Two Distinct DNA Binding Modes Guide Dual Roles of a CRISPR-Cas Protein
Blosser, T.R. ; Loeff, L. ; Westra, E.R. ; Vlot, M. ; Künne, T.A. ; Sobota, M. ; Dekker, C. ; Brouns, S.J.J. ; Joo, C. - \ 2015
Molecular Cell 58 (2015)1. - ISSN 1097-2765 - p. 60 - 70.
bacterial immune-system - in-vitro reconstitution - memory b-cells - surveillance complex - crystal-structure - streptococcus-thermophilus - adaptive immunity - escherichia-coli - seed sequence - rna
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets
Unravelling the structural and mechanistic basis of CRISPR-Cas systems
Oost, J. van der; Westra, E.R. ; Jackson, R.N. ; Wiedenheft, B. - \ 2014
Nature Reviews Microbiology 12 (2014)7. - ISSN 1740-1526 - p. 479 - 492.
short palindromic repeats - bacterial immune-system - rna silencing complex - processes pre-crrna - human gut virome - crystal-structure - escherichia-coli - streptococcus-thermophilus - thermus-thermophilus - interference complex
Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR–Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.
Functional genomics of lactic acid bacteria: from food to health
Douillard, F.P. ; Vos, W.M. de - \ 2014
Microbial Cell Factories 13 (2014)suppl.1. - ISSN 1475-2859
lactobacillus-rhamnosus gg - human gastrointestinal-tract - sortase-dependent proteins - johnsonii strain ncc533 - gram-positive bacteria - subsp cremoris mg1363 - lactococcus-lactis - in-vitro - streptococcus-thermophilus - enterococcus-faecalis
Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.
Production of aroma compounds in lactic fermentations
Smid, E.J. ; Kleerebezem, M. - \ 2014
Annual Review of Food Science and Technology 5 (2014). - ISSN 1941-1413 - p. 313 - 326.
cystathionine beta-lyase - citrate transporter citp - lactococcus-lactis - acid bacteria - flavor formation - lactobacillus-plantarum - starter cultures - streptococcus-thermophilus - proteolytic systems - food fermentations
This review describes recent scientific research on the production of aroma compounds by lactic acid bacteria (LAB) in fermented food products. We discuss the various precursor molecules for the formation of aroma compounds in connection with the metabolic pathways involved. The roles of nonmetabolic properties such as cell lysis are also described in relation to aroma formation. Finally, we provide an overview of the literature on methods to steer and control aroma formation by LAB in mixed culture fermentations. We demonstrate that the technological progress made recently in high-throughput analysis methods has been driving the development of new approaches to understand, control, and steer aroma formation in (dairy) fermentation processes. This currently entails proposing new rules for designing stable, high-performance mixed cultures constituting a selection of strains, which in concert and on the basis of their individual predicted gene contents deliver the required functionalities.
Degenerate target sites mediate rapid primed CRISPR adaptation
Fineran, P.C. ; Gerritzen, M.J.H. ; Suarez Diez, M. ; Künne, T.A. ; Boekhorst, J. ; Hijum, S.A.F.T. van; Staals, R.H.J. ; Brouns, S.J.J. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)16. - ISSN 0027-8424 - p. E1629 - E1638.
adaptive immune-systems - escherichia-coli - cas systems - streptococcus-thermophilus - bacterial immunity - defense system - foreign dna - dual-rna - recognition - interference
Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.
The impact of selected strains of probiotic bacteria on metabolite formation in set yoghurt
Settachaimongkon, S. ; Nout, M.J.R. ; Antunes Fernandes, E.C. ; Hooijdonk, A.C.M. van; Zwietering, M.H. ; Smid, E.J. ; Valenberg, H.J.F. van - \ 2014
International Dairy Journal 38 (2014)1. - ISSN 0958-6946 - p. 1 - 10.
nuclear-magnetic-resonance - delbrueckii subsp bulgaricus - lactic-acid bacteria - streptococcus-thermophilus - fermented milks - lactobacillus-acidophilus - functional foods - starter cultures - flavor compounds - dairy-products
The influence of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 in cofermentation with traditional starters on metabolite formation in set yoghurt was evaluated. Microbial activity during fermentation and refrigerated storage was investigated by monitoring bacterial population dynamics, milk acidification and overall changes in yoghurt metabolite profiles. A complementary metabolomics approach using solid-phase microextraction-gas chromatography/mass spectrometry and 1H nuclear magnetic resonance resulted in the identification of 37 volatile and 43 non-volatile metabolites, respectively. Results demonstrated that the two probiotic strains did not influence acidity and the key-aroma volatile metabolites of set yoghurt. However, a contribution by the presence of L. rhamnosus GG on the non-volatile metabolite profile of yoghurt was specifically noticed during storage. Multivariate analysis allowed yoghurts fermented by different starter combinations and different durations of storage to be differentiated according to their metabolite profiles. This provides new insights regarding the impact of probiotics on the metabolome of yoghurt.
Planting the seed: target recognition of short guide RNAs
Künne, T. ; Swarts, D.C. ; Brouns, S.J.J. - \ 2014
Trends in Microbiology 22 (2014)1. - ISSN 0966-842X - p. 74 - 83.
bacterial immune-system - staphylococcus-aureus reveals - binding small rnas - sm-like protein - messenger-rna - crispr rna - escherichia-coli - streptococcus-thermophilus - noncoding rnas - enzyme complex
Small guide RNAs play important roles in cellular processes such as regulation of gene expression and host defense against invading nucleic acids. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation, or replication. Several unrelated classes of small noncoding RNAs have been identified including eukaryotic RNA silencing-associated small RNAs, prokaryotic small regulatory RNAs (sRNAs), and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs (crRNAs). All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 612 nucleotides (nt) of the small RNA termed the seed plays a critical role. We review the concept of seed sequences and discuss its importance for initial target recognition and interference
Transcriptome-based characterization of interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in lactose-grown chemostat cocultures
Mendes, F. ; Sieuwerts, S. ; Hulster, E. de; Almering, M.J. ; Luttik, M.A.H. ; Pronk, J.T. ; Smid, E.J. ; Baron, P.A. ; Daran-Lapujade, P. - \ 2013
Applied and Environmental Microbiology 79 (2013)19. - ISSN 0099-2240 - p. 5949 - 5961.
lactic-acid bacteria - streptococcus-thermophilus - mixed-culture - kefiran production - oenococcus-oeni - fermentation - reveals - yeasts - carbon - temperature
Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations.
Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG
Douillard, F.P. ; Ribbera, A. ; Kant, R. ; Pietilä, T.E. ; Järvinen, H.M. ; Messing, M. ; Randazzo, C.L. ; Paulin, L. ; Laine, P.K. ; Ritari, J. ; Caggia, C. ; Lähteinen, T. ; Brouns, S.J.J. ; Satokari, R.M. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2013
Plos Genetics 9 (2013)8. - ISSN 1553-7404
lactic-acid bacteria - intestinal epithelial-cells - placebo-controlled trial - streptococcus-thermophilus - species identification - salmonella-typhimurium - gastrointestinal-tract - adaptive immunity - atopic disease - in-vitro
Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects
CRISPRTarget: bioinformatic prediction and analysis of crRNA targets
Biswas, A. ; Gagnon, J.N. ; Brouns, S.J.J. ; Fineran, P.C. ; Brown, C.M. - \ 2013
RNA Biology 10 (2013)5. - ISSN 1547-6286 - p. 817 - 827.
short palindromic repeats - bacterial immune-system - cas systems - pectobacterium-atrosepticum - streptococcus-thermophilus - salmonella-enterica - dynamic properties - antiviral defense - adaptive immunity - genome sequence
The bacterial and archaeal CRISPR/Cas adaptive immune system targets specific protospacer nucleotide sequences in invading organisms. This requires base pairing between processed CRISPR RNA and the target protospacer. For type I and II CRISPR/Cas systems, protospacer adjacent motifs (PAM) are essential for target recognition, and for type III, mismatches in the flanking sequences are important in the antiviral response. In this study, we examine the properties of each class of CRISPR. We use this information to provide a tool (CRISPRTarget) that predicts the most likely targets of CRISPR RNAs (http://bioanalysis.otago.ac.nz/CRISPRTarget). This can be used to discover targets in newly sequenced genomic or metagenomic data. To test its utility, we discover features and targets of well-characterized Streptococcus thermophilus and Sulfolobus solfataricus type II and III CRISPR/Cas systems. Finally, in Pectobacterium species, we identify new CRISPR targets and propose a model of temperate phage exposure and subsequent inhibition by the type I CRISPR/Cas systems
CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids
Westra, E.R. ; Staals, R.H.J. ; Gort, G. ; Høgh, S. ; Neumann, S. ; Cruz, F. ; Fineran, P.C. ; Brouns, S.J.J. - \ 2013
RNA Biology 10 (2013)5. - ISSN 1547-6286 - p. 749 - 761.
horizontal gene-transfer - protein h-ns - adaptive immune-systems - escherichia-coli - streptococcus-thermophilus - host-range - in-vitro - microbial communities - acquired-resistance - antiviral defense
Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid's mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the recipient cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the recipient cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against MOBF prototype plasmid F in Escherichia coli. Our results show that plasmid conjugation is effectively inhibited, but the level of immunity is not affected by targeting the plasmid in the leading or lagging region. Moreover, CRISPR-immunity levels do not depend on whether the incoming single-stranded plasmid DNA, or the DNA strand synthesized in the recipient is targeted. Our findings indicate that single-stranded DNA may not be a target for Type I-E CRISPR-Cas systems, and suggest that the protospacer distribution bias might be due to spacer acquisition preferences
Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition
Westra, E.R. ; Semenova, E. ; Datsenko, K.A. ; Jackson, R.N. ; Wiedenheft, B. ; Severinov, K. ; Brouns, S.J.J. - \ 2013
Plos Genetics 9 (2013)9. - ISSN 1553-7404
bacterial immune-system - processes pre-crrna - escherichia-coli - streptococcus-thermophilus - antiviral defense - seed sequence - foreign dna - invader dna - h-ns - rna
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism
Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC 533
Hertzberger, R.Y. ; Pridmore, R.D. ; Gysler, C. ; Kleerebezem, M. ; Teixeira de Mattos, M.J. - \ 2013
PLoS ONE 8 (2013)2. - ISSN 1932-6203 - 8 p.
population heterogeneity - transcriptome analysis - pyruvate oxidase - streptococcus-thermophilus - carbamoyl-phosphate - lactococcus-lactis - aerobic growth - plantarum - acid - bacteria
Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC 533. The probiotic Lactobacillus johnsonii NCC 533 is relatively sensitive to oxidative stress; the presence of oxygen causes a lower biomass yield due to early growth stagnation. We show however that oxygen can also be beneficial to this organism as it relieves the requirement for acetate and CO2 during growth. Both on agar- and liquid-media, anaerobic growth of L. johnsonii NCC 533 requires CO2 supplementation of the gas phase. Switching off the CO2 supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO2 dependency. Analogously, L. johnsonii NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth medium for lactobacilli already suffices for normal growth. Analogous to the CO2 requirement, oxygen supply relieves this acetate-dependency for growth. The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO2 and acetyl-CoA producing systems. Therefore, C1- and C2- compound production is predicted to largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/C1-generation is corroborated by the observation that in a POX deficient mutant of L. johnsonii NCC 533, oxygen is not able to overcome acetate dependency nor does it relieve the CO2 dependency.
Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution
Bachmann, H. ; Starrenburg, M.J.C. ; Molenaar, D. ; Kleerebezem, M. ; Hylckama Vlieg, J.E.T. van - \ 2012
Genome Research 22 (2012)1. - ISSN 1088-9051 - p. 115 - 124.
complete genome sequence - escherichia-coli populations - term experimental evolution - amino-acid biosynthesis - streptococcus-thermophilus - lactobacillus-bulgaricus - dairy environment - gene inactivation - mutator alleles - subsp lactis
Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and in dairy environments, and it is proposed that dairy strains originate from the plant niche. Here we investigate the adaptation of a L. lactis strain isolated from a plant to a dairy niche by propagating it for 1000 generations in milk. Two out of three independently evolved strains displayed significantly increased acidification rates and biomass yields in milk. Genome resequencing, revealed six, seven, and 28 mutations in the three strains, including point mutations in loci related to amino acid biosynthesis and transport and in the gene encoding MutL, which is involved in DNA mismatch repair. Two strains lost a conjugative transposon containing genes important in the plant niche but dispensable in milk. A plasmid carrying an extracellular protease was introduced by transformation. Although improving growth rate and growth yield significantly, the plasmid was rapidly lost. Comparative transcriptome and phenotypic analyses confirmed that major physiological changes associated with improved growth in milk relate to nitrogen metabolism and the loss or down-regulation of several pathways involved in the utilization of complex plant polymers. Reproducing the transition from the plant to the dairy niche through experimental evolution revealed several genome, transcriptome, and phenotype signatures that resemble those seen in strains isolated from either niche.
The rise and fall of CRISPRs - dynamics of spacer acquisition and loss
Westra, E.R. ; Brouns, S.J.J. - \ 2012
Molecular Microbiology 85 (2012)6. - ISSN 0950-382X - p. 1021 - 1025.
immune-system - streptococcus-thermophilus - acquired-resistance - antiviral defense - escherichia-coli - seed sequence - cas systems - rna - dna - repeats
Bacteria and Archaea are continuously exposed to mobile genetic elements (MGE), such as viruses and plasmids. MGEs may provide a selective advantage, may be neutral or may cause cell damage. To protect against invading DNA, prokaryotes utilize a number of defence systems, including the CRISPR/Cas system. CRISPR/Cas systems rely on integration of invader sequences (spacers) into CRISPR loci that act as a genetic memory of past invasions. Processed CRISPR transcripts are utilized as guides by Cas proteins to cleave complementary invader nucleic acids. In this issue, two groups report on spacer acquisition and turnover dynamics of CRISPR loci in a thermoacidophilic archeon and a pathogenic bacterium. Erdmann and Garrett demonstrate that three of the six CRISPR loci of Sulfolobus solfataricus rapidly acquire new spacer sequences from a conjugative plasmid present in a virus mixture. Intriguingly, two distinct mechanisms of spacer integration are utilized: leader adjacent and internal CRISPR spacer acquisition. Lopez-Sanchez and co-workers studied the type II system of Streptococcus agalactiae and observe heterogeneity in the bacterial population. A fraction of the population lost one or more anti-mobilome spacer sequences during its cultivation, allowing the transfer of a MGE in this subpopulation and a rapid response to altering selection pressures
Molecular biology. A Swiss army knife of immunity
Brouns, S.J.J. - \ 2012
Science 337 (2012)6096. - ISSN 0036-8075 - p. 808 - 809.
streptococcus-thermophilus - small rnas - bacteria - dna - resistance - systems - archaea
Selfish genetic elements are more than a daily nuisance in the life of prokaryotes. Whereas viruses can multiply by reprogramming host cells, or integrate in the host genome as “stowaways,” conjugative plasmids (transferrable extrachromosomal DNA) make cells addicted to plasmid-encoded antitoxin factors, thus preventing their disposal. Bacteria and archaea defend themselves against these invasive elements using an adaptive immune system based on clustered regularly interspaced short palindromic repeats (CRISPRs). On page 816 in this issue, Jinek et al. (1) show how the CRISPR effector enzyme Cas9 from bacteria is directed not by one, but two small RNAs to cleave invader DNA.
RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements
Jore, M.M. ; Brouns, S.J.J. ; Oost, J. van der - \ 2012
Cold Spring Harbor Perspectives in Biology 4 (2012)6. - ISSN 1943-0264
provides acquired-resistance - thermus-thermophilus hb8 - ray crystal-structure - escherichia-coli - streptococcus-thermophilus - sulfolobus-solfataricus - microbial communities - structural basis - dna - system
The CRISPR/Cas system in prokaryotes provides resistance against invading viruses and plasmids. Three distinct stages in the mechanism can be recognized. Initially, fragments of invader DNA are integrated as new spacers into the repetitive CRISPR locus. Subsequently, the CRISPR is transcribed and the transcript is cleaved by a Cas protein within the repeats, generating short RNAs (crRNAs) that contain the spacer sequence. Finally, crRNAs guide the Cas protein machinery to a complementary invader target, either DNA or RNA, resulting in inhibition of virus or plasmid proliferation. In this article, we discuss our current understanding of this fascinating adaptive and heritable defense system, and describe functional similarities and differences with RNAi in eukaryotes
CRISPR Interference Directs Strand Specific Spacer Acquisition
Swarts, D.C. ; Mosterd, C. ; Passel, M.W.J. van; Brouns, S.J.J. - \ 2012
PLoS ONE 7 (2012)4. - ISSN 1932-6203
escherichia-coli k-12 - immune-system - streptococcus-thermophilus - crystal-structure - h-ns - dna - rna - resistance - sequence - defense
BACKGROUND: CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference. RESULTS: We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner. CONCLUSIONS: The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster - in our case the first acquired spacer - and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect
Systems solutions by lactic acid bacteria: from paradigms to practice
Vos, W.M. de - \ 2011
Microbial Cell Factories 10 (2011)Suppl 1. - ISSN 1475-2859
complete genome sequence - lactobacillus-plantarum wcfs1 - lactococcus-lactis - serine proteinase - gene-expression - transcriptome analysis - streptococcus-thermophilus - molecular characterization - phosphotransferase system - gastrointestinal-tract
Lactic acid bacteria are among the powerhouses of the food industry, colonize the surfaces of plants and animals, and contribute to our health and well-being. The genomic characterization of LAB has rocketed and presently over 100 complete or nearly complete genomes are available, many of which serve as scientific paradigms. Moreover, functional and comparative metagenomic studies are taking off and provide a wealth of insight in the activity of lactic acid bacteria used in a variety of applications, ranging from starters in complex fermentations to their marketing as probiotics. In this new era of high throughput analysis, biology has become big science. Hence, there is a need to systematically store the generated information, apply this in an intelligent way, and provide modalities for constructing self-learning systems that can be used for future improvements. This review addresses these systems solutions with a state of the art overview of the present paradigms that relate to the use of lactic acid bacteria in industrial applications. Moreover, an outlook is presented of the future developments that include the transition into practice as well as the use of lactic acid bacteria in synthetic biology and other next generation applications
Structural basis for CRISPR RNA-guided DNA recognition by Cascade
Jore, M.M. ; Lundgren, N.M.J. ; Duijn, E. van; Bultema, J.B. ; Westra, E.R. ; Oost, J. van der; Brouns, S.J.J. ; Beijer, M.R. - \ 2011
Nature Structural and Molecular Biology 18 (2011)5. - ISSN 1545-9985 - p. 529 - 536.
of-flight instrument - mass-spectrometry - r-loops - streptococcus-thermophilus - archaeoglobus-fulgidus - immune-system - protein - defense - prokaryotes - bacteria
The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA