scholarly journals Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rafael Pinilla-Redondo ◽  
Saadlee Shehreen ◽  
Nicole D. Marino ◽  
Robert D. Fagerlund ◽  
Chris M. Brown ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Mobile Genetic Elements ◽  
Gene Clustering ◽  
Type I ◽  
Defense Gene ◽  
Genetic Elements ◽  
Defense Systems ◽  
Evasion Strategy ◽  
Genes Encoding ◽  
Potential Exploitation

Abstract Many prokaryotes employ CRISPR–Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have developed strategies to bypass immunity, including anti-CRISPR (Acr) proteins; yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we report the discovery of new Acrs by assaying candidate genes adjacent to a conserved Acr-associated (Aca) gene, aca5, against a panel of six type I systems: I–F (Pseudomonas, Pectobacterium, and Serratia), I–E (Pseudomonas and Serratia), and I–C (Pseudomonas). We uncover 11 type I–F and/or I–E anti-CRISPR genes encoded on chromosomal and extrachromosomal MGEs within Enterobacteriaceae and Pseudomonas, and an additional Aca (aca9). The acr genes not only associate with other acr genes, but also with genes encoding inhibitors of distinct bacterial defense systems. Thus, our findings highlight the potential exploitation of acr loci neighborhoods for the identification of previously undescribed anti-defense systems.

scholarly journals Discovery of multiple anti-CRISPRs uncovers anti-defense gene clustering in mobile genetic elements

2020 ◽  
Author(s):  
Rafael Pinilla-Redondo ◽  
Saadlee Shehreen ◽  
Nicole D. Marino ◽  
Robert D. Fagerlund ◽  
Chris M. Brown ◽  
...  
Keyword(s):  
Mobile Genetic Elements ◽  
Gene Clustering ◽  
Type I ◽  
Defense Gene ◽  
Genetic Elements ◽  
Defense Systems ◽  
Prokaryotic Genomes ◽  
New Type ◽  
Genomic Regions ◽  
Potential Exploitation

AbstractMany prokaryotes employ CRISPR-Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have evolved Anti-CRISPR (Acr) proteins to bypass this immunity, yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we uncover 11 new type I anti-CRISPR genes encoded on numerous chromosomal and extrachromosomal mobile genetic elements within Enterobacteriaceae and Pseudomonas. Candidate genes were identified adjacent to anti-CRISPR associated gene 5 (aca5) and assayed against a panel of six type I systems: I-F (Pseudomonas, Pectobacterium, and Serratia), I-E (Pseudomonas and Serratia), and I-C (Pseudomonas), revealing the type I-F and/or I-E acr genes and a new aca (aca9). We find that acr genes not only associate with other acr genes, but also with inhibitors of distinct bacterial defense systems. These genomic regions appear to be “anti-defense islands”, reminiscent of the clustered arrangement of “defense islands” in prokaryotic genomes. Our findings expand on the diversity of CRISPR-Cas inhibitors and reveal the potential exploitation of acr loci neighborhoods for identifying new anti-defense systems.

scholarly journals Recruitment of CRISPR-Cas systems by Tn7-like transposons

2017 ◽  
Author(s):  
Joseph E. Peters ◽  
Kira S. Makarova ◽  
Sergey Shmakov ◽  
Eugene V. Koonin
Keyword(s):  
Phylogenetic Analysis ◽  
Host Defense ◽  
Mobile Genetic Elements ◽  
Comparative Genomic ◽  
Type I ◽  
Genetic Elements ◽  
Defense Systems ◽  
Crispr Array ◽  
Target Binding ◽  
Chromosomal Sequences

AbstractA survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain ‘minimal’ type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f and cas6f genes, and a short CRISPR array. Additionally, several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas systems. This gene composition of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-crRNA processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Here we present phylogenetic analysis demonstrating that evolution of the CRISPR-Cas containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We further show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences, and in some cases, chromosomal sequences adjacent to the transposon. A hypothesis is proposed that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. This scenario fits the “guns for hire” concept whereby mobile genetic elements can capture host defense systems and repurpose them for different stages in the life cycle of the element.ImportanceCRISPR-Cas is an adaptive immunity system that protects bacteria and archaea from mobile genetic elements. We present comparative genomic and phylogenetic analysis of degenerate CRISPR-Cas variants associated with distinct families of transposable elements and develop the hypothesis that such repurposed defense systems contribute to the transposable element propagation by facilitating transposition into specific sites. Such recruitment of defense systems by mobile elements supports the “guns for hire” concept under which the same enzymatic machineries can be alternately employed for transposon proliferation or host defense.

scholarly journals Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
April Pawluk ◽  
Megha Shah ◽  
Marios Mejdani ◽  
Charles Calmettes ◽  
Trevor F. Moraes ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Repressor ◽  
Mobile Genetic Elements ◽  
Type I ◽  
Genetic Studies ◽  
Genetic Elements ◽  
Crispr System ◽  
Mechanistic Insight ◽  
Resistance To Invasion ◽  
Insight Into

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system.

scholarly journals Diverse enzymatic activities mediate antiviral immunity in prokaryotes

Science ◽  
2020 ◽  
Vol 369 (6507) ◽  
pp. 1077-1084 ◽  
Author(s):  
Linyi Gao ◽  
Han Altae-Tran ◽  
Francisca Böhning ◽  
Kira S. Makarova ◽  
Michael Segel ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Rna Editing ◽  
Satellite Dna ◽  
Gene Prediction ◽  
Mobile Genetic Elements ◽  
Enzymatic Activities ◽  
Antiviral Defense ◽  
Defense Gene ◽  
Defense Systems ◽  
Antiviral Gene

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses.

scholarly journals Shifts in Abundance and Diversity of Mobile Genetic Elements after the Introduction of Diverse Pesticides into an On-Farm Biopurification System over the Course of a Year

2014 ◽  
Vol 80 (13) ◽  
pp. 4012-4020 ◽  
Author(s):  
Simone Dealtry ◽  
Peter N. Holmsgaard ◽  
Vincent Dunon ◽  
Sven Jechalke ◽  
Guo-Chun Ding ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Mobile Genetic Elements ◽  
Replication Initiation ◽  
Genetic Elements ◽  
Class 1 ◽  
Genes Encoding ◽  
Abundance And Diversity ◽  
And Shifts

ABSTRACTBiopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1,intI2) and genes encoding resistance to sulfonamides (sul1,sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1trfAgene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1β and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

scholarly journals Disentangling the effects of selection and loss bias on gene dynamics

2017 ◽  
Author(s):  
Jaime Iranzo ◽  
José A. Cuesta ◽  
Susanna Manrubia ◽  
Mikhail I. Katsnelson ◽  
Eugene V. Koonin
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Genome Size ◽  
Mobile Genetic Elements ◽  
Loss Ratio ◽  
Effective Population ◽  
Strong Selection ◽  
Genetic Elements ◽  
Defense Systems ◽  
Parasite Defense

ABSTRACTWe combine mathematical modelling of genome evolution with comparative analysis of prokaryotic genomes to estimate the relative contributions of selection and intrinsic loss bias to the evolution of different functional classes of genes and mobile genetic elements (MGE). An exact solution for the dynamics of gene family size was obtained under a linear duplication-transfer-loss model with selection. With the exception of genes involved in information processing, particularly translation, which are maintained by strong selection, the average selection coefficient for most non-parasitic genes is low albeit positive, compatible with the observed positive correlation between genome size and effective population size. Free-living microbes evolve under stronger selection for gene retention than parasites. Different classes of MGE show a broad range of fitness effects, from the nearly neutral transposons to prophages, which are actively eliminated by selection. Genes involved in anti-parasite defense, on average, incur a fitness cost to the host that is at least as high as the cost of plasmids. This cost is probably due to the adverse effects of autoimmunity and curtailment of horizontal gene transfer caused by the defense systems and selfish behavior of some of these systems, such as toxin-antitoxin and restriction-modification modules. Transposons follow a biphasic dynamics, with bursts of gene proliferation followed by decay in the copy number that is quantitatively captured by the model. The horizontal gene transfer to loss ratio, but not the duplication to loss ratio, correlates with genome size, potentially explaining the increased abundance of neutral and costly elements in larger genomes.SIGNIFICANCEEvolution of microbes is dominated by horizontal gene transfer and the incessant host-parasite arms race that promotes the evolution of diverse anti-parasite defense systems. The evolutionary factors governing these processes are complex and difficult to disentangle but the rapidly growing genome databases provide ample material for testing evolutionary models. Rigorous mathematical modeling of evolutionary processes, combined with computer simulation and comparative genomics, allowed us to elucidate the evolutionary regimes of different classes of microbial genes. Only genes involved in key informational and metabolic pathways are subject to strong selection whereas most of the others are effectively neutral or even burdensome. Mobile genetic elements and defense systems are costly, supporting the understanding that their evolution is governed by the same factors.

scholarly journals Properties of genes encoding transfer RNAs as integration sites for genomic islands and prophages in Klebsiella pneumoniae

2020 ◽  
Author(s):  
Camilo Berríos-Pastén ◽  
Rodolfo Acevedo ◽  
Patricio Arros ◽  
Macarena A. Varas ◽  
Kelly L. Wyres ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Integration Site ◽  
Phylogenetic Analyses ◽  
Bacterial Genome ◽  
Mobile Genetic Elements ◽  
Genomic Islands ◽  
Genomic Context ◽  
Genetic Elements ◽  
Genes Encoding ◽  
Integration Sites

ABSTRACTThe evolution of traits including antibiotic resistance, virulence, and increased fitness in Klebsiella pneumoniae and related species has been linked to the acquisition of mobile genetic elements through horizontal transfer. Among them, genomic islands (GIs) preferentially integrating at genes encoding tRNAs and the tmRNA (t(m)DNAs) would be significant in promoting chromosomal diversity. Here, we studied the whole set of t(m)DNAs present in 66 Klebsiella chromosomes, investigating their usage as integration sites and the properties of the integrated GIs. A total of 5,624 t(m)DNAs were classified based on their sequence conservation, genomic context, and prevalence. 161 different GIs and prophages were found at these sites, hosting 3,540 gene families including various related to virulence and drug resistance. Phylogenetic analyses supported the acquisition of several of these elements through horizontal gene transfer, likely mediated by a highly diverse set of encoded integrases targeting specific t(m)DNAs and sublocations inside them. Only a subset of the t(m)DNAs had integrated GIs and even identical tDNA copies showed dissimilar usage frequencies, suggesting that the genomic context would influence the integration site selection. This usage bias, likely towards avoiding disruption of polycistronic transcriptional units, would be conserved across Gammaproteobacteria. The systematic comparison of the t(m)DNAs across different strains allowed us to discover an unprecedented number of K. pneumoniae GIs and prophages and to raise important questions and clues regarding the fundamental properties of t(m)DNAs as targets for the integration of mobile genetic elements and drivers of bacterial genome evolution and pathogen emergence.

scholarly journals Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Johann Peltier ◽  
Audrey Hamiot ◽  
Julian R. Garneau ◽  
Pierre Boudry ◽  
Anna Maikova ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Mobile Genetic Elements ◽  
Toxin Gene ◽  
Type I ◽  
Genetic Elements ◽  
Clostridioides Difficile ◽  
Bacterial Chromosomes ◽  
Toxin Protein

AbstractToxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.

scholarly journals Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements

2019 ◽  
Author(s):  
Aude Bernheim ◽  
David Bikard ◽  
Marie Touchon ◽  
Eduardo P C Rocha
Keyword(s):  
Mobile Genetic Elements ◽  
Type I ◽  
Large Set ◽  
Bacterial Genomes ◽  
Epistatic Interactions ◽  
Unique Type ◽  
Genetic Elements ◽  
Multiple Copies ◽  
In Trans ◽  
Genomic Locations

Abstract Prokaryotes use CRISPR–Cas systems for adaptive immunity, but the reasons for the frequent existence of multiple CRISPRs and cas clusters remain poorly understood. Here, we analysed the joint distribution of CRISPR and cas genes in a large set of fully sequenced bacterial genomes and their mobile genetic elements. Our analysis suggests few negative and many positive epistatic interactions between Cas subtypes. The latter often result in complex genetic organizations, where a locus has a single adaptation module and diverse interference mechanisms that might provide more effective immunity. We typed CRISPRs that could not be unambiguously associated with a cas cluster and found that such complex loci tend to have unique type I repeats in multiple CRISPRs. Many chromosomal CRISPRs lack a neighboring Cas system and they often have repeats compatible with the Cas systems encoded in trans. Phages and 25 000 prophages were almost devoid of CRISPR–Cas systems, whereas 3% of plasmids had CRISPR–Cas systems or isolated CRISPRs. The latter were often compatible with the chromosomal cas clusters, suggesting that plasmids can co-opt the latter. These results highlight the importance of interactions between CRISPRs and cas present in multiple copies and in distinct genomic locations in the function and evolution of bacterial immunity.

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