A Genomic Redefinition of <i>Pseudomonas avellanae</i> species

<div><p>The circumscription of bacterial species is a complex task. So far, DNA-DNA hybridization (DDH), 16S rRNA gene sequencing, and multiocus sequence typing analysis (MLSA) are currently the preferred techniques for their genetic determination. However, the average nucleotide identity (ANI) analysis of conserved and shared genes between two bacterial strains based on the pair-wise genome comparisons, with support of the tetranucleotide frequency correlation coefficients (TETRA) value, has recently been proposed as a reliable substitute for DDH. The species demarcation boundary has been set to a value of 95-96% of the ANI identity, with further confirmation through the assessment of the corresponding TETRA value. In this study, we performed a genome-wide MLSA of 14 phytopathogenic pseudomonads genomes, and assessed the ANI and TETRA values of 27 genomes, representing seven out of the nine genomospecies of <i>Pseudomonas</i> spp. sensu Gardan <i>et alii</i>, and their phylogenetic relationships using maximum likelihood and Bayesian approaches. The results demonstrate the existence of a well demarcated genomic cluster that includes strains classified as <i>P. avellanae, P. syringae</i> pv. <i>theae</i>, <i>P.</i> s. pv. <i>actinidiae</i> and one <i>P.</i> s. pv. <i>morsprunorum</i> strain all belonging to the single species <i>P. avellanae</i>. In addition, when compared with <i>P. avellanae</i>, five strains of <i>P.</i> s. pv. <i>tomato</i>, including the model strain DC3000, and one <i>P.</i> s. pv. <i>lachrymans</i> strain, appear as very closely related to <i>P. avellanae</i>, with ANI values of nearly 96% as confirmed by the TETRA analysis. Conversely, one representative strain, previously classified as <i>P. avellanae</i> and isolated in central Italy, is a genuine member of the <i>P. syringae</i> species complex and can be defined as <i>P.</i> s. pv. <i>avellanae</i>. Currently. The core and pan genomes of <i>P. avellanae</i> species consist of 3,995 and 5,410 putative protein-coding genes, respectively.</p> </div

Venn diagram of the type III effector gene complements of <i>Pseudomonas avellanae</i> species.

<p>The diagram is based on the comparison of the same complement of other sequenced plant pathogenic pseudomonads and showing full identity in the reciprocal comparison. The effector proteins conserved among the seven strains are indicated in the centre of the diagram.</p

The core genome of <i>Pseudomonas avellanae</i> species.

<p>Each strain is represented by an oval that is colored according to the current and traditional strain determination. The number of orthologous coding sequences (CDSs) shared by all strains (i.e., the core genome) is in the center (i.e., 3,995). Numbers in the non-overlapping portions of each oval show the number of CDSs unique to each strain.</p

Graphical representation of the average nucleotide identity (ANI) comparison between genomes of <i>Pseudomonas</i> strains.

<p>Graphical representation of the average nucleotide identity (ANI) comparison between genomes of <i>Pseudomonas</i> strains.</p

Split network of the concatenated 6,579 nucleotides for 29 <i>Pseudomonas</i> spp. genomes.

<div><p>A) Strains of genomospecies 3 (P. <i>s</i>. pv. <i>tomato</i>) and 2 (P. <i>s</i>. pv. <i>lachrymans</i>) and genomospecies 8 (<i>P. avellanae</i>, P. <i>s</i>. pv. <i>actinidiae, P</i>. <i>s</i>. pv. <i>theae</i>) sensu Gardan et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075794#B5" target="_blank">5</a>] as well as <i>P</i>. <i>s</i>. pv. <i>morsprunorum</i> M30228, clustered apart from the other representative strains of genomospecies 1, 4, 6 and 9. Bootstraps values higher than 65% are shown.</p> <p>B) Particular of the split network of Figure 5A regarding strains of <i>Pseudomonas avellanae</i> species and the closely-related <i>P. syringae</i> pv. <i>tomato</i> and <i>P</i>. <i>s</i>. pv. <i>lachrymans</i> strains. Bootstraps values higher than 65% are shown.</p></div

Bayesian phylogenetic tree showing relationships among representative strains of phytopathogenic <i>Pseudomonas</i> species and <i>P. syringae</i> pathovars.

<p>The phylogenetic tree was constructed using 2,193 concatenated amino acid of seven housekeeping genes (<i>argS, dnaQ, gltA, gyrB, recA, rpoB</i> and <i>rpoD</i>) with bootstrap values (100,000 generations) shown at the nodes. Strain members of genomospecies 8 (<i>P. avellanae</i>) sensu Gardan et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075794#B5" target="_blank">5</a>], including also <i>P</i>. <i>s</i>. pv. <i>morsprunorum</i> M302280, are shown in red, whereas strain members of genomospecies 2 (P. <i>s</i>. pv. <i>lachrymans</i> M302278) and 3 (P. <i>s</i>. pv. <i>tomato</i>) are in blue. <i>P. fluorescens</i> A506 and <i>P</i><i>. putida</i> UW4 were included as outgroups. To note that strains of genomospecies 2 and 3 are embedded into strains of genomospecies 8. The interior node values of the tree are clade credibility values based on the posterior credibility values produced by MrBayes. </p

Phylogenetic relationships among representative strains of phytopathogenic <i>Pseudomonas</i> species and <i>P. syringae</i> pathovars.

<p>The phylogenetic tree was constructed using 6,579 concatenated nucleotides of seven housekeeping genes (<i>argS, dnaQ, gltA, gyrB, recA, rpoB</i> and <i>rpoD</i>) with bootstrap values greater than 65 per cent (1,000 replicates) shown at the nodes. The phylogenetic relationships were inferred using the maximum likelihood (ML) method and the GTR + I + G as the best model with the PHYLIP package. Strain members of genomospecies 8 (<i>P. avellanae</i>) sensu Gardan et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075794#B5" target="_blank">5</a>], including also <i>P</i>. <i>s</i>. pv. <i>morsprunorum</i> M302280, are shown in red, whereas strain members of genomospecies 2 (P. <i>s</i>. pv. <i>lachrymans</i> M302278) and 3 (P. <i>s</i>. pv. <i>tomato</i>) are in blue. <i>P. fluorescens</i> A506 and <i>P</i><i>. putida</i> UW4 were included as outgroups.</p

The pan genome of <i>Pseudomonas avellanae</i> species.

<p>The <i>P. avellanae</i> pan genome consists of 5,410 ORFs. The graphic shows also the total putative proteins and protein family number found for each strain.</p

A Revertible, Autonomous, Self-Assembled DNA-Origami Nanoactuator

A DNA-origami actuator capable of autonomous internal motion in accord to an external chemical signal was designed, built, operated and imaged. The functional DNA nanostructure consists of a disk connected to an external ring in two, diametrically opposite points. A single stranded DNA, named probe, was connected to two edges of the disk perpendicularly to the axis of constrain. In the presence of a hybridizing target molecule, the probe coiled into a double helix that stretched the inner disk forcing the edges to move toward each other. The addition of a third single stranded molecule that displaced the target from the probe restored the initial state of the origami. Operation, dimension and shape were carefully characterized by combining microscopy and fluorescence techniques

Genomic-assisted characterisation of <i>Pseudomonas</i> sp. strain Pf4, a potential biocontrol agent in hydroponics

<p>In an attempt to select potential biocontrol agents against <i>Pythium</i> spp. and <i>Rhizoctonia</i> spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus <i>Enterobacter</i>, whereas nine strains belonged to the genus <i>Pseudomonas</i>. In <i>in vitro</i> assays, one strain of <i>Pseudomonas</i> sp., Pf4, closely related to <i>Pseudomonas protegens</i> (formerly <i>Pseudomonas fluorescens</i>), showed noteworthy antagonistic activity against two strains of <i>Pythium aphanidermatum</i> and two strains of <i>Rhizoctonia solani</i> AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for <i>in vivo</i> treatments on lamb’s lettuce against <i>R. solani</i> root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵ CFU g⁻¹ of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that <i>Pseudomonas</i> sp. Pf4 has the potential to produce an arsenal of secondary metabolites (<i>plt</i>, <i>phl</i>, <i>ofa</i> and <i>fit-rzx</i> gene clusters) very similar to that of the well-known biocontrol <i>P. protegens</i> strain Pf-5.</p