Linear display of pRSF-1b and locations of protospacers.

<p>The (+) and (−) strands and corresponding protospacers are coloured red and black, respectively. Kanamycin marker (Kan), Origin of replication (Ori) and <i>lacI</i> (LacI) are shown as arrows. Protospacers have an AAG PAM unless indicated otherwise.</p

PAM and repeat-end correlation.

<p>(A): PAMs of observed spacers and the co-occurring trinucleotide repeat-ends associated with these spacers. Notice that the spacer-proximal nucleotide of the repeat end is identical to the protospacer-proximal nucleotide of the PAM. (B): Schematic of the proposed mechanism for spacer acquisition during CRISPR adaptation. A protospacer with specific PAM is selected after which it is processed into the pre-spacer (at least 33–34 bp), which contains the last nucleotide of the PAM (the pre-spacer could be single-stranded or double-stranded). The pre-spacer is than integrated at the leader proximal end of the CRISPR locus. The nucleotide derived from the PAM forms the last nucleotide of the repeat. (C): R-loop formation by mature crRNA (61 nucleotides) during CRISPR interference. Notice that the last nucleotide of the repeat (the nucleotide derived from the PAM) is complementary to the target DNA sequence. It remains unknown whether base-pairing between these nucleotides is important for interference.</p

Model of the strand specific positive feedback loop.

<p>Cells with a spacer against a known and actively present invader DNA produce targeting Cascade complexes in the expression stage. In the interference stage, Cascade binds the target dsDNA after which the target is cleaved and degraded by Cas3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888-Westra1" target="_blank">[15]</a>. DNA degradation products generated by Cascade and Cas3 (which could be ssDNA or dsDNA) act as precursors for new spacers in the adaptation phase in a strand-specific manner. By integration of these strand-specific precursors, the spacer repertoire against an actively present invader is expanded, completing the positive feedback loop.</p

Effect of integrated spacers on retransformation efficiency.

<p>Transformation efficiencies of various PIMs and the control (Wild type <i>E. coli</i> K12 W3110) are given in a logarithmic scale as colony forming units (CFU) per µg of pRSF-1b plasmid DNA. For each PIM, the number of spacers integrated in either CRISPR locus 2.1 or 2.3 is given. All spacers have an AAG PAM, unless indicated otherwise. The exact spacer composition of each PIM is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>.</p

Graphical representation of AG and GC contents of each observed and possible spacer.

<p>Observed spacers (⧫) are spacers integrated in CRISPR loci 2.1 and 2.3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>). These spacers are 32 or 33-mers with various PAMs. Possible spacers (Ο) are all 32-mers found on pRSF-1b directly downstream of an AAG PAM.</p

Table_1_Environmental Surveillance of Zoonotic Francisella tularensis in the Netherlands.DOCX

<p>Tularemia is an emerging zoonosis caused by the Gram-negative bacterium Francisella tularensis, which is able to infect a range of animal species and humans. Human infections occur through contact with animals, ingestion of food, insect bites or exposure to aerosols or water, and may lead to serious disease. F. tularensis may persist in aquatic reservoirs. In the Netherland, no human tularemia cases were notified for over 60 years until in 2011 an endemic patient was diagnosed, followed by 17 cases in the 6 years since. The re-emergence of tularemia could be caused by changes in reservoirs or transmission routes. We performed environmental surveillance of F. tularensis in surface waters in the Netherlands by using two approaches. Firstly, 339 samples were obtained from routine monitoring -not related to tularemia- at 127 locations that were visited between 1 and 8 times in 2015 and 2016. Secondly, sampling efforts were performed after reported tularemia cases (n = 8) among hares or humans in the period 2013–2017. F. tularensis DNA was detected at 17% of randomly selected surface water locations from different parts of the country. At most of these positive locations, DNA was not detected at each time point and levels were very low, but at two locations contamination was clearly higher. From 7 out of the 8 investigated tularemia cases, F. tularensis DNA was detected in at least one surface water sample collected after the case. By using a protocol tailored for amplification of low amounts of environmental DNA, 10 gene targets were sequenced. Presence of F. tularensis subspecies holarctica was confirmed in 4 samples, and in 2 of these, clades B.12 and B.6 were identified. This study shows that for tularemia, information regarding the spatial and temporal distribution of its causative agent could be derived from environmental surveillance of surface waters. Tracking a particular strain in the environment as source of infection is feasible and could be substantiated by genotyping, which was achieved in water samples with only low levels of F. tularemia present. These techniques allow the establishment of a link between tularemia cases and environmental samples without the need for cultivation.</p

Schematic representation of the ODoSE workflow.

<p>Schematic representation of the ODoSE workflow.</p

Schematic representation of the flow device.

<p>A) Schematic representation of the flow device, with the dimensions in mm. Depicted in red and blue are the in- and outflow channels of the top compartment (light green). The respective in- and outflow channels of the lower compartment (yellow) are given in purple and dark green. B) Electron microscopy image of a microsieve. C) Electron microscopy image of a microdish. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036982#pone.0036982.s001" target="_blank">File S1</a> for more views of the device.</p

Fluorescent nematodes observed in the flow device.

<p>A) Nematodes floating over the wells while the chamber is filled with liquid. The fluorescent oesophagus in the front side of the nematode is clearly visible. B) Nematode trapped in a well filled with fluorescent <i>E. coli</i> cells after removing the liquid from the chamber. C) Next day: A nematode after consuming all fluorescent bacteria from the well, resulting in observable fluorescence in the nematode intestine.</p

Co-cultivation of cells separated by a microsieve.

<p>Increase of GFP expression of inducible cells on the sieve after inoculation of inducer cells below. Graph plotted with the image analysis and processing tool ImageJ. The x-axis corresponds to time and the y-axis shows the detected GFP signal (in arbitrary units). Below: a number of representative images of the microsieve. The time points at which the images were taken are indicated with an asterisk.</p