Thermal and Chemical Inactivation of Bacillus Phage BM-P1.

Bacillus spp. are often used as probiotics; however, they can be infected by phages, leading to significant economic losses. Biocidal and thermal treatments are considered rapid and effective methods for controlling microbial contamination. To prevent viral contamination in industrial dairy production, the impact of temperature and biocides on the viability of Bacillus methylotrophic phage BM-P1 was assessed. The results demonstrated that reconstituted skim milk (RSM) as a medium showed the most effective protective effect on phage BM-P1. Treatment at 90°C for 5 min or 72°C for 10 min inactivated it to nondetectable levels from the initial titer of 7.19 ± 0.11 log, regardless of the culture medium. Sodium hypochlorite exhibited the best inactivating effect, which could reduce the phage titer below the detection level in 4 min at 50 ppm. Additionally, treatment with 75% ethanol for 20 min or 50% isopropanol for 30 min could achieve inactivation to nondetectable levels. The inactivating effect of peracetic acid was limited; even when treated at the highest concentration (0.45%) for 60 min, only a 2.47 ± 0.17 log reduction was observed. This study may provide some theoretical basis and data support for establishing measures against Bacillus spp. phages.

Human Neutrophil Response to Pseudomonas Bacteriophage PAK_P1, a Therapeutic Candidate.

The immune system offers several mechanisms of response to harmful microbes that invade the human body. As a first line of defense, neutrophils can remove pathogens by phagocytosis, inactivate them by the release of reactive oxygen species (ROS) or immobilize them by neutrophil extracellular traps (NETs). Although recent studies have shown that bacteriophages (phages) make up a large portion of human microbiomes and are currently being explored as antibacterial therapeutics, neutrophilic responses to phages are still elusive. Here, we show that exposure of isolated human resting neutrophils to a high concentration of the Pseudomonas phage PAK_P1 led to a 2-fold increase in interleukin-8 (IL-8) secretion. Importantly, phage exposure did not induce neutrophil apoptosis or necrosis and did not further affect activation marker expression, oxidative burst, and NETs formation. Similarly, inflammatory stimuli-activated neutrophil effector responses were unaffected by phage exposure. Our work suggests that phages are unlikely to inadvertently cause excessive neutrophil responses that could damage tissues and worsen disease. Because IL-8 functions as a chemoattractant, directing immune cells to sites of infection and inflammation, phage-stimulated IL-8 production may modulate some host immune responses.

In Situ Structures of the Ultra-Long Extended and Contracted Tail of Myoviridae Phage P1.

The Myoviridae phage tail is a common component of contractile injection systems (CISs), essential for exerting contractile function and facilitating membrane penetration of the inner tail tube. The near-atomic resolution structures of the Myoviridae tail have been extensively studied, but the dynamic conformational changes before and after contraction and the associated molecular mechanism are still unclear. Here, we present the extended and contracted intact tail-structures of Myoviridae phage P1 by cryo-EM. The ultra-long tail of P1, 2450 Å in length, consists of a neck, a tail terminator, 53 repeated tail sheath rings, 53 repeated tube rings, and a baseplate. The sheath of the contracted tail shrinks by approximately 55%, resulting in the separation of the inner rigid tail tube from the sheath. The extended and contracted tails were further resolved by local reconstruction at 3.3 Å and 3.9 Å resolutions, respectively, allowing us to build the atomic models of the tail terminator protein gp24, the tube protein BplB, and the sheath protein gp22 for the extended tail, and of the sheath protein gp22 for the contracted tail. Our atomic models reveal the complex interaction network in the ultra-long Myoviridae tail and the novel conformational changes of the tail sheath between extended and contracted states. Our structures provide insights into the contraction and stabilization mechanisms of the Myoviridae tail.

Facile transduction with P1 phage in Escherichia coli associated with urinary tract infections.

We describe a modification of a standard method that efficiently transduces genes using P1 phage into uropathogenic Escherichia coli strains. This procedure allows utilization of the large KEIO mutant library for analysis of pathogenic E. coli strains.

P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri.

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.

The Role of O-antigen in P1 Transduction of Shigella flexneri and Escherichia coli with its Alternative S' Tail Fibre.

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.

Whole genome sequence analysis of bacteriophage P1 that infects the Lactobacillus plantarum.

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.

An endolysin Salmcide-p1 from bacteriophage fmb-p1 against gram-negative bacteria.

AIMS: A novel endolysin Salmcide-p1 was developed as a promising candidate of new preservative and a supplement to effective enzyme preparations against gram-negative bacterial contaminations. METHODS AND RESULTS: Salmcide-p1 was identified by complementing the genomic sequence of a virulent Salmonella phage fmb-p1. Salmcide-p1 of 112 µg ml-1 could quickly kill Salmonella incubated with 100 mmol l-1 EDTA, with no haemolytic activity. Meanwhile, Salmcide-p1 had a high activity of lysing Salmonella cell wall peptidoglycan. At different temperatures (4-75°C), pH (4-11) and NaCl concentration (10-200 mmol l-1 ), the relative activity of Salmcide-p1 was above 60%. At 4°C, the combination of Salmcide-p1 and EDTA-2Na could inhibit the number of Salmonella Typhimurium CMCC 50115 in skim milk to less than 4 log CFU ml-1 by 3 days, and the number of Shigella flexneri CMCC 51571 was lower than 4 log CFU ml-1 by 9 days. CONCLUSIONS: Salmcide-p1 had a wide bactericidal activity against gram-negative bacteria and showed a broader anti-Salmonella spectrum than the phage fmb-p1. The combination strategy of Salmcide-p1 and EDTA-2Na could significantly inhibit the growth of gram-negative bacteria inoculated in skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage endolysin as an antibacterial agent is considered to be a new strategy against bacterial contamination.

New Insights into the Structure and Assembly of Bacteriophage P1.

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.

Functional Dissection of P1 Bacteriophage Holin-like Proteins Reveals the Biological Sense of P1 Lytic System Complexity.

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.

Nearest-Neighbor Effects Modulate loxP Spacer DNA Chemical Shifts and Guide Oligonucleotide Design for Nuclear Magnetic Resonance Studies.

The Cre-loxP gene editing tool enables site-specific editing of DNA without leaving lesions that must be repaired by error-prone cellular processes. Cre recombines two 34-bp loxP DNA sites that feature a pair of palindromic recombinase-binding elements flanking an asymmetric 8-bp spacer region, via assembly of a tetrameric intasome complex and formation of a Holliday junction intermediate. Recombination proceeds by coordinated nucleophilic attack by pairs of catalytic tyrosine residues on specific phosphodiester bonds in the spacer regions of opposing strands. Despite not making base-specific contacts with the asymmetric spacer region of the DNA, Cre exhibits a preference for initial cleavage on one of the strands, suggesting that intrinsic properties of the uncontacted 8-bp spacer region give rise to this preference. Furthermore, little is known about the structural and dynamic features of the loxP spacer that make it a suitable target for Cre. To enable NMR spectroscopic studies of the spacer, we have aimed to identify a fragment of the 34-bp loxP site that retains the structural features of the spacer while minimizing the spectral crowding and line-broadening seen in longer oligonucleotides. Sequence-specific chemical shift differences between spacer oligos of different lengths, and of a mutant that inverts strand cleavage order, reveal how both nearest-neighbor and next-nearest-neighbor effects dominate the chemical environment experienced by the spacer. We have identified a 16-bp oligonucleotide that preserves the structural environment of the spacer, setting the stage for NMR-based structure determination and dynamics investigations.

Characterization of a P1-bacteriophage-like plasmid (phage-plasmid) harbouring bla CTX-M-15 in Salmonella enterica serovar Typhi.

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.

Interactions between Viral Regulatory Proteins Ensure an MOI-Independent Probability of Lysogeny during Infection by Bacteriophage P1.

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.

A Tail Fiber Engineering Platform for Improved Bacterial Transduction-Based Diagnostic Reagents.

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.

A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.

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.

Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities.

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.

Working with Bacterial Artificial Chromosomes (BACs) and Other High-Capacity Vectors.

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.

Bacteriophage P1 does not show spatial preference when infecting Escherichia coli.

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.

Diversity of P1 phage-like elements in multidrug resistant Escherichia coli.

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.

Productive infection and transduction by bacteriophage P1 in the species Salmonella bongori

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.