Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in...

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Autores principales:	Gary E. Heussler, Kyle C. Cady, Katja Koeppen, Sabin Bhuju, Bruce A. Stanton, George A. O’Toole
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1025da225b604a82ac05ace91f6d20c3
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spelling	oai:doaj.org-article:1025da225b604a82ac05ace91f6d20c32021-11-15T15:49:02ZClustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes10.1128/mBio.00129-152150-7511https://doaj.org/article/1025da225b604a82ac05ace91f6d20c32015-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00129-15https://doaj.org/toc/2150-7511ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. IMPORTANCE The various CRISPR/Cas systems found in both archaea and bacteria are incredibly diverse, and advances in understanding the complex mechanisms of these varied systems has not only increased our knowledge of host-virus interplay but has also led to a major advancement in genetic engineering. Recently, increasing evidence suggested that bacteria can co-opt the CRISPR system for functions besides adaptive immunity to phage infection. This study examined one such alternative function, and this report describes the mechanism of type 1-F CRISPR-dependent loss of the biofilm and swarming in the medically relevant opportunistic pathogen Pseudomonas aeruginosa. Since both biofilm formation and swarming motility are important in the virulence of P. aeruginosa, a full understanding of how the CRISPR system can regulate such group behaviors is fundamental to developing new therapeutics.Gary E. HeusslerKyle C. CadyKatja KoeppenSabin BhujuBruce A. StantonGeorge A. O’TooleAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 3 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Gary E. Heussler Kyle C. Cady Katja Koeppen Sabin Bhuju Bruce A. Stanton George A. O’Toole Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes
description	ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. IMPORTANCE The various CRISPR/Cas systems found in both archaea and bacteria are incredibly diverse, and advances in understanding the complex mechanisms of these varied systems has not only increased our knowledge of host-virus interplay but has also led to a major advancement in genetic engineering. Recently, increasing evidence suggested that bacteria can co-opt the CRISPR system for functions besides adaptive immunity to phage infection. This study examined one such alternative function, and this report describes the mechanism of type 1-F CRISPR-dependent loss of the biofilm and swarming in the medically relevant opportunistic pathogen Pseudomonas aeruginosa. Since both biofilm formation and swarming motility are important in the virulence of P. aeruginosa, a full understanding of how the CRISPR system can regulate such group behaviors is fundamental to developing new therapeutics.
format	article
author	Gary E. Heussler Kyle C. Cady Katja Koeppen Sabin Bhuju Bruce A. Stanton George A. O’Toole
author_facet	Gary E. Heussler Kyle C. Cady Katja Koeppen Sabin Bhuju Bruce A. Stanton George A. O’Toole
author_sort	Gary E. Heussler
title	Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes
title_short	Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes
title_full	Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes
title_fullStr	Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes
title_full_unstemmed	Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes
title_sort	clustered regularly interspaced short palindromic repeat-dependent, biofilm-specific death of <named-content content-type="genus-species">pseudomonas aeruginosa</named-content> mediated by increased expression of phage-related genes
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/1025da225b604a82ac05ace91f6d20c3
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Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Mediated by Increased Expression of Phage-Related Genes

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