scholarly journals Long-Term Persistence of Plasmids Targeted by CRISPR Interference in Bacterial Populations

2021 ◽  
Author(s):  
Viktor Mamontov ◽  
Alexander Martynov ◽  
Natalia Morozova ◽  
Anton Bukatin ◽  
Dmitry B. Staroverov ◽  
...  
Keyword(s):  
Complex Dynamics ◽  
Mobile Genetic Elements ◽  
Genetic Alterations ◽  
Common Mechanism ◽  
Type I ◽  
Bacterial Populations ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Rapid Changes

CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy, and microfluidics analyses we demonstrated that plasmids can persist in Escherichia coli cells at conditions of continuous targeting by the type I-E CRISPR-Cas system without acquiring any genetic alterations. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to the presence of mobile genetic elements in some cells, leading to diversification of phenotypes in the entire community and allowing rapid changes in the population structure to meet the demands of a changing environment.

scholarly journals Bacterial Transformation Buffers Environmental Fluctuations through the Reversible Integration of Mobile Genetic Elements

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Gabriel Carvalho ◽  
David Fouchet ◽  
Gonché Danesh ◽  
Anne-Sophie Godeux ◽  
Maria-Halima Laaberki ◽  
...  
Keyword(s):  
Horizontal Gene Transfer ◽  
Resistance Genes ◽  
Natural Transformation ◽  
Bacterial Genome ◽  
Mobile Genetic Elements ◽  
Bacterial Populations ◽  
Genetic Elements ◽  
Intermediate Transformation ◽  
Bacterial Fitness

ABSTRACT Horizontal gene transfer (HGT) promotes the spread of genes within bacterial communities. Among the HGT mechanisms, natural transformation stands out as being encoded by the bacterial core genome. Natural transformation is often viewed as a way to acquire new genes and to generate genetic mixing within bacterial populations. Another recently proposed function is the curing of bacterial genomes of their infectious parasitic mobile genetic elements (MGEs). Here, we propose that these seemingly opposing theoretical points of view can be unified. Although costly for bacterial cells, MGEs can carry functions that are at points in time beneficial to bacteria under stressful conditions (e.g., antibiotic resistance genes). Using computational modeling, we show that, in stochastic environments, an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes, although these MGEs are costly for host cell replication. Based on this dual function (MGE acquisition and removal), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, and this would explain its striking conservation. IMPORTANCE Natural transformation is the acquisition, controlled by bacteria, of extracellular DNA and is one of the most common mechanisms of horizontal gene transfer, promoting the spread of resistance genes. However, its evolutionary function remains elusive, and two main roles have been proposed: (i) the new gene acquisition and genetic mixing within bacterial populations and (ii) the removal of infectious parasitic mobile genetic elements (MGEs). While the first one promotes genetic diversification, the other one promotes the removal of foreign DNA and thus genome stability, making these two functions apparently antagonistic. Using a computational model, we show that intermediate transformation rates, commonly observed in bacteria, allow the acquisition then removal of MGEs. The transient acquisition of costly MGEs with resistance genes maximizes bacterial fitness in environments with stochastic stress exposure. Thus, transformation would ensure both a strong dynamic of the bacterial genome in the short term and its long-term stabilization.

scholarly journals Black Queen Evolution and Trophic Interactions Determine Plasmid Survival after the Disruption of the Conjugation Network

mSystems ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Johannes Cairns ◽  
Katariina Koskinen ◽  
Reetta Penttinen ◽  
Tommi Patinen ◽  
Anna Hartikainen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Communities ◽  
Resistance Mechanism ◽  
Real Life ◽  
Ecological Factors ◽  
Mobile Genetic Elements ◽  
Resistance Mechanisms ◽  
Antibiotics Resistance ◽  
Bacterial Populations ◽  
Genetic Elements

ABSTRACTMobile genetic elements such as conjugative plasmids are responsible for antibiotic resistance phenotypes in many bacterial pathogens. The ability to conjugate, the presence of antibiotics, and ecological interactions all have a notable role in the persistence of plasmids in bacterial populations. Here, we set out to investigate the contribution of these factors when the conjugation network was disturbed by a plasmid-dependent bacteriophage. Phage alone effectively caused the population to lose plasmids, thus rendering them susceptible to antibiotics. Leakiness of the antibiotic resistance mechanism allowing Black Queen evolution (i.e. a “race to the bottom”) was a more significant factor than the antibiotic concentration (lethal vs sublethal) in determining plasmid prevalence. Interestingly, plasmid loss was also prevented by protozoan predation. These results show that outcomes of attempts to resensitize bacterial communities by disrupting the conjugation network are highly dependent on ecological factors and resistance mechanisms.IMPORTANCEBacterial antibiotic resistance is often a part of mobile genetic elements that move from one bacterium to another. By interfering with the horizontal movement and the maintenance of these elements, it is possible to remove the resistance from the population. Here, we show that a so-called plasmid-dependent bacteriophage causes the initially resistant bacterial population to become susceptible to antibiotics. However, this effect is efficiently countered when the system also contains a predator that feeds on bacteria. Moreover, when the environment contains antibiotics, the survival of resistance is dependent on the resistance mechanism. When bacteria can help their contemporaries to degrade antibiotics, resistance is maintained by only a fraction of the community. On the other hand, when bacteria cannot help others, then all bacteria remain resistant. The concentration of the antibiotic played a less notable role than the antibiotic used. This report shows that the survival of antibiotic resistance in bacterial communities represents a complex process where many factors present in real-life systems define whether or not resistance is actually lost.

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 Oscillatory dynamics in a bacterial cross-protection mutualism

2016 ◽  
Vol 113 (22) ◽  
pp. 6236-6241 ◽  
Author(s):  
Eugene Anatoly Yurtsev ◽  
Arolyn Conwill ◽  
Jeff Gore
Keyword(s):  
Evolutionary Dynamics ◽  
Cooperative Behavior ◽  
Cross Protection ◽  
Common Mechanism ◽  
Natural Environments ◽  
Term Survival ◽  
Bacterial Populations ◽  
Oscillatory Dynamics ◽  
Protection Mutualism

Cooperation between microbes can enable microbial communities to survive in harsh environments. Enzymatic deactivation of antibiotics, a common mechanism of antibiotic resistance in bacteria, is a cooperative behavior that can allow resistant cells to protect sensitive cells from antibiotics. Understanding how bacterial populations survive antibiotic exposure is important both clinically and ecologically, yet the implications of cooperative antibiotic deactivation on the population and evolutionary dynamics remain poorly understood, particularly in the presence of more than one antibiotic. Here, we show that two Escherichia coli strains can form an effective cross-protection mutualism, protecting each other in the presence of two antibiotics (ampicillin and chloramphenicol) so that the coculture can survive in antibiotic concentrations that inhibit growth of either strain alone. Moreover, we find that daily dilutions of the coculture lead to large oscillations in the relative abundance of the two strains, with the ratio of abundances varying by nearly four orders of magnitude over the course of the 3-day period of the oscillation. At modest antibiotic concentrations, the mutualistic behavior enables long-term survival of the oscillating populations; however, at higher antibiotic concentrations, the oscillations destabilize the population, eventually leading to collapse. The two strains form a successful cross-protection mutualism without a period of coevolution, suggesting that similar mutualisms may arise during antibiotic treatment and in natural environments such as the soil.

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 CRISPR-interference-based modulation of mobile genetic elements in bacteria

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Ákos Nyerges ◽  
Balázs Bálint ◽  
Judit Cseklye ◽  
István Nagy ◽  
Csaba Pál ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Complete Removal ◽  
Mobile Genetic Elements ◽  
Industrial Biotechnology ◽  
Chromosome Engineering ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Promising Tool ◽  
Single Plasmid

Abstract Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of engineered functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5 and IS150 at up to 38 chromosomal loci in Escherichia coli, in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. The fitness cost of multiple IS-knockdown, detectable in flask-and-shaker systems was readily outweighed by the less frequent inactivation of the transgene, as observed in green fluorescent protein (GFP)-overexpression experiments. In addition, global transcriptomics analysis revealed only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multiple E. coli strains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

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.

scholarly journals Genomic instability and mobile genetic elements in regions surrounding two discoidin I genes of Dictyostelium discoideum.

1984 ◽  
Vol 4 (4) ◽  
pp. 671-680 ◽  
Author(s):  
S J Poole ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Base Pair ◽  
Wild Type Strain ◽  
Mobile Genetic Elements ◽  
Wild Type ◽  
Repeat Elements ◽  
Genetic Elements ◽  
Rapid Changes ◽  
Genomic Regions ◽  
Derivative Strain

We have found that the genomic regions surrounding the linked discoidin I genes of various Dictyostelium discoideum strains have undergone rapid changes. Wild-type strain NC-4 has three complete discoidin I genes; its axenic derivative strain Ax-3L has duplicated a region starting approximately 1 kilobase upstream from the two linked genes and extending for at least 8 kilobases past the genes. A separately maintained stock, strain Ax-3K, does not have this duplication but has undergone a different rearrangement approximately 3 kilobases farther upstream. We show that there are repeat elements in these rapidly changing regions. At least two of these elements, Tdd-2 and Tdd-3, have characteristics associated with mobile genetic elements. The Tdd-3 element is found in different locations in related strains and causes a 9- to 10-base-pair duplication of the target site DNA. The Tdd-2 and Tdd-3 elements do not cross-hybridize, but they share a 22-base-pair homology near one end. At two separate sites, the Tdd-3 element has transposed into the Tdd-2 element, directly adjacent to the 22-base-pair homology. The Tdd-3 element may use this 22-base-pair region as a preferential site of insertion.

scholarly journals Comparative prevalence of superantigenic toxin genes in meticillin-resistant and meticillin-susceptible Staphylococcus aureus isolates

2008 ◽  
Vol 57 (9) ◽  
pp. 1106-1112 ◽  
Author(s):  
Dong-Liang Hu ◽  
Katsuhiko Omoe ◽  
Fumio Inoue ◽  
Takesi Kasai ◽  
Minoru Yasujima ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mobile Genetic Elements ◽  
Toxin Gene ◽  
Type I ◽  
Gene Encoding ◽  
Genetic Elements ◽  
Toxin Genes ◽  
Time Period ◽  
Enterotoxin Genes ◽  
Toxic Shock Syndrome Toxin

A total of 118 meticillin-resistant Staphylococcus aureus (MRSA) and 140 meticillin-susceptible S. aureus (MSSA) isolates from different patients in the same time period were comprehensively searched using a multiplex PCR for the classical and recently described superantigenic toxin gene family comprising the staphylococcal enterotoxin genes sea to ser and the toxic shock syndrome toxin 1 gene, tst-1. Both MRSA and MSSA isolates carried a number of superantigenic toxin genes, but the MRSA isolates harboured more superantigenic toxin genes than the MSSA isolates. The most frequent genotype of the MRSA isolates was sec, sell and tst-1 together with the gene combination seg, sei, selm, seln and selo, which was found strictly in combination in 69.5 % of the isolates tested. In contrast, possession of the sec, sell and tst-1 genes in MSSA isolates was significantly less than in MRSA (2.1 vs 77.1 %, respectively), although they also often contained the combination genes (25.0 %). This notable higher prevalence in MRSA isolates indicated that possession of the sec, sell and tst-1 genes in particular appeared to be a habitual feature of MRSA. Moreover, these were mainly due to the fixed combinations of the mobile genetic elements type I νSa4 encoding sec, sell and tst-1, and type I νSaβ encoding seg, sei, selm, seln and selo. Analysis of the relationship between toxin genotypes and the toxin gene-encoding profiles of mobile genetic elements has a possible role in determining superantigenic toxin genotypes in S. aureus.

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.

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