TY - BOOK

T1 - Plasmids, CRISPR-Cas and Anti-CRIPSRs

T2 - Uncovering the secret lives of mobile genetic elements

AU - Pinilla Redondo, Rafael

PY - 2020

Y1 - 2020

N2 - Horizontal gene transfer plays a fundamental role in bacterial adaptation to changing environments. The vehicles that facilitate genetic exchange among cells are known as mobile genetic elements—semi-independent genetic entities that require a host cell to propagate (e.g. plasmids and bacteriophages). Although mobile genetic elements can be advantageous to their cell hosts by carrying beneficial traits and acting as valuable sources of evolutionary innovation, their replicative self-interests inherently imply a fitness cost. The competing yet interdependent relationships between mobile genetic elements and their hosts result in complex co-evolutionary dynamics which, among other things, have led to the evolution of a diverse panel of defense and anti-defense mechanisms.Mobile genetic elements have been at the core of microbiological investigations for several decades, yet many aspects of their ecology and evolution remain obscure. The overarching aim of this Ph.D. Thesis work has been to study the double-edged nature of mobile genetic elements and to obtain knowledge on the molecular and genetic mechanisms underlying the arms races that emerge between such entities and their bacterial hosts, as well as with other mobile genetic elements. On a second plane, an important motivation has been the discovery of natural phenomena with potential biotechnological applications. Altogether, the work presented here compiles 6 manuscripts (three published, two under peer-review, and one in preparation for submission) that have been divided into three main parts according to their subject matter.Part I is centered on the study of plasmid transfer dynamics within complex microbial communities. Plasmids are effective vehicles of horizontal gene transfer that are involved in the dissemination of antibiotic resistance among bacteria. The forces that govern plasmid transfer in the environment are poorly understood, largely due to the challenging design of relevant experimental setups. In Manuscript 1, we review the current approaches for investigating the transfer frequencies and taxonomic host ranges of plasmids, with a particular focus on critically discussing the benefits and pitfalls of high-throughput fluorometric detection techniques. In Manuscript 2, we use the latter approach to investigate the conjugative transfer dynamics of four natural plasmids among bacteria from groundwater-fed rapid sand filter communities, revealing high transfer efficiencies across distantly related taxa.Part II investigates CRISPR-Cas systems on plasmids. These systems have been extensively studied for providing many bacteria with adaptive immune protection against invasive mobile genetic elements. Certain mobile genetic elements have independently recruited CRISPR-Cas components, but with few exceptions, their diversity and functions remain unknown. In Manuscripts 3 and 4 we systematically investigate the distribution, prevalence, diversity, and putative functions of plasmid-encoded CRISPRCas systems. Our analyses revealed a broad diversity of plasmid-encoded CRISPR-Cas systems that are widespread across taxa and primarily involved in plasmid-plasmid competition dynamics.Part III focuses on the identification of novel anti-CRISPR proteins and their biotechnological applications. Many mobile genetic elements have evolved protein inhibitors in order to bypass CRISPR-Cas-based host immunity. In Manuscript 5, we describe the discovery of 11 novel type I anti-CRISPR families and one new anti-CRISPR-associated gene. These genes are widely distributed among mobile genetic elements harbored by diverse bacterial taxa, highlighting their broad mechanisms of action and horizontal gene transfer potential. Here we also propose the concept of “anti-defense islands” to refer to discrete clusters of anti-defense genes within the genomes of diverse mobile genetic elements. Finally, Manuscript 6 reviews the numerous applications of anti- CRISPRs for controlling endogenous and heterologous CRISPR-Cas systems utilized in research and biotechnology.

AB - Horizontal gene transfer plays a fundamental role in bacterial adaptation to changing environments. The vehicles that facilitate genetic exchange among cells are known as mobile genetic elements—semi-independent genetic entities that require a host cell to propagate (e.g. plasmids and bacteriophages). Although mobile genetic elements can be advantageous to their cell hosts by carrying beneficial traits and acting as valuable sources of evolutionary innovation, their replicative self-interests inherently imply a fitness cost. The competing yet interdependent relationships between mobile genetic elements and their hosts result in complex co-evolutionary dynamics which, among other things, have led to the evolution of a diverse panel of defense and anti-defense mechanisms.Mobile genetic elements have been at the core of microbiological investigations for several decades, yet many aspects of their ecology and evolution remain obscure. The overarching aim of this Ph.D. Thesis work has been to study the double-edged nature of mobile genetic elements and to obtain knowledge on the molecular and genetic mechanisms underlying the arms races that emerge between such entities and their bacterial hosts, as well as with other mobile genetic elements. On a second plane, an important motivation has been the discovery of natural phenomena with potential biotechnological applications. Altogether, the work presented here compiles 6 manuscripts (three published, two under peer-review, and one in preparation for submission) that have been divided into three main parts according to their subject matter.Part I is centered on the study of plasmid transfer dynamics within complex microbial communities. Plasmids are effective vehicles of horizontal gene transfer that are involved in the dissemination of antibiotic resistance among bacteria. The forces that govern plasmid transfer in the environment are poorly understood, largely due to the challenging design of relevant experimental setups. In Manuscript 1, we review the current approaches for investigating the transfer frequencies and taxonomic host ranges of plasmids, with a particular focus on critically discussing the benefits and pitfalls of high-throughput fluorometric detection techniques. In Manuscript 2, we use the latter approach to investigate the conjugative transfer dynamics of four natural plasmids among bacteria from groundwater-fed rapid sand filter communities, revealing high transfer efficiencies across distantly related taxa.Part II investigates CRISPR-Cas systems on plasmids. These systems have been extensively studied for providing many bacteria with adaptive immune protection against invasive mobile genetic elements. Certain mobile genetic elements have independently recruited CRISPR-Cas components, but with few exceptions, their diversity and functions remain unknown. In Manuscripts 3 and 4 we systematically investigate the distribution, prevalence, diversity, and putative functions of plasmid-encoded CRISPRCas systems. Our analyses revealed a broad diversity of plasmid-encoded CRISPR-Cas systems that are widespread across taxa and primarily involved in plasmid-plasmid competition dynamics.Part III focuses on the identification of novel anti-CRISPR proteins and their biotechnological applications. Many mobile genetic elements have evolved protein inhibitors in order to bypass CRISPR-Cas-based host immunity. In Manuscript 5, we describe the discovery of 11 novel type I anti-CRISPR families and one new anti-CRISPR-associated gene. These genes are widely distributed among mobile genetic elements harbored by diverse bacterial taxa, highlighting their broad mechanisms of action and horizontal gene transfer potential. Here we also propose the concept of “anti-defense islands” to refer to discrete clusters of anti-defense genes within the genomes of diverse mobile genetic elements. Finally, Manuscript 6 reviews the numerous applications of anti- CRISPRs for controlling endogenous and heterologous CRISPR-Cas systems utilized in research and biotechnology.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/1pioq0f/alma99123792449505763

M3 - Ph.D. thesis

BT - Plasmids, CRISPR-Cas and Anti-CRIPSRs

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -