Table_1_Comparative Genomic Analysis of 130 Bacteriophages Infecting Bacteria in the Genus Pseudomonas.XLSX

<p>Bacteria of the genus Pseudomonas are genetically diverse and ubiquitous in the environment. Like other bacteria, those of the genus Pseudomonas are susceptible to bacteriophages which can significantly affect their host in many ways, ranging from cell lysis to major changes in morphology and virulence. Insights into phage genomes, evolution, and functional relationships with their hosts have the potential to contribute to a broader understanding of Pseudomonas biology, and the development of novel phage therapy strategies. Here we provide a broad-based comparative and evolutionary analysis of 130 complete Pseudomonas phage genome sequences available in online databases. We discovered extensive variation in genome size (ranging from 3 to 316 kb), G + C percentage (ranging from 37 to 66%), and overall gene content (ranging from 81–96% of genome space). Based on overall nucleotide similarity and the numbers of shared gene products, 100 out of 130 genome sequences were grouped into 12 different clusters; 30 were characterized as singletons, which do not have close relationships with other phage genomes. For 5/12 clusters, constituent phage members originated from two or more different Pseudomonas host species, suggesting that phage in these clusters can traverse bacterial species boundaries. An analysis of CRISPR spacers in Pseudomonas bacterial genome sequences supported this finding. Substantial diversity was revealed in analyses of phage gene families; out of 4,462 total families, the largest had only 39 members and there were 2,992 families with only one member. An evolutionary analysis of 72 phage gene families, based on patterns of nucleotide diversity at non-synonymous and synonymous sites, revealed strong and consistent signals for purifying selection. Our study revealed highly diverse and dynamic Pseudomonas phage genomes, and evidence for a dominant role of purifying selection in shaping the evolution of genes encoded in them.</p

Table_2_Comparative Genomic Analysis of 130 Bacteriophages Infecting Bacteria in the Genus Pseudomonas.XLSX

<p>Bacteria of the genus Pseudomonas are genetically diverse and ubiquitous in the environment. Like other bacteria, those of the genus Pseudomonas are susceptible to bacteriophages which can significantly affect their host in many ways, ranging from cell lysis to major changes in morphology and virulence. Insights into phage genomes, evolution, and functional relationships with their hosts have the potential to contribute to a broader understanding of Pseudomonas biology, and the development of novel phage therapy strategies. Here we provide a broad-based comparative and evolutionary analysis of 130 complete Pseudomonas phage genome sequences available in online databases. We discovered extensive variation in genome size (ranging from 3 to 316 kb), G + C percentage (ranging from 37 to 66%), and overall gene content (ranging from 81–96% of genome space). Based on overall nucleotide similarity and the numbers of shared gene products, 100 out of 130 genome sequences were grouped into 12 different clusters; 30 were characterized as singletons, which do not have close relationships with other phage genomes. For 5/12 clusters, constituent phage members originated from two or more different Pseudomonas host species, suggesting that phage in these clusters can traverse bacterial species boundaries. An analysis of CRISPR spacers in Pseudomonas bacterial genome sequences supported this finding. Substantial diversity was revealed in analyses of phage gene families; out of 4,462 total families, the largest had only 39 members and there were 2,992 families with only one member. An evolutionary analysis of 72 phage gene families, based on patterns of nucleotide diversity at non-synonymous and synonymous sites, revealed strong and consistent signals for purifying selection. Our study revealed highly diverse and dynamic Pseudomonas phage genomes, and evidence for a dominant role of purifying selection in shaping the evolution of genes encoded in them.</p

Mitonuclear hybrid strains more often resemble their mitochondrial parental isolate.

<p>Averages of maximum pharyngeal bulb fluorescence for mitochondrial (PB800 and HK105) and nuclear (AF16) parent isolates are on either side of the two hybrid strains (AFPB800 and AFHK105) (Fig. 1). Letters denote significantly different groups as determined by Tukey HSD analysis. Bars show one SEM for 15–20 independent samples.</p

Natural and experimental <i>C. briggsae</i> strains and description of the <i>nad5Δ</i> mtDNA deletion.

<p>A. Phylogenetic relationship and <i>nad5Δ</i> heteroplasmy level of <i>C. briggsae</i> isolates studied here. GL = global superclade; KE = Kenya clade; TE and TR = temperate and tropical subclades of GL; C(+) = isolates bearing compensatory Ψ<i>nad5Δ</i>-2 allele; C(-) = isolates bearing ancestral alleles. <i>nad5Δ</i> heteroplasmy categories were assigned to each <i>C. briggsae</i> natural isolate for statistical analysis following Estes et al. (2011): High = underlined font, medium = italicized, low = regular, and zero-<i>nad5Δ</i>="N/A”. Note that we assayed the natural HK104 isolate here instead of the mutation-accumulation line progenitor reported in Estes et al. (2011), which had evolved high <i>nad5Δ</i> levels in the lab (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043837#s2" target="_blank">Materials and Methods</a>). B. Positions of the <i>nad5Δ</i> deletion (dashed line at top) and Ψ<i>nad5Δ</i>-2 elements in the mitochondrial genome. Primers are indicated by arrows (adapted from Howe and Denver, 2008). C. Mitochondrial and nuclear parent isolates for each mitochondrial-nuclear hybrid. <i>nad5Δ</i> heteroplasmy for each hybrid strain matches that of the maternal isolates as expected. Mitochondrial phenotypes are expected to match those of the maternal isolate if measured traits are predominantly determined by the mitochondrial genotype.</p

<em>In Vivo</em> Quantification Reveals Extensive Natural Variation in Mitochondrial Form and Function in <em>Caenorhabditis briggsae</em>

<div><p>We have analyzed natural variation in mitochondrial form and function among a set of <em>Caenorhabditis briggsae</em> isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic <em>nad5</em> gene deletion (<em>nad5Δ</em>) that correlates negatively with organismal fitness. We performed <em>in vivo</em> quantification of 24 mitochondrial phenotypes including reactive oxygen species level, membrane potential, and aspects of organelle morphology, and observed significant among-isolate variation in 18 traits. Although several mitochondrial phenotypes were non-linearly associated with <em>nad5Δ</em> levels, most of the among-isolate phenotypic variation could be accounted for by phylogeographic clade membership. In particular, isolate-specific mitochondrial membrane potential was an excellent predictor of clade membership. We interpret this result in light of recent evidence for local adaptation to temperature in <em>C. briggsae</em>. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. This study demonstrates that multicellular eukaryotic species are capable of extensive natural variation in organellar phenotypes and highlights the potential of integrating evolutionary and cell biology perspectives.</p> </div

Associations between mitochondrial function and morphology traits and isolate-specific <i>nad5Δ</i> level.

<p>Natural variation among <i>C. briggsae</i> isolates in (A) the total area of functional mitochondria, (B) the average area of individual non-functional mitochondria, (C) the total area of non-functional mitochondria, the (D) aspect ratio, (E) circularity, (F) circularity variance of non-functional mitochondria, in (G) relative ΔΨM, (I) the ratio of functional to non-functional organelles, and (H) relative ROS levels. Column colors corresponding to phylogenetic clade (orange = Kenya, white = Temperate, blue = Tropical), and isolates are ordered by deletion frequency along the x-axis. ED3101 and ED3092 do not experience the deletion and were assigned arbitrary x-values of −7 and −5, respectively, for this figure. Averages of maximum pharyngeal bulb fluorescence in <i>C. briggsae</i> natural isolates are plotted in relative fluorescence units (RFU). Bars represent one SEM for 15–20 independent samples.</p

Assigned labels and descriptions of all mitochondrial traits measured for <i>C. briggsae</i> natural isolates.

<p>The grand mean, F-ratio and degrees of freedom for one-way ANOVA testing for phenotypic differences among <i>C. briggsae</i> isolates. Bold font identifies the nine traits retained in the classification tree analysis when using categories based on isolate-specific <i>nad5Δ</i> % (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043837#pone.0043837.s003" target="_blank">Table S3</a>). *, **, and *** denote p<0.05, 0.01, 0.001, respectively. Subscripts N, F, and T indicate whether the measure refers to Non-functional, Functional, or Total mitochondria. Subscript P and V denote that the measure refers to the entire mitochondrial population (not individual mitochondria), or the average individual variance in that trait, respectively.</p

Genome skimming schematic.

<p>Boxes progressing diagonally from top left to bottom right show steps typical of conventional genome projects. Grey boxes show steps shared by genome skimming and conventional genome projects. Red boxes, arrows, and Xs show conventional genome project steps eliminated in the genome skimming approach. Green boxes show analyses specific to our genome skimming strategy.</p

Genome skimming summary information and effector gene hits.

<p>Genome skimming summary information and effector gene hits.</p

Genome Skimming: A Rapid Approach to Gaining Diverse Biological Insights into Multicellular Pathogens

<p>Genome Skimming: A Rapid Approach to Gaining Diverse Biological Insights into Multicellular Pathogens</p