Insertion mutants in the c1AH10 <i>pdl1</i> gene are defective in Tcd supernatant release.

<p>Mean weight gain of cohorts of <i>M. sexta</i> neonates fed dilutions of washed cells or supernatants from 72 hour old cultures of <i>E. coli</i> containing the c1AH10 cosmid with transposon inserts in either the <i>pdl1</i> gene (<i>pdl1</i> KO1-mutant), white, or the pWEB cosmid vector backbone insertion (CVI-wt), shaded. Error bars represent the standard error. The more potent the toxic effect, the smaller the mean larval weight. Note the loss of toxicity at higher dilutions of the supernatants of the <i>pdl1</i> knock out cosmid strain. The figures in white and black above the bars represent larval mortality numbers during the 7 day assay.</p

Pdl1 increases TcdB release into the supernatant.

<p>(<b>A</b>) Western blot analysis of culture supernatants (sup), and cellular membrane (mem) and soluble fractions, which includes cell cytosol and periplasm fractions (sol). Preparations from 24 h cultures of <i>E. coli</i> containing c1AH10 with transposon insertions into either the pWEB vector backbone (CVI-wt) or the <i>pdl1</i> gene (<i>pdl1</i> KO-mutant). X = marker and Vec = whole cell pWEB negative vector control. The presence or absence of Tcd is visualised using the antibody raised against the C-terminus of the B-subunit. Protein from an induced clone containing a B and C-subunit expression construct (pBC+) is included as a positive control. (<b>B</b>) An anti-β-Lactamase western blot was performed to ensure no contamination of soluble material in the membrane fraction. (<b>C</b>) An anti-SecG western blot was performed to ensure no contamination of membrane material in the soluble fraction.</p

Pdl1 exhibits some haemolytic activity.

<p>A weak haemolytic phenotype displayed by the <i>E. coli</i> heterologous pBAD30-<i>pdl1</i> based expression strain. The un-induced (<b>A</b>) and arabinose induced (<b>B</b>) expression strain on agar plates containing sheep red blood cells. Note the diffuse zone of haemolysis surrounding the induced <i>E. coli</i> expressing Pdl1 (arrow). (<b>C</b>) Haemolysis of sheep red blood cells by heterologously expressed Pdl1 in a liquid assay. Controls include, PBS alone, LB medium alone, LB supplemented with 0.2% w/v arabinose and the un-induced pBAD30-<i>pdl1</i> expression strain. Results are expressed as a percentage haemolysis, where 100% corresponds to the amount of haemoglobin released by complete lysis of the red blood cells using water.</p

Pdl1 and Orf53 expression in strain W14 increases and decreases Tc toxin release respectively.

<p><i>M. sexta</i> oral bioassay of dilutions (0.1×, 0.05× and 0.025×) of cells and supernatants from <i>P. luminescens</i> W14 cultures over-expressing C-terminally his-tagged Pdl1 (<i>pdl1</i>) or Orf53 (<i>o53</i>) from the pBAD30 expression vector. <i>Pl</i> W14 harbouring the induced empty pBAD30 expression vector (Vec) provides a control against which the data has been normalised. Bioassays were conducted using samples taken at 24 and 48 hours post induction and the mean Relative Weight Gain (RWG) of larvae is shown, in relation to the negative controls (Vec) which therefore give a RWG of 1. Note these are the same cultures examined by western blot in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002692#ppat.1002692.s005" target="_blank">Fig. S5</a>. Each data point is derived from the mean weight of 10 neonate larvae after seven days consumption. RWG-standard error bars are also shown.</p

The genus <i>Photorhabdus</i> contains three predominant species.

<p>A stylized representation of a previous six gene MLST phylogeny (<i>adk</i>, <i>ghd</i>, <i>mdk</i>, <i>ndh</i>, <i>pgm</i> and <i>recA</i>) of <i>Photorhabdus</i> (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144937#pone.0144937.ref005" target="_blank">5</a>]) is shown. The grey areas indicate species that consist of multiple strains, the majority of which are unable to grow above 34°C, with only a few <i>P</i>. <i>luminescens</i> strains capable of growth at temperatures up to 37°C. Example strains are <i>P</i>. <i>luminescens</i>^TT01 and <i>P</i>. <i>temperata</i>^K122. The clinical strains adapted to 37°C are boxed. The stars and circles indicate the potential historical timing of temperature adaptation, which could have occurred ancestrally (star) or independently (circles) in different geographical isolates.</p

A schematic summarising some key differences in metabolism at 37°C compared to 28°C, centred on glutamate/asparagine metabolism and the TCA cycle.

<p>This model is predicted by integrating data from the RNA-seq, proteomics and phenotype microarray studies. Intermediates (boxes) and pathways (arrows) predicted to be down regulated at 37°C are in red while those up regulated are in green. Data suggests TCA cycle intermediates (back boxes) would be relatively isolated from glutamate/asparagine metabolism and could be maintained via the conversion of L-serine into citrate via pyruvate. Black arrows indicate certain potential enzyme pathways that are present and predicted to be unchanged at 37°C. The data suggests a central role for imported peptides and amino acids in metabolism at 37°C. Opp/Dpp represent oligo- and di-peptide importers, TCT represents tricarboxylic acid and PEP is Phosphoenolpyruvate.</p

Clinical <i>Photorhabdus</i> isolates are able to survive exposure to higher temperatures than most non-clinical isolates.

<p>The optical density achieved by representative strains after overnight growth in static conditions (at 28°C in LB medium) after prior 18 h exposure to a range of temperatures. A range of clinical (N. American and Australian) and non-clinical (European) strains of <i>P</i>. <i>asymbiotica (Pa)</i> were tested, and the well-studied <i>P</i>. <i>luminescens</i> strain (<i>Pl</i>^TT01) was included for comparison. Green stars and red diamonds indicate thermal tolerance and intolerance respectively. <i>Pa</i> strain designations are indicated as superscripts.</p

The secreted metalloprotease PrtA is one of the most highly up regulated genes at 37°C.

<p>An Artemis view of mapped RNA-seq data showing higher transcription of the <i>prtA</i> gene at 37°C compared to 28°C. A slight increase is also seen in the associated ABC transporter genes, <i>prtBCD</i>, and the predicted inhibitor gene <i>inh</i>.</p

The expression and function of the <i>Photorhabdus</i> natural product rhabduscin.

<p>(A) Artemis views of the RNA-seq reads of the three replicates mapped onto the <i>Pa</i>^ATCC43949 operons responsible for rhabduscin synthesis. The <i>isnAB</i> genes are responsible for synthesis of the aglycon precursor shown above the left panel. The PAU_02755–7 genes encode glycosidase enzymes that add the sugar groups to produce the final rhabduscin molecule. Note PAU_02756 is unique to the <i>P</i>. <i>asymbiotica</i> (replaced by a transposase in <i>Pl</i>^TT01) and so the final <i>Pa</i>^ATCC43949 rhabduscin structure from <i>Pa</i>^ATCC43949 may not be the same as that shown from <i>Pl</i>^TT01 (above the right panel). (B) The purified aglycon precursor of rhabduscin (shown above the key) is able to completely inhibit the human alternative complement pathway. (C) Cell free supernatants from <i>Pa</i>^ATCC43949 (PaATCC43949), <i>Pa</i>^Kingscliff (Pa Kc) and <i>Pl</i>^TT01 (Pl TT01) can all inhibit the human alternative complement pathway (AP). Note the classical (CP) is only partially inhibited, while LB alone also inhibits the Maltose binding lectin (MBLP) pathway to some extent.</p