The T3SS effector protein ExoT is produced and detected in mucoid <i>P. aeruginosa</i> biofilms.

<p>ExoT was tagged with a C-terminal tetracysteine motif that can be bound by a fluorescein-based biarsenical dye (FlAsH reagent). Biofilms of non-mucoid strains PAO1 and FRD2 and mucoid strain FRD1 encoding this chromosomal ExoT<i>-</i>CCPGCC construct were cultivated at 37°C for 2 days and incubated with the FlAsH reagent. Fluorescence microscopy shows the levels of ExoT production in (A) PAO1, (D) FRD2, (G) FRD1 and (J) FRD1 grown in the presence of 10 mM exogenous calcium as well as the biofilm matrix counterstained for exopolysaccharides (B, E, H, K). The merged images are provided in the right column (C, F, I, L). In each case, representative images of x-y (large panel), x-z (bottom panel), and y-z (right panel) slices are displayed. Scale bar, 15 µm. Mucoid FRD1 biofilms were cultivated then supplemented with excess 10 mM Ca²⁺ (J–L) to examine the effect on ExoT production. Each experiment was performed at least three times and representative images are shown.</p

Calcium chelation by exogenous DNA and alginate induces expression of the T3SS in <i>P. aeruginosa</i>.

<p><i>P. aeruginosa</i> PAO1 <i>exoS-lux</i> was used as a reporter for the T3SS. Planktonic cultures were supplemented with (A) exogenous DNA or (B) alginate and various concentrations of calcium and magnesium to BM2 with a baseline concentration of 0.2 mM calcium. Gene expression was measured every 20 minutes for 18 hours and the maximal gene expression is shown. The values shown are the means from experiments done in triplicate and the error bars represent the standard deviation. Values that differ significantly (p<0.02 by unpaired <i>t</i>-test) from the controls (BM2 alone) are marked with an asterisk. Each experiment was performed at least three times and representative values are shown.</p

Exogenous alginate and DNA induce expression of the T3SS in <i>P. aeruginosa</i> planktonic cultures.

<p><i>P. aeruginosa</i> PAO1 <i>exoS-lux</i>, <i>exoT-lux</i> and <i>exoY-lux</i> were used as reporter strains to monitor expression of the T3SS. (A) Bioluminescence gene expression assays were used to measure <i>exoS-lux</i> expression in planktonic cultures of PAO1 grown in BM2 with 0.2 mM Ca²⁺ and 0.5 mM Mg²⁺ and supplemented with various concentrations of NTA, exogenous DNA or alginate. (B) Gene expression assays were used to measure <i>exoT-lux</i> and <i>exoY-lux</i> expression in planktonic cultures of PAO1 grown in BM2 with 0.2 mM Ca²⁺ and 0.5 mM Mg²⁺ and supplemented with various concentrations of alginate (C) or (D) extracellular DNA. Gene expression was measured every 20 minutes for 18 hours and the maximal gene expression is shown. The values shown are the means from triplicate experiments and the error bars represent the standard deviation. Values that differ significantly (p<0.02 by unpaired <i>t</i>-test) from the controls (BM2 alone) are marked with an asterisk. Each experiment was performed at least three times and representative values are shown.</p

<i>ExoS-lux</i> expression in mucoid and non-mucoid isolates grown in planktonic cultures and as agar colonies.

<p>(A) Mucoid (FRD1, PAO1<i>mucA22</i>) and non-mucoid (FRD2, PAO1) <i>P. aeruginosa</i> strains encoding a chromosomal <i>exoS-lux</i> fusion as a reporter for the T3SS were grown in LB and (B) BM2 (0.5 mM Mg²⁺, 0.2 mM Ca²⁺) planktonic cultures. Gene expression was measured every 20 minutes for 18 hours and the maximal gene expression is shown. The values shown are the means from experiments done in triplicate and the error bars represent the standard deviation. All values from mucoid strains (FRD1, mucA22) differ significantly (p<0.02 by unpaired <i>t</i>-test) from the corresponding non-mucoid controls (FRD2, PAO1). (C,E) This strain panel was plated on Pseudomonas Isolation Agar (PIA) and (D,F) LB and incubated at 37°C for 2 days. Bioluminescence was detected in Bio-Rad XRS Chemidoc Imaging system. The colonies are depicted using epi white illumination (left panel) and corresponding <i>exoS-lux</i> expression using bioluminescence imaging (lower panel). Each experiment was performed at least three times and representative values and images are shown.</p

FlAsH detection of ExoT-Cys₄ in an aggregate of the mucoid isolate FRD1.

<p>Phase contrast images of (A) PAO1, (B) FRD1 and (C) FRD2 mid-log planktonic cultures. Fluorescence microscopy shows a mid-log culture of mucoid FRD1 stained with (D) FlAsH to detect the production of ExoT-Cys₄ and (E) a membrane-specific dye FM 4–64 (red). The merged image is shown in (F). Each experiment was performed at least three times and representative images are shown.</p

Circos [16]plot detailing HaRNAV sequence recovery.

<p>The red and blue lines represent reads aligning on the minus and plus strand, respectively. The Heterosigma akashiwo RNA virus has an 8,587 bp ss-RNA linear genome with a single CDS, shown in green on the circos plot. The read depth of coverage is shown in the centre of the plot. The genome is depicted by alternating black-white arcs of 500 bp in size.</p

Sequence summary and detection of viral DNA.

<p>*ppm: pairs per million.</p

Assembly of the Complete Sitka Spruce Chloroplast Genome Using 10X Genomics’ GemCode Sequencing Data

<div><p>The linked read sequencing library preparation platform by 10X Genomics produces barcoded sequencing libraries, which are subsequently sequenced using the Illumina short read sequencing technology. In this new approach, long fragments of DNA are partitioned into separate micro-reactions, where the same index sequence is incorporated into each of the sequencing fragment inserts derived from a given long fragment. In this study, we exploited this property by using reads from index sequences associated with a large number of reads, to assemble the chloroplast genome of the Sitka spruce tree (<i>Picea sitchensis)</i>. Here we report on the first Sitka spruce chloroplast genome assembled exclusively from <i>P</i>. <i>sitchensis</i> genomic libraries prepared using the 10X Genomics protocol. We show that the resulting 124,049 base pair long genome shares high sequence similarity with the related white spruce and Norway spruce chloroplast genomes, but diverges substantially from a previously published <i>P</i>. <i>sitchensis- P</i>. <i>thunbergii</i> chimeric genome. The use of reads from high-frequency indices enabled separation of the nuclear genome reads from that of the chloroplast, which resulted in the simplification of the de Bruijn graphs used at the various stages of assembly.</p></div