Image_1_Construction of a Shuttle Vector Using an Endogenous Plasmid From the Cyanobacterium Synechocystis sp. PCC6803.PDF

<p>To advance synthetic biology in the photosynthetic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we constructed a shuttle vector with some versatile features. This shuttle vector, pSCB-YFP, consists of a putative replicon identified on the plasmid pCC5.2, the origin of replication of pMB1 from E. coli, as well as the YFP reporter gene and a spectinomycin/streptomycin resistance cassette. pSCB-YFP is stably maintained in Syn6803M (a motile strain that lacks the endogenous pCC5.2) and expresses YFP. In addition, we engineered a fragment into pSCB-YFP that has multiple cloning sites and other features such that this plasmid can also be used as an expression vector (pSCBe). The shuttle vector pSCB-YFP can be stably maintained for at least 50 generations without antibiotic selection. It is a high copy number plasmid and can stably co-exist with the RSF1010-based pPMQAK1-GFP.</p

Diversity in a Polymicrobial Community Revealed by Analysis of Viromes, Endolysins and CRISPR Spacers

<div><p>The polymicrobial biofilm communities in Mushroom and Octopus Spring in Yellowstone National Park (YNP) are well characterized, yet little is known about the phage populations. Dominant species, <i>Synechococcus sp</i>. JA-2-3B'a(2–13), <i>Synechococcus</i> sp. JA-3-3Ab, <i>Chloroflexus sp</i>. <i>Y-400-fl</i>, and <i>Roseiflexus sp</i>. <i>RS-1</i>, contain multiple CRISPR-Cas arrays, suggesting complex interactions with phage predators. To analyze phage populations from Octopus Spring biofilms, we sequenced a viral enriched fraction. To assemble and analyze phage metagenomic data, we developed a custom module, VIRITAS, implemented within the MetAMOS framework. This module bins contigs into groups based on tetranucleotide frequencies and CRISPR spacer-protospacer matching and ORF calling. Using this pipeline we were able to assemble phage sequences into contigs and bin them into three clusters that corroborated with their potential host range. The virome contained 52,348 predicted ORFs; some were clearly phage-like; 9319 ORFs had a recognizable Pfam domain while the rest were hypothetical. Of the recognized domains with CRISPR spacer matches, was the phage endolysin used by lytic phage to disrupt cells. Analysis of the endolysins present in the thermophilic cyanophage contigs revealed a subset of characterized endolysins as well as a Glyco_hydro_108 (PF05838) domain not previously associated with sequenced cyanophages. A search for CRISPR spacer matches to all identified phage endolysins demonstrated that a majority of endolysin domains were targets. This strategy provides a general way to link host and phage as endolysins are known to be widely distributed in bacteriophage. Endolysins can also provide information about host cell wall composition and have the additional potential to be used as targets for novel therapeutics.</p></div

Breakdown of ORFs Containing CRISPR Spacer Matches by Bin.

<p>A) Pfam distribution across Clusters 1–3 and contigs under 1Kb visualized as a heat map. Colour corresponds to count, with black = 0, medium grey = 1, and light grey = 2 or more. B) ORFs with known predictions containing CRISPR spacer matches from contigs over 1Kb (Cluster 1, 2 & 3) as well as under 1Kb (shown in purple). Glyco_hydro_108 domains are marked with a purple star.</p

OS-V-09 454Ti Run Statistics.

<p>OS-V-09 454Ti Run Statistics.</p

ESOM of Assembled Viral Contigs.

<p>A) The tetranucleotide signature for viral contigs greater than 1Kb (navy), as well as 5K fragments from five genomes from fully sequenced mat species <i>Synechococcus</i> sp. JA-2-3B'a(2–13) (light pink), <i>Synechococcus</i> sp. JA-3-3Ab (salmon pink), <i>Meiothermus silvanus</i> (light grey), <i>Chloroflexus sp</i>. Y-400-fl (mint green) and <i>Roseiflexus</i> sp. RS-1 (yellow), was calculated via scripts from Dick et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160574#pone.0160574.ref059" target="_blank">59</a>]. Viral contigs clustered into three major groups (Cluster 1–3). B) Viral contigs with at least one CRISPR spacer hit re-coloured to reflect their host as shown in part a. Legend represents tetranucleotide frequency distances from valleys (blue) to peaks (white).</p

Distribution of Endolysin Catalytic Domains in Sequenced Cyanophages and OS-V-09.

<p>CIRCOS plot depicting the distribution of Endolysin Catalytic Domains (shown in red) found in OS-V-09 (shown in green) and in annotated cyanophage genomes from IMG (shown in teal). A subset of domains (PF00182, PF05838, PF01464 and PF01551) were found in thermophilic phage as compared to cyanophages. In addition, the Glyco_hydro_108 (PF05838) indicated with a purple star was only found in OS-V-09.</p

Endolysin CRISPR hit distribution.

<p>Endolysin CRISPR hit distribution.</p

SPADes Assembly Statistics.

<p>SPADes Assembly Statistics.</p

Biophysical model of EPS-based mobility enhancement captures the observed fingering behavior during phototaxis.

<p>A) As shown schematically, each cyanobacterium (green) is assumed to undergo a biased random walk toward the light source (LED). Each cell secretes EPS (blue), whose local concentration increases the cell's mobility <i>M</i> (red). B) The cell deposition, crescent formation, and finger formation observed in experiments (top) are recapitulated by simulations using our model (bottom) with and ; cells are shown in green, EPS in red. Immediately after deposition (left), cells are distributed randomly and the boundary with the substrate is smooth (inset). Cells collect at the edge of the initial deposition area as they migrate toward the light source (middle, 20 hr), with small variations at the interface (inset). The front matures into discrete groups of cells that separately migrate toward the light source (right, 45 hr). Scale bars are 1 mm.</p

Cells secrete an extracellular substance that enhances their motility.

<p>A) Typical fingering of a <i>Synechocystis</i> community toward a light source during a phototaxis assay. The contrasted inset shows EPS deposited by motile groups. B) Phase-contrast microscopy of motile groups immediately before, and then at specified intervals after a change in the light direction (orange arrows). The white dashed lines indicate the bounds of a region through which a finger recently passed. The motile group changes course after a change in the light direction, and upon intersection with the EPS trail of a neighboring finger, group speed toward the light increases and the cells become more dispersed. C) Median velocity of cells in the motile group in both the <i>x</i> and <i>y</i> directions with respect to the coordinate system shown in (B); dashed horizontal lines indicate 95% confidence intervals. Left: prior to the change in light direction, cells have positive velocity toward the light source (top), and approximately zero net velocity perpendicular to the light (bottom). Middle: following the change in light direction, the cells reorient and velocity in the <i>x</i> direction rises to a value comparable with the <i>y</i> velocity prior to the light change (bottom), while the net y velocity approaches zero (top). Right: when the group of cells merges with the trail of the neighboring finger (dashed vertical lines), the spread in <i>y</i> velocities increases (top) and the median <i>x</i> velocity increases by approximately three-fold (bottom). D) Histograms of speeds for the same cells (<i>n</i> = 95) before and soon after merging with the trail of secreted extracellular substance, with mean speed and standard deviation indicated in the legend. E) Individual cells experience an increase in speed after the group merges with the trail of another finger, indicating that the change in group dynamics upon merging is coupled to a change in the motility of individual cells.</p