Distribution of motility halo diameter in the two selection treatments and the ancestral lineage.

<p>Distribution of motility halo diameter in the two selection treatments and the ancestral lineage.</p

Analysis of insect survival after they are injected with 2000 bacteria cells.

<p>In this analysis, only the 20 lineages we used in the dilution experiment and for phenotypic measurements are present. In addition to the insect weight and the number of CFU present in the injected solution, we considered the median lag, the phenotypic distance to the ancestral lineage, the proportion of non-motile clones and the proportion of clones with the total haemolysis phenotype as explanatory variables. The analysis of all lineages show that the factor that explains the best the highest virulence of selected lineages is the increase in the proportion of total haemolysis, although this effect is marginal. The analysis of HDI lineages demonstrated no significant correlation between the virulence and the phenotypes we have measured. Conversely, in LDI lineages, we found a highly significant correlation between the virulence and the median lag time, i.e. the time that elapses before the absorbance of 50% of the bacterial populations of a lineage overreach that of empty wells. Contrary to our expectations, though, the LDI bacteria that kill insects the fastest are those which take the longest to start growing.</p

Phylogenetic analysis of CdiI^FRM16 orthologous sequences

<p>Phylogenetic trees were constructed by the maximum likelihood (ML) method, with bootstrap values indicated at the nodes. The branch length scale bar below the phylogenetic tree reflects the numbers of amino-acid substitutions per site. The protein sequences are split into three clades, I, II and III, indicated at the right of the tree. Accession numbers of the sequences are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167443#pone.0167443.s002" target="_blank">S2 Table</a>.</p

Figure 1

<p><b>A.</b> The absorbance () of each culture as a function of time. For each curve, we analyzed separately the time that elapses before the absorbance starts increasing and the variation in absorbance after this first increase has occurred. <b>B.</b> In spite of the great variance observed in A, the shape of the growth curve each culture follows is fairly stereotyped. This is represented here by the humped relationship that exists between the increase in absorbance () and current absorbance (). The curves in this figure represent the prediction of a GAM adjusted for each treatment on all replicate experiments (see text). The maximum increase in absorbance is significantly lower and occurs for higher absorbance in selected bacteria than in the ancestral lineages. <b>C.</b> Proportion of cultures which absorbance has not yet increased above that of wells containing only LB, as a function of time, for the ancestral lineage, the HDI treatment and the LDI treatment lines. This time varies a lot from one culture to another, but always positively correlates to the number of cells present in the culture after 20 hours. Overall, we found that absorbance starts increasing sooner in selected bacteria than in the ancestral lineage, although this difference was not found in one of the three replicate experiments.</p

Proportion of surviving insects as a function of the time that elapsed since they were injected with bacteria.

<p>PBS injected insects serve here as a negative control, as they were not injected with bacteria. 46 hours after injection, more than 95% of all insects injected with bacteria were dead.</p

Analysis of insect survival after they are injected with 2000 bacteria cells.

<p>This analysis was performed using a non-parametrical Cox proportional hazard model of survival. In this analysis, we controlled for insect weight and for the number of injected CFU which were found to have no significant effect on survival. A total of trials are analyzed here, with the differences between the lots of insects used in this experiment modeled as a random block factor (with an estimated variance of 0.008). The analysis demonstrates that insects injected with selected bacteria die earlier than those injected with the ancestral lineage.</p

Inventory of <i>cdiBCAI</i>-type loci in <i>Xenorhabdus</i> and <i>Photorhabdus</i> genomes.

<p>Inventory of <i>cdiBCAI</i>-type loci in <i>Xenorhabdus</i> and <i>Photorhabdus</i> genomes.</p

Median lethal time (i.e. the time at which 50% of the insects injected with a particular lineage are dead) as a function of median lag time (i.e. the time at which 50% of the populations of a particular lineage have overreached the absorbance of an empty well).

<p>The estimation of these times are performed using a parametric survival regression method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015872#s2" target="_blank">method</a>). Horizontal and vertical bars correspond to the standard error of these estimates. In LDI lineages, the two quantities are negatively correlated, indicating that the fastest growing LDI lineages are the least virulent. No relation could be found between medium lag time and medium lethal time in HDI lineages. The size of symbols indicates the proportion of clones that display the total haemolysis phenotype. When analyzing both ancestral and selected lineages, we found a marginally significant link between this variable and virulence.</p

A New Member of the Growing Family of Contact-Dependent Growth Inhibition Systems in <i>Xenorhabdus doucetiae</i> - Fig 1

<p><b>The <i>cdiBCAI</i> locus of <i>Xenorhabdus doucetiae</i> FRM16 A</b>. <b>Genetic organization of the <i>cdiBCAI</i> locus</b> Boxes represent genes. Gene labels are shown above the boxes. The putative <i>cdiA</i> and <i>cdiB</i> genes are shown in blue. The <i>cdiA</i>-CT region is indicated. The location of the nucleotide sequence encoding the VENN motif is indicated in yellow. <b>B. The <i>cdiBCAI</i> locus is located in an integrative conjugative element (ICE).</b> The <i>cdi</i> locus is shown in blue. The conserved genes of the ICE, as defined in a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167443#pone.0167443.ref040" target="_blank">40</a>], are highlighted in green. Notable cargo genes (e.g. potentially involved in host interactions) are highlighted in red. The label numbers of the genes are indicated above the locus. The ICE is embedded in a large genomic island inserted between the <i>fba</i> and <i>mltC</i> genes (black boxes). The genetic content of the genomic island is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167443#pone.0167443.s003" target="_blank">S3 Table</a>.</p

Analysis of the time that elapses before absorbance starts increasing.

<p>This analysis was performed using a non-parametrical Cox model of survival. The total number of kinetics that are analyzed here is . The variation between the three replicate experiments is modeled as resulting from a random factor. The possible influence of initial bacterial density is controlled by considering the initial absorbance as a covariate. This analysis shows that selected lines, LDI or HDI, reach the threshold absorbance faster than the ancestral lineage.</p