Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants.

Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.

Complete genome sequences of novel Berlinvirus and novel Certrevirus lytic for Pectobacterium sp. causing soft rot and black leg disease of potato.

Two novel dsDNA bacteriophages named Pectobacterium virus CB251 (PcCB251) and Pectobacterium virus CB7V (PcCB7V) targeting plant pathogen Pectobacterium parmentieri have been isolated and sequenced. The PcCB251 genome consists of 40,557 bp with G+C content of 48.6% and contains 47 predicted genes on a single strand. The phage is classified in genus Berlinvirus, family Autographiviridae. The PcCB7V phage has a circular dsDNA genome of 146,054 bp with G+C content of 50.4% and contains 269 predicted protein genes on both strands and 13 tRNA genes. The PcCB7V phage can be classified in genus Certrevirus, subfamily Vequintavirinae. Both novel bacteriophages have narrow host ranges, but they extend the list of candidates for phage-based control of pectolytic bacteria causing soft rot disease of potato.

Biochemical and Molecular Characteristics of Pc1 Virulent Phage Isolate Infecting Pectobacterium carotovorum.

BACKGROUND AND OBJECTIVE: Pectobacterium carotovorum subsp. carotovorum is a plant-pathogenic bacterium. It is a post-harvest pathogen and causes soft rot diseases in infected plants. Different virulent bacteriophages have been isolated from different regions in the world. These bacteriophages were tolerant to high concentrations of calcium chloride and magnesium chloride. Whereas, the high concentrations of zinc chloride and aluminum chloride decreased the activity and stability of phages. Therefore, the present research aimed to study the biology of P. carotovorum phage (Pc1) by using a one-step growth experiment, its stability to different concentrations of some chemicals and molecular characteristics of this phage isolate. MATERIALS AND METHODS: One step growth experiment, chemical stability, and molecular characteristics by using RAPD-PCR of P. carotovorum phage (Pc1) were studied. RESULTS: The P. carotovorum phage (Pc1) isolate was found to have a latent period of 20 min and its burst size is about 92 pfu cell-1. Calcium chloride, magnesium chloride, and copper sulphate (from 0.1-0.5 mM) increased the infectivity of Pc1 phage, while, zinc chloride in the same concentrations reduced its infectivity. RAPD-PCR amplification was indicated that the total amplified products were 32 bands with size ranged from 0.179-2.365 Kbp. CONCLUSION: Since, zinc chloride (at concentrations of 0.1-0.5 mM) reduced infectivity of Pc1 phage isolate, therefore, any chemical compounds containing zinc must be avoided in designing biocontrol strategy by using phages against soft rot bacterium (P. carotovorum) in potatoes.

Type I-F CRISPR-Cas resistance against virulent phages results in abortive infection and provides population-level immunity.

Type I CRISPR-Cas systems are abundant and widespread adaptive immune systems in bacteria and can greatly enhance bacterial survival in the face of phage infection. Upon phage infection, some CRISPR-Cas immune responses result in bacterial dormancy or slowed growth, which suggests the outcomes for infected cells may vary between systems. Here we demonstrate that type I CRISPR immunity of Pectobacterium atrosepticum leads to suppression of two unrelated virulent phages, ɸTE and ɸM1. Immunity results in an abortive infection response, where infected cells do not survive, but viral propagation is severely decreased, resulting in population protection due to the reduced phage epidemic. Our findings challenge the view of CRISPR-Cas as a system that protects the individual cell and supports growing evidence of abortive infection by some types of CRISPR-Cas systems.

A novel six-phage cocktail reduces Pectobacterium atrosepticum soft rot infection in potato tubers under simulated storage conditions.

Pectobacterium atrosepticum is a species of plant pathogenic bacteria responsible for significant losses in potato production worldwide. Pectobacterium atrosepticum can cause blackleg disease on potato stems as well as the tuber disease termed potato soft rot. Methods for the effective control of these diseases are limited and are primarily based on good agricultural practices. Bacteriophages, viruses of bacteria, could be used as an alternative, environmentally friendly, control measure. Here, we describe the isolation and characterization of 29 phages virulent to P. atrosepticum. The phages belong to 12 different species based on a 95% sequence identity cut-off. Furthermore, based on sequence diversity and propagation results, we selected six of these phages to form a phage cocktail. The phages in the cocktail was tested on a number of P. atrosepticum strains in order to determine their host range. The phages was found to lyse 93% of the tested strains. The cocktail was subsequently tested for its effectiveness in combatting potato soft rot under simulated storage conditions. Use of the phage cocktail reduced both disease incidence and disease severity by 61% and 64%, respectively, strongly indicating that phage biocontrol has the potential to reduce the economic impact of soft rot in potato production.

Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These 'spacers' provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Imprecise Spacer Acquisition Generates CRISPR-Cas Immune Diversity through Primed Adaptation.

Many prokaryotes possess CRISPR-Cas adaptive immune systems to defend against viruses and invading mobile genetic elements. CRISPR-Cas immunity relies on genetic memories, termed spacers, for sequence-specific recognition of infections. The diversity of spacers within host populations is important for immune resilience, but we have limited understanding of how CRISPR diversity is generated. Type I CRISPR-Cas systems use existing spacers to enhance the acquisition of new spacers through primed CRISPR adaptation (priming). Here, we present a pathway to priming that is stimulated by imprecisely acquired (slipped) spacers. Slipped spacers are less effective for immunity but increase priming compared with canonical spacers. The benefits of slipping depend on the relative rates of phage mutation and adaptation during defense. We propose that slipped spacers provide a route to increase population-level spacer diversity that pre-empts phage escape mutant proliferation and that the trade-off between adaptation and immunity is important in diverse CRISPR-Cas systems.

High genomic variability in the plant pathogenic bacterium Pectobacterium parmentieri deciphered from de novo assembled complete genomes.

BACKGROUND: Pectobacterium parmentieri is a newly established species within the plant pathogenic family Pectobacteriaceae. Bacteria belonging to this species are causative agents of diseases in economically important crops (e.g. potato) in a wide range of different environmental conditions, encountered in Europe, North America, Africa, and New Zealand. Severe disease symptoms result from the activity of P. parmentieri virulence factors, such as plant cell wall degrading enzymes. Interestingly, we observe significant phenotypic differences among P. parmentieri isolates regarding virulence factors production and the abilities to macerate plants. To establish the possible genomic basis of these differences, we sequenced 12 genomes of P. parmentieri strains (10 isolated in Poland, 2 in Belgium) with the combined use of Illumina and PacBio approaches. De novo genome assembly was performed with the use of SPAdes software, while annotation was conducted by NCBI Prokaryotic Genome Annotation Pipeline. RESULTS: The pan-genome study was performed on 15 genomes (12 de novo assembled and three reference strains: P. parmentieri CFBP 8475T, P. parmentieri SCC3193, P. parmentieri WPP163). The pan-genome includes 3706 core genes, a high number of accessory (1468) genes, and numerous unique (1847) genes. We identified the presence of well-known genes encoding virulence factors in the core genome fraction, but some of them were located in the dispensable genome. A significant fraction of horizontally transferred genes, virulence-related gene duplications, as well as different CRISPR arrays were found, which can explain the observed phenotypic differences. Finally, we found also, for the first time, the presence of a plasmid in one of the tested P. parmentieri strains isolated in Poland. CONCLUSIONS: We can hypothesize that a large number of the genes in the dispensable genome and significant genomic variation among P. parmentieri strains could be the basis of the potential wide host range and widespread diffusion of P. parmentieri. The obtained data on the structure and gene content of P. parmentieri strains enabled us to speculate on the importance of high genomic plasticity for P. parmentieri adaptation to different environments.

Pectobacterium atrosepticum Phage vB_PatP_CB5: A Member of the Proposed Genus 'Phimunavirus'.

Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (≥80%) with P. atrosepticum phages φM1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal⁻arrest⁻release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, φM1, and PP90 to a separate new genus designated Phimunavirus.

CRISPR-Cas-Mediated Phage Resistance Enhances Horizontal Gene Transfer by Transduction.

A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum, CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted.IMPORTANCE The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution.

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

The bacterial Type III toxin-antitoxin system, ToxIN, is a dynamic protein-RNA complex with stability-dependent antiviral abortive infection activity.

Bacteria have evolved numerous defense systems to protect themselves from viral (bacteriophage) infection. The ToxIN system of Pectobacterium atrosepticum is a Type III toxin-antitoxin complex and "altruistic suicide" anti-phage system, which kills phage-infected cells through the release of a ribonuclease toxin, ToxN. ToxN is counteracted by a co-transcribed antitoxic RNA pseudoknot, ToxI, which self-assembles with ToxN into an inactive 3 ToxI:3 ToxN complex in vitro. However it is not known whether this complex is predominant in vivo, or how the complex is disassembled following infection to trigger a lethal, "altruistic" response. In this study, we characterise ToxI turnover and folding, and explore the link between complex stability and anti-phage activity, with a view to understanding events that lead to ToxN-mediated suicide following phage infection. We present evidence that ToxN constantly cleaves fresh ToxI in vivo rather than staying associated with pre-processed antitoxin, and that the ToxI antitoxin can partially fold spontaneously using conserved nucleotides. We also show that reducing the stability of the ToxIN complex can increase the strength of the antiviral response in a phage-dependent manner. Based on this information, we propose a revised model for ToxN inhibition, complex assembly and activation by infecting bacteriophage.

The viability of lytic bacteriophage ΦD5 in potato-associated environments and its effect on Dickeya solani in potato (Solanum tuberosum L.) plants.

Dickeya solani is one of the most important pectinolytic phytopathogens responsible for high losses in potato, especially in seed potato production in Europe. Lytic bacteriophages can affect the structure of the host population and may influence spread, survival and virulence of the pathogen and in consequence, infection of the plant. In this study, we aimed to acquire information on the viability of the broad host lytic bacteriophage ΦD5 on potato, as well as to apprehend the specific effect of this bacteriophage on its host D. solani type-strain in different settings, as a preliminary step to target co-adaptation of phages and host bacteria in plant environment. Viability of the ΦD5 phage in tuber extract, on tuber surface, in potting compost, in rainwater and on the leaf surface, as well as the effect of copper sulfate, were examined under laboratory conditions. Also, the interaction of ΦD5 with the target host D. solani in vitro and in compost-grown potato plants was evaluated. ΦD5 remained infectious in potato tuber extract and rain water for up to 72 h but was inactivated in solutions containing 50 mM of copper. The phage population was stable for up to 28 days on potato tuber surface and in potting compost. In both, tissue culture and compost-grown potato plants, ΦD5 reduced infection by D. solani by more than 50%. The implications of these findings are discussed.

Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

Bacteriophage vB_PcaP_PP2 (PP2) is a novel virulent phage that infects the plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. PP2 phage has a 41,841-bp double-stranded DNA encoding 47 proteins, and it was identified as a member of the family Podoviridae by transmission electron microscopy. Nineteen of its open reading frames (ORFs) show homology to functional proteins, and 28 ORFs have been characterized as hypothetical proteins. PP2 phage is homologous to Cronobacter phage vB_CskP_GAP227 and Dev-CD-23823. Based on phylogenetic analysis, PP2 and its homologous bacteriophages form a new group within the subfamily Autographivirinae in the family Podoviridae, suggesting the need to establish a new genus. No lysogenic-cycle-related genes or bacterial toxins were identified.

Evolution of Pectobacterium Bacteriophage ΦM1 To Escape Two Bifunctional Type III Toxin-Antitoxin and Abortive Infection Systems through Mutations in a Single Viral Gene.

Some bacteria, when infected by their viral parasites (bacteriophages), undergo a suicidal response that also terminates productive viral replication (abortive infection [Abi]). This response can be viewed as an altruistic act protecting the uninfected bacterial clonal population. Abortive infection can occur through the action of type III protein-RNA toxin-antitoxin (TA) systems, such as ToxINPa from the phytopathogen Pectobacterium atrosepticum Rare spontaneous mutants evolved in the generalized transducing phage ΦM1, which escaped ToxINPa-mediated abortive infection in P. atrosepticum ΦM1 is a member of the Podoviridae and a member of the "KMV-like" viruses, a subset of the T7 supergroup. Genomic sequencing of ΦM1 escape mutants revealed single-base changes which clustered in a single open reading frame. The "escape" gene product, M1-23, was highly toxic to the host bacterium when overexpressed, but mutations in M1-23 that enabled an escape phenotype caused M1-23 to be less toxic. M1-23 is encoded within the DNA metabolism modular section of the phage genome, and when it was overexpressed, it copurified with the host nucleotide excision repair protein UvrA. While the M1-23 protein interacted with UvrA in coimmunoprecipitation assays, a UvrA mutant strain still aborted ΦM1, suggesting that the interaction is not critical for the type III TA Abi activity. Additionally, ΦM1 escaped a heterologous type III TA system (TenpINPl) from Photorhabdus luminescens (reconstituted in P. atrosepticum) through mutations in the same protein, M1-23. The mechanistic action of M1-23 is currently unknown, but further analysis of this protein may provide insights into the mode of activation of both systems.IMPORTANCE Bacteriophages, the viral predators of bacteria, are the most abundant biological entities and are important factors in driving bacterial evolution. In order to survive infection by these viruses, bacteria have evolved numerous antiphage mechanisms. Many of the studies involved in understanding these interactions have led to the discovery of biotechnological and gene-editing tools, most notably restriction enzymes and more recently the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Abortive infection is another such antiphage mechanism that warrants further investigation. It is unique in that activation of the system leads to the premature death of the infected cells. As bacteria infected with the virus are destined to die, undergoing precocious suicide prevents the release of progeny phage and protects the rest of the bacterial population. This altruistic suicide can be caused by type III toxin-antitoxin systems, and understanding the activation mechanisms involved will provide deeper insight into the abortive infection process.

Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts.

This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer's buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

Genomic, proteomic and morphological characterization of two novel broad host lytic bacteriophages ΦPD10.3 and ΦPD23.1 infecting pectinolytic Pectobacterium spp. and Dickeya spp.

Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-field gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.

Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

A processed noncoding RNA regulates an altruistic bacterial antiviral system.

The ≥ 10³° bacteriophages on Earth relentlessly drive adaptive coevolution, forcing the generation of protective mechanisms in their bacterial hosts. One such bacterial phage-resistance system, ToxIN, consists of a protein toxin (ToxN) that is inhibited in vivo by a specific RNA antitoxin (ToxI); however, the mechanisms for this toxicity and inhibition have not been defined. Here we present the crystal structure of the ToxN-ToxI complex from Pectobacterium atrosepticum, determined to 2.75-Å resolution. ToxI is a 36-nucleotide noncoding RNA pseudoknot, and three ToxI monomers bind to three ToxN monomers to generate a trimeric ToxN-ToxI complex. Assembly of this complex is mediated entirely through extensive RNA-protein interactions. Furthermore, a 2'-3' cyclic phosphate at the 3' end of ToxI, and catalytic residues, identify ToxN as an endoRNase that processes ToxI from a repetitive precursor but is regulated by its own catalytic product.

Phage-selected lipopolysaccharide mutants of Pectobacterium atrosepticum exhibit different impacts on virulence.

AIMS: To positively select Pectobacterium atrosepticum (Pa) mutants with cell surface defects and to assess the impact of these mutations on phytopathogenesis. METHODS AND RESULTS: Several phages were isolated from treated sewage effluent and were found to require bacterial lipopolysaccharide (LPS) for infection. Two strains with distinct mutations in LPS were obtained by transposon mutagenesis. Along with a third LPS mutant, these strains were characterized with respect to various virulence-associated phenotypes, including growth rate, motility and exoenzyme production, demonstrating that LPS mutations are pleiotropic. Two of the strains were deficient in the synthesis of the O-antigen portion of LPS, and both were less virulent than the wild type. A waaJ mutant, which has severe defects in LPS biosynthesis, was dramatically impaired in potato tuber rot assays. The infectivity of these novel phages on 32 additional strains of Pa was tested, showing that most Pa isolates were sensitive to the LPS-dependent phages. CONCLUSIONS: Native LPS is crucial for optimal growth, survival and virulence of Pa in vivo, but simultaneously renders such strains susceptible to phage infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the power of phages to select and identify the virulence determinants on the bacterial surface, and as potential biocontrol agents for Pa infections.