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The P. aeruginosa CRISPR/Cas system and its role in bacteria-bacteriophage interactions

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Author	Cady, Kyle C.
Title	The P. aeruginosa CRISPR/Cas system and its role in bacteria-bacteriophage interactions
Date	2012
Abstract	CRISPR/Cas systems are a diverse family of small RNA pathways that are commonly described as a resistance mechanism to infection by lytic bacteriophage and invasion by conjugative plasmids. In the current work we demonstrate that CRISPR/Cas systems are capable of multiple phenotypic outputs, drastically altering our perception of their role in prokaryotic biology. We begin with the initial observation that the P. aeruginosa Yersinia subtype CRISPR/Cas system interacts with chromosomally integrated lysogenic bacteriophage DMS3 to inhibit biofilm formation and swarming motility, but in its native form, does NOT mediate bacteriophage resistance. Genetic analysis established for the first time that the CRISPR associated ( cas ) genes: csy1 , csy2 , csy3 , csy4 , and cas3 are required for activity of the Yersinia subtype CRISPR/Cas system. I also provide the first evidence for Cas1-mediated new spacer insertion in the Yersinia subtype CRISPR/Cas system. Using mutational analysis I showed that the CRISPR derived small RNA crRNAC2S1 directly and site-specifically interacts with bacteriophage DMS3 to inhibit biofilm formation. The relative low level of complementary required for crRNAC2S1 to interact with DMS3 has helped fundamentally change the view of crRNA-target interactions, leading to new models to explain the targeting of crRNAs that lack 100% identity to their target sequences. Systematic chromosomal mutation of the bacteriophage target sequence provided further insights into crRNA-target determinants required for CRISPR/Cas interactions. Importantly, I found that a single nucleotide mutation in the bacteriophage target of the crRNA C2S1 sequence altered the output of this CRISPR/Cas system from biofilm inhibition to complete resistance to bacteriophage infection. Using the discrete crRNAC2S1 phenotypic outputs of biofilm inhibition and bacteriophage resistance we established that DNA is targeted by the Yersinia subtype CRISPR/Cas system to mediate both biofilm inhibition and bacteriophage resistance. Use of classic reporter fusions demonstrated that crRNA targeting of WT bacteriophage DMS3 results in misregulation of late bacteriophage gene expression, providing the first concrete evidence for crRNA-mediated gene regulation. Surprisingly, in contrast with the current model of CRISPR/Cas system function, no cleavage of bacteriophage DNA was observed for either crRNA C2S1 -mediated biofilm inhibition or bacteriophage resistance phenotypes. I propose a model in which crRNAC2S1 and associated Cas proteins recruit RNA polymerase to the target site, resulting in either active transcription, or alternatively, binding of RNA polymerase in the absence of transcription resulting in a block in DNA replication. This work provides new mechanistic insights into one of the 8 CRISPR/Cas subtypes, and fundamentally alters the current narrow view of how CRISPR/Cas systems modulate microbial biology.
Description	vii, 290 leaves : ill. (some col.) ; 28 cm.
Format	application/pdf
Advisor	O'Toole, George A.; Myers, Lawrence C.; Guerinot, Mary Lou; Terns, Michael
College	Dartmouth College -- Department of Microbiology and Immunology.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.420

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	The P. aeruginosa CRISPR/Cas system and its role in bacteria-bacteriophage interactions A Thesis Submitted to the Faculty In Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy in Microbiology and Immunology By Kyle C. Cady DARTMOUTH COLLEGE Hanover, New Hampshire May 2012 Examining Committee: (Chair) George A. O’Toole Lawrence C. Myers Mary Lou Guerinot Michael Terns Brian Pogue, Ph.D. Dean of Graduate Studies

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