ORCID Profile
0000-0002-5741-9457
Current Organisations
Wageningen University and Research Centre
,
Wageningen University & Research
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Publisher: The Royal Society
Date: 25-03-2019
Abstract: CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These ‘spacers’ provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic ersity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
Publisher: Springer Science and Business Media LLC
Date: 13-06-2016
DOI: 10.1038/NMICROBIOL.2016.85
Abstract: CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids(1,2). They are present in approximately half of all sequenced prokaryotes(3) and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function(4,5). We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of erse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with ergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.
Publisher: Cold Spring Harbor Laboratory
Date: 21-06-2019
DOI: 10.1101/679308
Abstract: Type I CRISPR-Cas systems are the most abundant and widespread adaptive immune systems of bacteria and can greatly enhance bacterial survival in the face of temperate phage infection. However, it is less clear how these systems protect against virulent phages. Here we experimentally show that type I CRISPR immunity of Pectobacterium atrosepticum leads to rapid suppression of two unrelated virulent phages, ΦTE and ΦM1. However, unlike the case where bacteria are infected with temperate phages, this is the result of an abortive infection-like phenotype, where infected cells do not survive the infection but instead become metabolically inactive and lose their membrane integrity. Our findings challenge the view of CRISPR-Cas as a system that protects the in idual cell and supports growing evidence of an Abi-like function for some types of CRISPR-Cas systems.
Publisher: Elsevier BV
Date: 12-2016
Publisher: Wiley
Date: 07-07-2005
Publisher: Proceedings of the National Academy of Sciences
Date: 07-04-2014
Abstract: Bacteria are constantly exposed to foreign elements, such as bacteriophages and plasmids. The CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated) adaptive immune systems provide heritable sequence-specific protection against these invaders. To develop immunity, bacteria add segments of foreign nucleic acid to their CRISPR memory. However, phage and plasmid mutants can evade CRISPR-Cas recognition by altering their targeted sequence. CRISPR-Cas responds to evasion by quickly generating immunity by acquiring new pieces of invader genome. We determined that this rapid generation of resistance is promiscuous, with recognition of highly erged or related elements eliciting new immunity. Our results demonstrate that CRISPR-Cas systems are more robust than previously thought and, not only have a highly specific resistance memory, but also have a broad ability to identify ergent genetic elements.
Publisher: Wiley
Date: 24-05-2016
Publisher: Springer Science and Business Media LLC
Date: 03-10-2016
DOI: 10.1038/NCOMMS12853
Abstract: CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum . Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition’) is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.
Publisher: Springer Science and Business Media LLC
Date: 17-11-2015
Publisher: Oxford University Press (OUP)
Date: 02-07-2014
DOI: 10.1093/NAR/GKU527
Publisher: Proceedings of the National Academy of Sciences
Date: 13-06-2017
Abstract: CRISPR-Cas systems provide prokaryotic adaptive immunity against invading genetic elements. For immunity, fragments of invader DNA are integrated into CRISPR arrays by Cas1 and Cas2 proteins. Type I-F systems contain a unique fusion of Cas2 to Cas3, the enzyme responsible for destruction of invading DNA. Structural, biophysical, and biochemical analyses of Cas1 and Cas2-3 from Pectobacterium atrosepticum demonstrated that they form a 400-kDa complex with a Cas1 4 :Cas2-3 2 stoichiometry. Cas1–Cas2-3 binds, processes, and catalyzes the integration of DNA into CRISPR arrays independent of Cas3 activity. The arrangement of Cas3 in the complex, together with its redundant role in processing and integration, supports a scenario where Cas3 couples invader destruction with immunization—a process recently demonstrated in vivo.
Publisher: Springer Science and Business Media LLC
Date: 17-05-2016
Publisher: Informa UK Limited
Date: 18-09-2018
Publisher: American Society for Microbiology
Date: 07-03-2018
Abstract: A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum , CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted. IMPORTANCE The generation of genetic ersity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution.
Publisher: Informa UK Limited
Date: 27-03-2013
DOI: 10.4161/RNA.24202
Publisher: Springer Science and Business Media LLC
Date: 04-12-2019
DOI: 10.1038/S41467-019-13445-2
Abstract: Type I CRISPR-Cas systems are abundant and widespread adaptive immune systems in bacteria and can greatly enhance bacterial survival in the face of phage infection. Upon phage infection, some CRISPR-Cas immune responses result in bacterial dormancy or slowed growth, which suggests the outcomes for infected cells may vary between systems. Here we demonstrate that type I CRISPR immunity of Pectobacterium atrosepticum leads to suppression of two unrelated virulent phages, ɸTE and ɸM1. Immunity results in an abortive infection response, where infected cells do not survive, but viral propagation is severely decreased, resulting in population protection due to the reduced phage epidemic. Our findings challenge the view of CRISPR-Cas as a system that protects the in idual cell and supports growing evidence of abortive infection by some types of CRISPR-Cas systems.
Publisher: Portland Press Ltd.
Date: 08-04-2016
DOI: 10.1042/BCJ20160078
Abstract: CRISPR–Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many erse types of CRISPR–Cas systems, including type I-F systems, which are typified by a unique Cas1–Cas2–3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR–Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity.
Location: Netherlands
No related grants have been discovered for Raymond Staals.