Phage Genes Research Articles

The novel anti-phage system Shield co-opts an RmuC domain to mediate phage defense across Pseudomonas species.

Competitive bacteria-bacteriophage interactions have resulted in the evolution of a plethora of bacterial defense systems preventing phage propagation. In recent years, computational and bioinformatic approaches have underpinned the discovery of numerous novel bacterial defense systems. Anti-phage systems are frequently encoded together in genomic loci termed defense islands. Here we report the identification and characterisation of a novel anti-phage system, that we have termed Shield, which forms part of the Pseudomonas defensive arsenal. The Shield system comprises the core component ShdA, a membrane-bound protein harboring an RmuC domain. Heterologous production of ShdA alone is sufficient to mediate bacterial immunity against several phages. We demonstrate that Shield and ShdA confer population-level immunity and that they can also decrease transformation efficiency. We further show that ShdA homologues can degrade DNA in vitro and, when expressed in a heterologous host, can alter the organisation of the host chromosomal DNA. Use of comparative genomic approaches identified how Shield can be divided into four subtypes, three of which contain additional components that in some cases can negatively affect the activity of ShdA and/or provide additional lines of phage defense. Collectively, our results identify a new player within the Pseudomonas bacterial immunity arsenal that displays a novel mechanism of protection, and reveals a role for RmuC domains in phage defense.

Uncovering the virome and its interaction with antibiotic resistome during compost fertilization

Antibiotic resistance is a pressing global health issue, leading to increased illnesses and fatalities. The contribution of viruses to the acquisition, preservation, and dissemination of antibiotic resistance genes (ARGs) is not yet fully understood. By using a high-throughput functional gene-based microarray (GeoChip 5.0), this study examines the prevalence and relative abundance of bacteriophage and eukaryotic viral genes in swine manure, compost, compost-amended agricultural soil, and unamended soil from suburban regions of Beijing, China. Our findings reveal a significantly elevated presence of biomarker viral genes in compost-amended soils compared to unamended soils, suggesting potential health risks associated with compost fertilization. We also observed stronger ecological interactions between ARGs and viral genes in manure and compost than in soils. Network analysis identified arabinose efflux permeases and EmrB/QacA resistance genes, linked to CRISPR encoding sequences, as keystone nodes, indicating possible ARG acquisition via virus infections. Moreover, positive correlations were found between viral genes, antibiotic concentrations, and ARG diversity in manure, compost, and compost-amended soils, highlighting a likely pathway for virus-mediated ARG transfer. In summary, our results indicate that use of compost as a fertilizer in agricultural settings could facilitate the spread of ARGs through viral mechanisms, allowing for time-delayed genetic exchanges over broader temporal and spatial scales than ARGs within bacterial genomes.

Isolation and Characterization of Phages That Bypass the Requirement for RNA-Mediated Antitermination.

The rpoCY75N mutation in the zinc-binding domain of the β' subunit of Escherichia coli RNA polymerase blocks the RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022. Mutant phages that overcome the block imposed by the rpoCY75N mutation are described. These phages, designated "orc" (overcomes rpoC), carry mutations that create new promoters. Promoter activity was assessed by cloning the respective regions from the wild-type and orc phages into a promoterless lacZ reporter vector. Reporter assays showed that the sequence originating from orc phages had significant promoter activity when compared with the equivalent sequence cloned from the parental phage. The newly created promoters facilitate the expression of phage genes that are essential for growth on the rpoCY75N strain by bypassing transcription terminators. The small plaque phenotype of orc phages, when grown on the mutant host, suggests that suppression of the rpoCY75N mutation is incomplete.

Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.

Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.

MgCod: Gene Prediction in Phage Genomes with Multiple Genetic Codes

Massive sequencing of microbiomes has led to the discovery of a large number of phage genomes with intermittent stop codon recoding. We have developed a computational tool, MgCod, that identifies genomic regions (blocks) with distinct stop codon recoding simultaneously with the prediction of protein-coding regions. When MgCod was used to scan a large volume of human metagenomic contigs hundreds of viral contigs with intermittent stop codon recoding were revealed. Many of these contigs originated from genomes of known crAssphages. Further analyses had shown that intermittent recoding was associated with subtle patterns in the organization of protein-coding genes, such as ‘single-coding’ and ‘dual-coding’. The dual-coding genes, clustered into blocks, could be translated by two alternative codes producing nearly identical proteins. It was observed that the dual-coded blocks were enriched with the early-stage phage genes, while the late-stage genes were residing in the single-coded blocks. MgCod can identify types of stop codon recoding in novel genomic sequences in parallel with gene prediction. It is available for download from https://github.com/gatech-genemark/MgCod.

Siphophage 0105phi7-2 of Bacillus thuringiensis: Novel Propagation, DNA, and Genome-Implied Assembly.

Diversity of phage propagation, physical properties, and assembly promotes the use of phages in ecological studies and biomedicine. However, observed phage diversity is incomplete. Bacillus thuringiensis siphophage, 0105phi-7-2, first described here, significantly expands known phage diversity, as seen via in-plaque propagation, electron microscopy, whole genome sequencing/annotation, protein mass spectrometry, and native gel electrophoresis (AGE). Average plaque diameter vs. plaque-supporting agarose gel concentration plots reveal unusually steep conversion to large plaques as agarose concentration decreases below 0.2%. These large plaques sometimes have small satellites and are made larger by orthovanadate, an ATPase inhibitor. Phage head-host-cell binding is observed by electron microscopy. We hypothesize that this binding causes plaque size-increase via biofilm evolved, ATP stimulated ride-hitching on motile host cells by temporarily inactive phages. Phage 0105phi7-2 does not propagate in liquid culture. Genomic sequencing/annotation reveals history as temperate phage and distant similarity, in a virion-assembly gene cluster, to prototypical siphophage SPP1 of Bacillus subtilis. Phage 0105phi7-2 is distinct in (1) absence of head-assembly scaffolding via either separate protein or classically sized, head protein-embedded peptide, (2) producing partially condensed, head-expelled DNA, and (3) having a surface relatively poor in AGE-detected net negative charges, which is possibly correlated with observed low murine blood persistence.

Identification of Toxic Proteins Encoded by Mycobacteriophage TM4 Using a Next-Generation Sequencing-Based Method.

Mycobacteriophages are viruses that specifically infect mycobacteria and which, due to their diversity, represent a large gene pool. Characterization of the function of these genes should provide useful insights into host-phage interactions. Here, we describe a next-generation sequencing (NGS)-based, high-throughput screening approach for the identification of mycobacteriophage-encoded proteins that are toxic to mycobacteria. A plasmid-derived library representing the mycobacteriophage TM4 genome was constructed and transformed into Mycobacterium smegmatis. NGS and growth assays showed that the expression of TM4 gp43, gp77, -78, and -79, or gp85 was toxic to M. smegmatis. Although the genes associated with bacterial toxicity were expressed during phage infection, they were not required for lytic replication of mycobacteriophage TM4. In conclusion, we describe here an NGS-based approach which required significantly less time and resources than traditional methods and allowed the identification of novel mycobacteriophage gene products that are toxic to mycobacteria. IMPORTANCE The wide spread of drug-resistant Mycobacterium tuberculosis has brought an urgent need for new drug development. Mycobacteriophages are natural killers of M. tuberculosis, and their toxic gene products might provide potential anti-M. tuberculosis candidates. However, the enormous genetic diversity of mycobacteriophages poses challenges for the identification of these genes. Here, we used a simple and convenient screening method, based on next-generation sequencing, to identify mycobacteriophage genes encoding toxic products for mycobacteria. Using this approach, we screened and validated several toxic products encoded by mycobacteriophage TM4. In addition, we also found that the genes encoding these toxic products are nonessential for lytic replication of TM4. Our work describes a promising method for the identification of phage genes that encode proteins that are toxic to mycobacteria and which might facilitate the identification of novel antimicrobial molecules.

Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug-resistant Salmonella enterica serovar Kentucky.

Salmonella enterica serovar Kentucky is one of the food-borne zoonotic pathogens which is isolated in high frequency from poultry meat in the recent decades and is known for its multidrug resistance. The current study was aimed to isolate and characterize a bacteriophage against S. enterica serovar Kentucky isolate, 5925, which showed resistance to at least seven antibiotics and to study its efficiency to decontaminate S. Kentucky from chicken skin. The bacteriophage against S. enterica serovar Kentucky was isolated and was named vB_SenS_Ib_psk2 representing the place, source, and host. Electron microscopy revealed that the phage possesses isometric head and contractile tail, indicative of Siphoviridae family. Molecular detection of major capsid protein E gene yielded 511bp, and NCBI blast analysis revealed that the phage belonged to the genus chivirus. The optimum temperature and pH for phage survival and multiplication were found to be - 20 to 42°C and 6-10, respectively. One-step growth curve experiment of vB_SenS_Ib_psk2 revealed a latent period of 20min and burst size of 253 phages/bacterial cell. The host susceptibility studies revealed that 83% of MDR isolates of S. enterica were susceptible to vB_SenS_Ib_psk2. Artificial spiking studies on chicken skin revealed that high multiplicity of infection (MOI) of phages of 106 pfu/mL is required for significant reduction (p ≤ 0.01) of bacterial concentration (0.14 ± 0.04) after 24-h incubation at 8°C compared to group 1 (2.55 ± 0.89cfu/mL).

Engineered Superinfective Pf Phage Prevents Dissemination of Pseudomonas aeruginosa in a Mouse Burn Model.

Pf is a filamentous bacteriophage integrated in the chromosome of most clinical isolates of Pseudomonas aeruginosa. Under stress conditions, mutations occurring in the Pf genome result in the emergence of superinfective variants of Pf (SI-Pf) that are capable of circumventing phage immunity; therefore, SI-Pf can even infect Pf-lysogenized P. aeruginosa. Here, we identified specific mutations located between the repressor and the excisionase genes of Pf4 phage in the P. aeruginosa PAO1 strain that resulted in the emergence of SI-Pf. Based on these findings, we genetically engineered an SI-Pf (eSI-Pf) and tested it as a phage therapy tool for the treatment of life-threatening burn wound infections caused by PAO1. In validation experiments, eSI-Pf was able to infect PAO1 grown in a lawn as well as biofilms formed in vitro on polystyrene. eSI-Pf also infected PAO1 present in burned skin wounds on mice but was not capable of maintaining a sustained reduction in bacterial burden beyond 24 h. Despite not lowering bacterial burden in burned skin tissue, eSI-Pf treatment completely abolished the capability of P. aeruginosa to disseminate from the burn site to internal organs. Over the course of 10 days, this resulted in bacterial clearance and survival of all treated mice. We subsequently determined that eSI-Pf induced a small-colony variant of P. aeruginosa that was unable to disseminate systemically. This attenuated phenotype was due to profound changes in virulence determinant production and altered physiology. Our results suggest that eSI-Pf has potential as a phage therapy against highly recalcitrant antimicrobial-resistant P. aeruginosa infections of burn wounds. IMPORTANCE Pseudomonas aeruginosa is a major cause of burn-related infections. It is also the most likely bacterial infection to advance to sepsis and result in burn-linked death. Frequently, P. aeruginosa strains isolated from burn patients display a multidrug-resistant phenotype necessitating the development of new therapeutic strategies and prophylactic treatments. In this context, phage therapy using lytic phages has demonstrated exciting potential in the control P. aeruginosa infection. However, lytic phages can present a set of drawbacks during phage therapy, including the induction of bacterial resistance and limited bacteria-phage interactions in vivo. Here, we propose an alternative approach to interfere with P. aeruginosa pathogenesis in a burn infection model, i.e., by using an engineered superinfective filamentous phage. Our study demonstrates that treatment with the engineered Pf phage can prevent sepsis and death in a burn mouse model.

Optimization of Escherichia coli Bacteriophages Production

Aims: Phage therapy may be the best alternative to antibiotics in poultry. There is a need for optimization of phage production conditions to produce more Escherichia coli specific phages for poultry applications.
 Methodology: An E. coli λ bacteriophage isolated in our laboratory was used for optimization studies. An optimization of the E. coli λ bacteriophage production process was carried out by conventional methods to identify the factors, and the Thaguchi process was used for the optimization of conditions for increased bacteriophage production. With four factors each at three levels L9, experimental runs were carried out.
 Results: In the conventional method, 8 factors were studied and 4 selected for statistical optimization by Taguchi. At optimized conditions of modified nutrient broth with 1% glycerol, temperature 37oC, 100 rpm for 12 hours of phage multiplication, an improvement of the final titer was achieved from a control 2x109 to optimized 5x1012 Plauque formation Units (PFU)/mL.
 Conclusion: Phage production is dependent on bacterial growth and bacteriophage multiplication in bacteria. In such a complicated system, the Thaguchi method will be a convenient method of phage production optimization.

Complete genome of Sphingomonas paucimobilis ZJSH1, an endophytic bacterium from Dendrobium officinale with stress resistance and growth promotion potential.

Sphingomonas paucimobilis ZJSH1 is an endophytic bacterium isolated from the roots of Dendrobium officinale with the ability to promote plant growth. It was found that the genome of strain ZJSH1 had gene fragment rearrangement compared with the genomes of the other four strains of S. paucimobilis, and the genome was integrated with phage genes. Functional analysis showed that the strain contained colonization-related genes, chemotaxis and invasion. A variety of genes encoding active materials, such as hormones (IAA, SA, ABA and zeaxanthin), phosphate cycle, antioxidant enzymes, and polysaccharides were identified which provide the strain with growth promotion and stress-resistant characteristics. Experiments proved that S. paucimobilis ZJSH1 grew well in media containing 80g/L sodium chloride, 240g/L polyethylene glycol and 800μmol/L Cd2+, indicating its potential for resistance to stresses of salt, drought and cadmium, respectively. S. paucimobilis ZJSH1 is the only endophytic bacterium of this species that has been reported to promote plant growth. The analysis of its genome is conducive to understanding its growth-promoting mechanism and laying a foundation for the development and utilization of this species in the field of agriculture.

ShigaPass: an in silico tool predicting Shigella serotypes from whole-genome sequencing assemblies.

Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase's MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

Bacteriophage antidefense genes that neutralize TIR and STING immune responses

Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.

Optimizing Protein Production in Therapeutic Phages against a Bacterial Pathogen, Mycobacterium abscessus

Therapeutic phages against pathogenic bacteria should kill the bacteria efficiently before the latter evolve resistance against the phages. While many factors contribute to phage efficiency in killing bacteria, such as phage attachment to host, delivery of phage genome into the host, phage mechanisms against host defense, phage biosynthesis rate, and phage life cycle, this paper focuses only on the optimization of phage mRNA for efficient translation. Phage mRNA may not be adapted to its host translation machinery for three reasons: (1) mutation disrupting adaptation, (2) a recent host switch leaving no time for adaptation, and (3) multiple hosts with different translation machineries so that adaptation to one host implies suboptimal adaptation to another host. It is therefore important to optimize phage mRNAs in therapeutic phages. Theoretical and practical principles based on many experiments were developed and applied to phages engineered against a drug-resistant Mycobacterium abscessus that infected a young cystic fibrosis patient. I provide a detailed genomic evaluation of the three therapeutic phages with respect to translation initiation, elongation, and termination, by making use of both experimental results and highly expressed genes in the host. For optimizing phage genes against M. abscessus, the start codon should be AUG. The DtoStart distance from base-pairing between the Shine-Dalgarno (SD) sequence and the anti-SD sequence should be 14–16. The stop codon should be UAA. If UAG or UGA is used as a stop codon, they should be followed by nucleotide U. Start codon, SD, or stop codon should not be embedded in a secondary structure that may obscure the signals and interfere with their decoding. The optimization framework should be generally applicable to developing therapeutic phages against bacterial pathogens.

Efficient traceless modification of the P1 bacteriophage genome through homologous recombination with enrichment in double recombinants: A new perspective on the functional annotation of uncharacterized phage genes.

The advent of high-throughput omic technologies has caused unprecedented progress in research on bacteriophages, the most abundant and still the least explored entities on earth. Despite the growing number of phage genomes sequenced and the rejuvenation of interest in phage therapy, the progress in the functional analysis of phage genes is slow. Simple and efficient techniques of phage genome targeted mutagenesis that would allow one to knock out particular genes precisely without polar effects in order to study the effect of these knock-outs on phage functions are lacking. Even in the case of model phages, the functions of approximately half of their genes are unknown. P1 is an enterobacterial temperate myophage of clinical significance, which lysogenizes cells as a plasmid. It has a long history of studies, serves as a model in basic research, is a gene transfer vector, and is a source of genetic tools. Its gene products have structural homologs in several other phages. In this perspective article, we describe a simple and efficient procedure of traceless P1 genome modification that could also serve to acquire targeted mutations in the genomes of certain other temperate phages and speed up functional annotations of phage genes.

Microbial source tracking of untreated human wastewater and animal scats in urbanized estuarine waters

The study assessed the performance characteristics of host sensitivity, host specificity and concentration for seven human wastewater- and six animal scat-associated marker genes by analysing human wastewater and animal scat samples from urban catchments of the mega-coastal city of Sydney, Australia. Absolute host sensitivity was exhibited across three criteria used to assess seven human wastewater-associated marker genes of cross-assembly phage (CrAssphage), human adenovirus (HAdV), Bacteroides HF183 (HF183), human polyomavirus (HPyV), Lachnospiraceae (Lachno3), Methnobrevibacter smithii nifH (nifH) and pepper mild mottle virus (PMMoV). In contrast, only the horse scat-associated marker gene Bacteroides HoF597 (HoF597) exhibited absolute host sensitivity. The absolute host specificity value of 1.0 was returned for the wastewater-associated marker genes of HAdV, HPyV, nifH and PMMoV for each of the three applied host specificity calculation criteria, while values of >0.9 were returned for CrAssphage and Lachno3. Ruminants and cow scat-associated marker genes of BacR and CowM2, respectively exhibited the absolute host specificity value of 1.0. Concentrations of Lachno3 were greater in most human wastewater samples followed by CrAssphage, HF183, nifH, HPyV, PMMoV and HAdV. Human wastewater marker genes were detected in several scat samples from cats and dogs, and this suggests concordant sampling of animal scat-associated marker genes and at least two human wastewater-associated marker genes will be required to assist in interpretation of fecal sources in environmental waters. A greater prevalence, together with several samples with greater concentrations of human wastewater-associated marker genes PMMoV and CrAssphage warrant consideration by water quality managers for the detection of diluted human fecal pollution in estuarine waters.

The RNA-RNA interactome between a phage and its satellite virus reveals a small RNA that differentially regulates gene expression across both genomes.

Satellite viruses are present across all domains of life, defined as subviral parasites that require infection by another virus for satellite progeny production. Phage satellites exhibit various regulatory mechanisms to manipulate phage gene expression to the benefit of the satellite, redirecting resources from the phage to the satellite, and often inhibiting phage progeny production. While small RNAs (sRNAs) are well documented as regulators of prokaryotic gene expression, they have not been shown to play a regulatory role in satellite-phage conflicts. Vibrio cholerae encodes the phage inducible chromosomal island-like element (PLE), a phage satellite, to defend itself against the lytic phage ICP1. Here, we use Hi-GRIL-seq to identify a complex RNA-RNA interactome between PLE and ICP1. Both inter- and intragenome RNA interactions were detected, headlined by the PLE sRNA, SviR. SviR is involved in regulating both PLE and ICP1 gene expression uniquely, decreasing ICP1 target translation and affecting PLE transcripts. The striking conservation of SviR across all known PLEs suggests the sRNA is deeply rooted in the PLE-ICP1 conflict and implicates sRNAs as unidentified regulators of gene expression in phage-satellite interactions.

Unveil the Secret of the Bacteria and Phage Arms Race

Bacteria have developed different mechanisms to defend against phages, such as preventing phages from being adsorbed on the surface of host bacteria; through the superinfection exclusion (Sie) block of phage's nucleic acid injection; by restricting modification (R-M) systems, CRISPR-Cas, aborting infection (Abi) and other defense systems to interfere with the replication of phage genes in the host; through the quorum sensing (QS) enhancement of phage's resistant effect. At the same time, phages have also evolved a variety of counter-defense strategies, such as degrading extracellular polymeric substances (EPS) that mask receptors or recognize new receptors, thereby regaining the ability to adsorb host cells; modifying its own genes to prevent the R-M systems from recognizing phage genes or evolving proteins that can inhibit the R-M complex; through the gene mutation itself, building nucleus-like compartments or evolving anti-CRISPR (Acr) proteins to resist CRISPR-Cas systems; and by producing antirepressors or blocking the combination of autoinducers (AIs) and its receptors to suppress the QS. The arms race between bacteria and phages is conducive to the coevolution between bacteria and phages. This review details bacterial anti-phage strategies and anti-defense strategies of phages and will provide basic theoretical support for phage therapy while deeply understanding the interaction mechanism between bacteria and phages.

Restriction endonuclease cleavage of phage DNA enables resuscitation from Cas13-induced bacterial dormancy.

Type VI CRISPR systems protect against phage infection using the RNA-guided nuclease Cas13 to recognize viral messenger RNA. Upon target recognition, Cas13 cleaves phage and host transcripts non-specifically, leading to cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is unclear. Here we show that type VI CRISPR and DNA-cleaving restriction-modification (RM) systems frequently co-occur and synergize to clear phage infections and resuscitate cells. In the natural type VI CRISPR host Listeria seeligeri, we show that RM cleaves the phage genome, thus removing the source of phage transcripts and enabling cells to recover from Cas13-induced cellular dormancy. We find that phage infections are neutralized more effectively when Cas13 and RM systems operate together. Our work reveals that type VI CRISPR immunity is cell-autonomous and non-abortive when paired with RM, and hints at other synergistic roles for the diverse host-directed immune systems in bacteria.

Use of a phage cocktail to reduce the numbers of seven Escherichia coli strains belonging to different STEC serogroups applied to fresh produce and seeds

AbstractThe aims of this research were to evaluate the effectiveness of a phage cocktail at reducing seven Shiga toxigenic Escherichia coli (STEC) serogroups on different food matrixes: mung bean sprouts (MBP), lettuce, and mung bean seeds (MBS) and to test the phage cocktail effectiveness to reduce E. coli O157 on Romaine and iceberg lettuce. To study the effect of the type of food matrix on the STEC phage cocktail effectiveness, a mixture of seven highly sensitive STEC strains designated as phage propagation strains (PPS) were used to adulterate Romaine lettuce, MBP, and MBS matrixes at a concentration of 105 logs CFU/g. A subsample of the treated MBS was germinated to assess STEC survival. Recovered STEC strains were confirmed using latex agglutination and PCR. To test the phage cocktail effectiveness to reduce E. coli O157:H7 on Romaine and iceberg lettuce, a mixture of four STEC strains (different than phage propagation strains, non‐PPS) at both low (103 CFU/g) and high (105 CFU/g) concentrations were used to spike the samples in scaled up trials for the purpose of potential commercialization. Phage treatments including a combination of STEC phage cocktail and chlorinated water treatment were then applied to lettuce in a simulated scaled‐up trial. STEC was assessed on the treated samples at different storage time and temperatures (0, 24, 48, and 72 hr at 2, 10 and 25°C). In the food matrix trial, the combination of STEC phage cocktail and chlorinated water‐reduced PPS (p < 0.001) STEC on lettuce by 2.1 log10 CFU/g and on MBP by 2.2 log10 CFU/g. However, isolates from all 7 STEC serogroups remained viable after phage treatment in both lettuce and MBP; particularly those associated with serogroup O111, O121, O103, and O145, while only a few colonies of serogroup O26, O45, and O157 were detected. Lettuce adulterated with low levels of 4 non‐PPS E. coli O157:H7 (103 CFU/g) achieved a reduction of 2.6–3.2 logs. While a reduction 1.7–2.3 logs was achieved by the phage cocktail when lettuce was inoculated with 105 CFU/g. Overall phage performance was more effective at 2 and 10°C and improved over storage time up to 72 hr. However, for MBS, the phage cocktail was not able to kill any of the STEC populations as all of them recovered during germination.