Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates

Abstract

Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33 %) were more prevalent than the Escherichia CRISPR region subtype (6 %) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100 % identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100 % identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.

Fig. 1.

Organization and conservation of P. aeruginosa CRISPR regions. (a) Organization of cas genes and flanking CRISPR are shown for the Escherichia subtype (top) and Yersinia subtype (bottom). Spacers are shown as diamonds and repeats as rectangles. Arrows indicate cas genes and their predicted direction of transcription. (b) Repeat sequences of the Yersinia and Escherichia subtypes (differences are underlined).

Fig. 2.

Location of spacers 100 % identical to common temperate P. aeruginosa bacteriophage. Boxes denote known or putative bacteriophage ORFs that have been published previously. Forward oriented ORFs are depicted above those ORFs that are divergently transcribed. Each arrow signifies the site of a proto-spacer sequence 100 % identical to a spacer identified in our analysis, with Yersinia subtype spacer matches shown as black triangles and Escherichia subtype spacer matche shown as open triangles.

Fig. 3.

Characterization of CRISPR-positive and -negative P. aeruginosa clinical isolates. Northern blot analysis of small crRNA production for the wild-type lab strain and six clinical isolates (left) and their ΔCRISPR derivatives (right). In this experiment, the three spacers and two repeats closest to the cas1 gene of CRISPR2 of P. aeruginosa UCBPP-PA14 were used as the probe.