, we built a simple genetic model to elucidate this MOI independency based on the gene-regulatory circuitry of P1. We also proposed that the effective communication between coinfecting phages contributes to the lysis-lysogeny decision-making of P1 and highlighted the significance of spatial organization in the process of cell fate determination in a single-cell environment. Finally, our work provides new insights into different strategies acquired by viruses to interact with their bacterial hosts in different scenarios for their optimal survival.


Subject(s)
Bacteria/virology , Bacteriophage P1/genetics , Bacteriophage P1/metabolism , Gene Expression Regulation, Viral , Lysogeny/genetics , Microbial Interactions , Viral Regulatory and Accessory Proteins/genetics , Bacteriophage P1/chemistry , Lysogeny/physiology , Viral Regulatory and Accessory Proteins/metabolism
8.
ACS Synth Biol ; 10(6): 1292-1299, 2021 06 18.
Article in English | MEDLINE | ID: mdl-33983709

ABSTRACT

Bacterial transduction particles were critical to early advances in molecular biology and are currently experiencing a resurgence in interest within the diagnostic and therapeutic fields. The difficulty of developing a robust and specific transduction reagent capable of delivering a genetic payload to the diversity of strains constituting a given bacterial species or genus is a major impediment to their expanded utility as commercial products. While recent advances in engineering the reactivity of these reagents have made them more attractive for product development, considerable improvements are still needed. Here, we demonstrate a synthetic biology platform derived from bacteriophage P1 as a chassis to target transduction reagents against four clinically prevalent species within the Enterobacterales order. Bacteriophage P1 requires only a single receptor binding protein to enable attachment and injection into a target bacterium. By engineering and screening particles displaying a diverse array of chimeric receptor binding proteins, we generated a potential transduction reagent for a future rapid phenotypic carbapenem-resistant Enterobacterales diagnostic assay.


Subject(s)
Bacteriophage P1/genetics , Carbapenem-Resistant Enterobacteriaceae/genetics , Enterobacteriaceae Infections/diagnosis , Genetic Engineering/methods , Viral Tail Proteins/genetics , Anti-Bacterial Agents/pharmacology , Carbapenem-Resistant Enterobacteriaceae/drug effects , Enterobacteriaceae Infections/microbiology , Ertapenem/pharmacology , Microbial Sensitivity Tests/methods , Phenotype , Synthetic Biology/methods , Transduction, Genetic/methods , beta-Lactam Resistance/drug effects , beta-Lactam Resistance/genetics
9.
Mol Microbiol ; 115(2): 255-271, 2021 02.
Article in English | MEDLINE | ID: mdl-32985020

ABSTRACT

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K-12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K-12, the 4.88 Mbp Fec10 genome is characterized by distinct single-nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two-component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand-alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI-Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI-SG17M. Furthermore, we noted a unique contribution of ClpGGI-Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food-borne heat-resistant strains in the human gut.


Subject(s)
Escherichia coli/metabolism , Thermotolerance/genetics , Thermotolerance/physiology , Bacteriophage P1/genetics , Bacteriophages/genetics , Escherichia coli/genetics , Genome, Bacterial , Genomic Islands , Humans , Oxygen Consumption/physiology , Plasmids/genetics , Symbiosis/physiology
10.
Cold Spring Harb Protoc ; 2020(10)2020 10 01.
Article in English | MEDLINE | ID: mdl-33004554

ABSTRACT

Genetic targeting of specific cell types is fundamentally important for modern molecular-genetic studies. The development of simple methods to engineer high-capacity vectors-in particular, bacterial artificial chromosomes (BACs)-for the preparation of transgenic lines that accurately express a gene of interest has resulted in commonplace usage of transgenic techniques in a wide variety of experimental systems. Here we provide a brief description of each of the four major types of large-capacity vectors, with a focus on the use of BAC vectors.


Subject(s)
Bacteriophage P1/genetics , Chromosomes, Artificial, Bacterial/genetics , Chromosomes, Artificial, Yeast/genetics , Genetic Vectors/genetics , Animals , Escherichia coli/genetics , Gene Transfer Techniques , Genes, Reporter/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice, Transgenic , Models, Genetic , Recombination, Genetic/genetics , Transgenes/genetics
11.
J Biol Chem ; 295(50): 17298-17309, 2020 12 11.
Article in English | MEDLINE | ID: mdl-33055234

ABSTRACT

The faithful segregation, or "partition," of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a "super-repressor." In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.


Subject(s)
Bacteriophage P1/metabolism , Chromosomes, Bacterial/metabolism , DNA, Bacterial/metabolism , Escherichia coli/metabolism , Viral Proteins/metabolism , Amino Acid Substitution , Bacteriophage P1/chemistry , Bacteriophage P1/genetics , Chromosomes, Bacterial/chemistry , Chromosomes, Bacterial/genetics , DNA Primase/chemistry , DNA Primase/genetics , DNA Primase/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/virology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutation, Missense , Operator Regions, Genetic , Viral Proteins/chemistry , Viral Proteins/genetics
12.
Virology ; 542: 1-7, 2020 03.
Article in English | MEDLINE | ID: mdl-31957661

ABSTRACT

To begin its infection, a bacteriophage first needs to adsorb to cells. The adsorption site on the cell surface may influence viral DNA injection, gene expression and cell-fate development. Here, we study the early steps of the infection cycle of coliphage P1, focusing on their correlation with spatial locations at the single-cell level. By fluorescently labeling P1 virions, we found that P1 shows no spatial preference on cell surface adsorption. In addition, live-cell phage DNA imaging revealed that adsorption sites do not affect the success rate for P1 in injecting its DNA into the cell. Furthermore, the lysis-lysogeny decision of P1 does not depend on the adsorption site, based on fluorescence reporters for the lytic and lysogenic pathways. These findings highlight the different infection strategies used by the two paradigmatic coliphages differ from those found in the paradigmatic phage lambda, highlighting that different infection strategies are used by phages.


Subject(s)
Bacteriophage P1/pathogenicity , Escherichia coli/virology , Adsorption , Bacteriophage P1/genetics , Bacteriophage P1/physiology , Capsid Proteins/genetics , Capsid Proteins/physiology , Cell Membrane/virology , Cytoplasm/virology , DNA, Viral/genetics , DNA, Viral/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lysogeny , Microscopy, Fluorescence , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Single-Cell Analysis , Virus Attachment
13.
Sci Rep ; 9(1): 18861, 2019 12 11.
Article in English | MEDLINE | ID: mdl-31827120

ABSTRACT

The spread of multidrug resistance via mobile genetic elements is a major clinical and veterinary concern. Pathogenic Escherichia coli harbour antibiotic resistance and virulence genes mainly on plasmids, but also bacteriophages and hybrid phage-like plasmids. In this study, the genomes of three E. coli phage-like plasmids, pJIE250-3 from a human E. coli clinical isolate, pSvP1 from a porcine ETEC O157 isolate, and pTZ20_1P from a porcine commensal E. coli, were sequenced (PacBio RSII), annotated and compared. All three elements are coliphage P1 variants, each with unique adaptations. pJIE250-3 is a P1-derivative that has lost lytic functions and contains no accessory genes. In pTZ20_1P and pSvP1, a core P1-like genome is associated with insertion sequence-mediated acquisition of plasmid modules encoding multidrug resistance and virulence, respectively. The transfer ability of pTZ20_1P, carrying antibiotic resistance markers, was also tested and, although this element was not able to transfer by conjugation, it was able to lysogenize a commensal E. coli strain with consequent transfer of resistance. The incidence of P1-like plasmids (~7%) in our E. coli collections correlated well with that in public databases. This study highlights the need to investigate the contribution of phage-like plasmids to the successful spread of antibiotic resistant pathotypes.


Subject(s)
Bacteriophage P1 , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli/genetics , Genetic Variation , Genome, Bacterial , Animals , Bacteriophage P1/genetics , Coliphages/genetics , Escherichia coli/physiology , Humans , Sequence Analysis, DNA , Swine
14.
Electron. j. biotechnol ; 41: 9-12, sept. 2019. tab, ilus
Article in English | LILACS | ID: biblio-1053556

ABSTRACT

Background: Horizontal gene transfer (HGT) is the most important mechanism in the evolution of new genetic capabilities in bacteria, including specific degradative pathways, virulence factors, and resistance to antibiotics. Among the processes involved in HGT, transduction is noteworthy. This is a mechanism for gene transmission mediated by a bacteriophage that functions both as a reservoir and as a vector of exogenous genes, which remain protected from environmental effects in the bacteriophage's capsid. Within this context, this investigation aimed to evaluate the ability of the generalized transducing bacteriophage P1 to productively infect and transduce in the bacterial species Salmonella bongori. Results: We could establish that a derivative of bacteriophage P1, P1Cm, infects strains of S. bongori with frequencies of lysogenization in the order of ~10−2 lysogens/UFP. Through thermal induction, infective viral progeny was obtained, and we could show that P1Cm readily formed plaques on S. bongori lawns, a phenomenon thus far not reported for other members of the genus Salmonella. Finally, we showed P1Cm-mediated transduction of the model plasmid RP4 at frequencies of ~10−7 transductants/donor. Conclusion: Therefore, bacteriophage P1 can be used as a tool for the genetic manipulation in the species S. bongori.


Subject(s)
Salmonella , Transduction, Genetic , Bacteriophage P1/genetics , Bacteriophage P1/pathogenicity , Capsid , Gene Transfer, Horizontal , Escherichia coli , Lysogeny
15.
Proc Natl Acad Sci U S A ; 115(2): 337-342, 2018 01 09.
Article in English | MEDLINE | ID: mdl-29259110

ABSTRACT

Bacteria form colonies and secrete extracellular polymeric substances that surround the individual cells. These spatial structures are often associated with collaboration and quorum sensing between the bacteria. Here we investigate the mutual protection provided by spherical growth of a monoclonal colony during exposure to phages that proliferate on its surface. As a proof of concept we exposed growing colonies of Escherichia coli to a virulent mutant of phage P1. When the colony consists of less than [Formula: see text]50,000 members it is eliminated, while larger initial colonies allow long-term survival of both phage-resistant mutants and, importantly, colonies of mostly phage-sensitive members. A mathematical model predicts that colonies formed solely by phage-sensitive bacteria can survive because the growth of bacteria throughout the colony exceeds the killing of bacteria on the surface and pinpoints how the critical colony size depends on key parameters in the phage infection cycle.


Subject(s)
Bacteriophage P1/pathogenicity , Escherichia coli/growth & development , Escherichia coli/virology , Bacterial Load , Bacterial Physiological Phenomena , Bacteriophage P1/genetics , Ecosystem , Escherichia coli/genetics , Host-Pathogen Interactions , Microbial Viability/genetics , Mutation , Quorum Sensing/genetics , Virulence/genetics
16.
Mol Microbiol ; 105(3): 399-412, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28509398

ABSTRACT

Bacterial Type I restriction-modification (R-M) systems present a major barrier to foreign DNA entering the bacterial cell. The temperate phage P1 packages several proteins into the virion that protect the phage DNA from host restriction. Isogenic P1 deletion mutants were used to reconstitute the previously described restriction phenotypes associated with darA and darB. While P1ΔdarA and P1ΔdarB produced the expected phenotypes, deletions of adjacent genes hdf and ddrA also produced darA-like phenotypes and deletion of ulx produced a darB-like phenotype, implicating several new proteins of previously unknown function in the P1 dar antirestriction system. Interestingly, disruption of ddrB decreased P1's sensitivity to EcoB and EcoK restriction. Proteomic analysis of purified virions suggests that packaging of antirestriction components into P1 virions follows a distinct pathway that begins with the incorporation of DarA and Hdf and concludes with DarB and Ulx. Electron microscopy analysis showed that hdf and darA mutants also produce abnormally high proportions of virions with aberrant small heads, which suggests Hdf and DarA play a role in capsid morphogenesis. The P1 antirestriction system is more complex than previously realized and is comprised of multiple proteins including DdrA, DdrB, Hdf, and Ulx in addition to DarA and DarB.


Subject(s)
Bacteriophage P1/metabolism , Capsid/physiology , Bacterial Proteins/metabolism , Bacteriophage P1/genetics , Bacteriophages/genetics , DNA Restriction-Modification Enzymes/genetics , DNA, Viral/metabolism , Escherichia coli/genetics , Morphogenesis , Proteomics , Viral Proteins/metabolism , Virion/genetics
17.
Curr Protoc Mouse Biol ; 7(1): 1-12, 2017 Mar 02.
Article in English | MEDLINE | ID: mdl-28252198

ABSTRACT

The cre-loxP-mediated recombination system (the "cre-loxP system") is an integral experimental tool for mammalian genetics and cell biology. Use of the system has greatly expanded our ability to precisely interrogate gene function in the mouse, providing both spatial and temporal control of gene expression. This has been largely due to the simplicity of its use and its adaptability to address diverse biological questions. While the use of the cre-loxP system is becoming increasingly widespread, in particular because of growing availability of conditional mouse mutants, many considerations need to be taken into account when utilizing the cre-loxP system. This review provides an overview of the cre-loxP system and its various permutations. It addresses the limitations of cre-loxP technology and related considerations for experimental design, and it discusses alternative strategies for site-specific genetic recombination and integration. © 2017 by John Wiley & Sons, Inc.


Subject(s)
Attachment Sites, Microbiological/genetics , Genetic Engineering/methods , Integrases/genetics , Recombination, Genetic , Animals , Anti-Bacterial Agents/pharmacology , Bacteriophage P1/genetics , Base Sequence , Binding Sites/genetics , Doxycycline/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Ligands , Mice , Models, Genetic , Receptors, Estrogen/genetics , Reproducibility of Results
19.
Nat Chem Biol ; 12(7): 490-6, 2016 07.
Article in English | MEDLINE | ID: mdl-27159580

ABSTRACT

Conditional cooperativity is a common mechanism involved in transcriptional regulation of prokaryotic type II toxin-antitoxin operons and is intricately related to bacterial persistence. It allows the toxin component of a toxin-antitoxin module to act as a co-repressor at low doses of toxin as compared to antitoxin. When toxin level exceeds a certain threshold, however, the toxin becomes a de-repressor. Most antitoxins contain an intrinsically disordered region (IDR) that typically is involved in toxin neutralization and repressor complex formation. To address how the antitoxin IDR is involved in transcription regulation, we studied the phd-doc operon from bacteriophage P1. We provide evidence that the IDR of Phd provides an entropic barrier precluding full operon repression in the absence of Doc. Binding of Doc results in a cooperativity switch and consequent strong operon repression, enabling context-specific modulation of the regulatory process. Variations of this theme are likely to be a common mechanism in the autoregulation of bacterial operons that involve intrinsically disordered regions.


Subject(s)
Antitoxins/metabolism , Entropy , Allosteric Regulation , Antitoxins/genetics , Bacteriophage P1/genetics , Bacteriophage P1/metabolism , Operon/genetics
20.
PLoS Genet ; 12(1): e1005797, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26765929

ABSTRACT

Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.


Subject(s)
Ciprofloxacin/pharmacology , DNA-Binding Proteins/genetics , Endonucleases/genetics , Rec A Recombinases/genetics , Viral Proteins/genetics , Bacteriophage P1/genetics , DNA Damage/drug effects , DNA Damage/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/genetics , Humans , Lysogeny/genetics , Neisseria gonorrhoeae/drug effects , SOS Response, Genetics , Staphylococcus aureus/drug effects
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