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 Recruitment of CRISPR-Cas systems by Tn7-like transposons

2017 ◽  
Vol 114 (35) ◽  
pp. E7358-E7366 ◽  
Author(s):  
Joseph E. Peters ◽  
Kira S. Makarova ◽  
Sergey Shmakov ◽  
Eugene V. Koonin
Keyword(s):  
Phylogenetic Analysis ◽  
Host Defense ◽  
Small Groups ◽  
Temperate Phage ◽  
Type I ◽  
Defense Systems ◽  
Crispr Array ◽  
Minimal Type ◽  
Target Binding ◽  
Chromosomal Sequences

A 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. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates 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 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. We hypothesize 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. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

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 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 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 High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages inClostridioides difficile

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Julian R. Garneau ◽  
Ognjen Sekulovic ◽  
Bruno Dupuy ◽  
Olga Soutourina ◽  
Marc Monot ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Mobile Genetic Elements ◽  
Large Chromosome ◽  
Comparative Genomic ◽  
Content Type ◽  
Genetic Elements ◽  
Clostridioides Difficile ◽  
A Genome ◽  
Depth Analysis ◽  
High Prevalence

ABSTRACTClostridioides difficile(formerlyClostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified inC. difficileso far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infectingLactococcus lactis, and phage c-st, infectingClostridium botulinum. A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and acas3gene. We screened 2,584C. difficilegenomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected inC. difficilestrains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficileis a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of differentC. difficilestrains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infectC. difficileso far. Screening 2,584C. difficilegenomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages inC. difficileto better understand the epidemiology of this clinically important human pathogen.

scholarly journals Evidence of an epidemic spread of KPC-producing Enterobacterales in Czech hospitals

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lucie Kraftova ◽  
Marc Finianos ◽  
Vendula Studentova ◽  
Katerina Chudejova ◽  
Vladislav Jakubu ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Infection Control ◽  
Mobile Genetic Elements ◽  
Epidemic Spread ◽  
Sequencing Data ◽  
Genetic Elements ◽  
Long Read ◽  
Genetic Rearrangements

AbstractThe aim of the present study is to describe the ongoing spread of the KPC-producing strains, which is evolving to an epidemic in Czech hospitals. During the period of 2018–2019, a total of 108 KPC-producing Enterobacterales were recovered from 20 hospitals. Analysis of long-read sequencing data revealed the presence of several types of blaKPC-carrying plasmids; 19 out of 25 blaKPC-carrying plasmids could be assigned to R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups. Five of the remaining blaKPC-carrying plasmids were multireplicon, while one plasmid couldn’t be typed. Additionally, phylogenetic analysis confirmed the spread of blaKPC-carrying plasmids among different clones of diverse Enterobacterales species. Our findings demonstrated that the increased prevalence of KPC-producing isolates was due to plasmids spreading among different species. In some districts, the local dissemination of IncR and IncN plasmids was observed. Additionally, the ongoing evolution of blaKPC-carrying plasmids, through genetic rearrangements, favours the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reporting KPC-producing strains.

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 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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