Genome, proteome and structure of a T7-like bacteriophage of the kiwifruit canker phytopathogen pseudomonas syringae pv. actinidiae

La pseudomonas syringae pv. actinidiae es un patógeno responsable significativo de la afta bacteriana severa del kiwi (Actinidia sp.). Los bacteriófagos infectados de este fitopatógeno tienen potencial como agentes de control biológico como parte de un enfoque integrado de la gestión del cancro bacteriano, y para su uso como herramientas molecular para el estudio de esta bacteria. Una variedad de bacteriófagos fueron previamente aislados, antes de ser infectados con P. syringae pv. Actinidiae; y sus propiedades básicas fueron caracterizadas para proporcionar un marco para la formulación de estos fagos, como agentes de biocontrol. Aquí, hemos examinado con más detalle el φPsa17, un fago con la capacidad de infectar a una amplia gama de cepas P. syringae pv. Actinidiae, único miembro de la Podoviridae en esta colección. La morfología de partículas fue visualizada mediante criomicroscopía electrónica, el genoma fue secuenciado, y sus proteínas estructurales fueron analizados usando shotgun proteomics. Estos estudios demostraron que 40,525 φPsa17 tiene un genoma de BP, es un miembro de género T7likevirus y está estrechamente relacionada con la pseudomonada llamada fágicas φPSA2 y GH-1. Once proteínas estructurales (andamios) fueron detectados por la proteómica y φPsa17 tiene una cápside de aproximadamente 60 nm de diámetro. No fueron identificados genes indicativos de un ciclo de vida lisogénica, sugiriendo que el fago es necesariamente lítico. Estas características indican que φPsa17 pueden ser adecuadas para la formulación como un agente de biocontrol de P. syringae pv. actinidiaePseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiaeTrabajo patrocinado por. Royal Society. Fellowship Rutherford, para Peter C. Fineran Otago School of Medical Sciences Summer Research Scholarship, para Danni ChenpeerReviewe

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ABSTRACT. Objective. Ileocolitis is a recognized feature of ankylosing spondylitis (AS) and is likely to play a role in the pathogenesis of AS, in conjunction with the normal intestinal microbiota. In order to investigate the host immune response in AS, we measured cytokines in tissue culture following exposure of peripheral blood mononuclear cells (PBMC) to autologous colonic bacteria. Methods. Twenty-one patients with AS and 21 matched controls were recruited. Subjects in the AS group were assessed clinically. Bacteroides species belonging to the B. fragilis group were selectively cultured from stool samples and paired with blood samples from each participant. Ten cultures of autologous Bacteroides were randomly selected from cultures grown from the fecal specimens of each of the 21 patients with AS and 21 controls. These were then tested for reactivity with PBMC and the cytokines produced by proliferating lymphocytes [interleukin 10 (IL-10), IL-17, interferon-γ, tumor necrosis factor-α] were measured in cell culture supernatants. Differences between groups were analyzed using censored normal regression analysis. Results. The patients with AS had severe active AS with Bath AS Disease Activity Index 5.5 (± 1.6) and C-reactive protein (mg/l) 13.8 (± 12.2) (mean ± standard deviation). IL-10 concentrations in ex vivo assay supernatants were lower in the AS group compared with controls (p = 0.047). There were no statistically significant differences between the groups for other cytokines. In ankylosing spondylitis (AS) there is evidence that an environmental trigger, originating in the gastrointestinal tract, is an etiological factor. This has been demonstrated in an animal model, where rats transgenic for human HLA-B27 have been shown to develop a disease characterized by inflammation of the spine and joints and colitis 1 . However, if these rats are derived under germ-free conditions they remain healthy. Subsequent colonization of the gut with normal commensals, particularly Bacteroides vulgatus, leads to the development of disease in these rats 2 . These observations indicate that interactions between a dysfunctional immune system and the bowel microbiota are fundamental aspects of the pathogenesis of AS. Further, in human populations, a strong association with Crohn's disease 3 is well established and ileocolitis has been observed in two-thirds of patients with AS 4 . We have shown previously that the proportions of the major bacterial phylotypes comprising the fecal microbiota did not differ between subjects with AS and healthy controls 5 . Moreover, populations of B. vulgatus, a bacterium shown to promote colitis in HLA-B27 transgenic rats 6 , did not differ in size between patients with AS and controls. We therefore postulated that the nature of the host immune response to autologous bowel bacteria may be of greater importance than the presence or absence of particular bacterial species. To investigate this, we measured the cytokine profile produced by peripheral blood mononuclear cells (PBMC) from patients with AS when exposed to the cells of autologous Bacteroides

Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species

CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids1,2. They are present in approximately half of all sequenced prokaryotes3 and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function4,5. We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.</p

Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.</p

Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands

<div><p>In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In <i>Pectobacterium atrosepticum</i> the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the <i>cas</i> operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.</p></div

Chromosomal targeting results in cell elongation indicative of DNA damage.

<p>Plasmids containing no spacers (control; pC1-16) or one spacer targeting <i>expI</i> (anti-<i>expI</i>; pE1-16) were repressed or induced in the WT and then visualised by (A) LIVE/DEAD staining and fluorescence microscopy (white scale bar; 20 µm; all images to scale) or (B) transmission electron microscopy (TEM) (black scale bars; 2 µm) (C) Quantification of cell lengths of 60 cells from each treatment as assessed by TEM. ns, not significant; *, p-value of <0.0001 when assessed by unpaired two-tailed t-test (PRISM).</p

A single nucleotide PAM mutation enables escape from native CRISPR/Cas targeting.

<p>(A) <i>P. atrosepticum</i> CRISPR/Cas genomic organisation. The <i>cas1</i> and the <i>cas2</i>-<i>cas3</i> hybrid genes are shown in blue, <i>csy1</i>-<i>3</i> in pale blue, <i>cas6f</i> in orange and the CRISPRs as grey arrows in the direction of transcription. CRISPR2 and 3 are separated by a toxin/antitoxin system (white arrows). (B) Spacer 6 of CRISPR2 (from leader) contains deviations from the repeat consensus (shown in blue) and (C) has a 100% match to a protospacer (red) within <i>eca0560</i> in HAI2 in the <i>P. atrosepticum</i> genome. The protospacer matching spacer 6 contains a non-consensus 5′-protospacer-TG-3′ PAM (green). (D) Toxicity assays in the WT with plasmids containing consensus repeats and either no spacer (pC1-16), a single native spacer 6 (5′-protospacer-TG-3′ PAM; pTraGS6-16) or an engineered spacer (5′-protospacer-GG-3′ PAM; pTraG1-16) that targets <i>eca0560</i>. The protospacer locations in <i>eca0560</i> of the native (pTraGS6-16) and engineered (pTraG1-16) spacers are shown below in red. (E) Sequence and pairing of the engineered anti-<i>eca0560</i> spacer (in pTraG1-16) with the consensus 5′-protospacer-GG-3′ PAM (green).</p